Aconitase (aconitate hydratase; ) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function that arises when the cellular environment changes, such as when iron levels drop [, ]. Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal alpha/beta/alpha domains, a linker region, followed by a C-terminal 'swivel' domain with a beta/beta/alpha structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three alpha/beta/alpha domains (HEAT-4-1-2-3) [].Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic form of cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) [, ]. As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism - it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated [].IRP2 is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis []. Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes []. In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica [, ]. In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances theamount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes the synthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.This entry represents Aconitate hydratase A []found predominantly in bacteria. Iron-responsive element-binding protein 2 []and Cytoplasmic aconitate hydratase []from animals; and Aconitate hydratase []from plants also belong to this family.
This group of cysteine aminopeptidases belong to the peptidase family C5 (adenain family, clan CE). Several adenovirus proteins are synthesised as precursors, requiringprocessing by a protease before the virion is assembled [, ]. Untilrecently, the adenovirus endopeptidase was classified as a serine protease,having been reported to be inhibited by serine protease inhibitors [, ].However, it has since been shown to be inhibited by cysteine proteaseinhibitors, and the catalytic residues are believed to be His-54 andCys-104 [, ].A cysteine peptidase is a proteolytic enzyme that hydrolyses a peptide bond using the thiol group of a cysteine residue as a nucleophile. Hydrolysis involves usually a catalytic triad consisting of the thiol group of the cysteine, the imidazolium ring of a histidine, and a third residue, usually asparagine or aspartic acid, to orientate and activate the imidazolium ring. In only one family of cysteine peptidases, is the role of the general base assigned to a residue other than a histidine: in peptidases from family C89 (acid ceramidase) an arginine is the general base. Cysteine peptidases can be grouped into fourteen different clans, with members of each clan possessing a tertiary fold unique to the clan. Four clans of cysteine peptidases share structural similarities with serine and threonine peptidases and asparagine lyases. From sequence similarities, cysteine peptidases can be clustered into over 80 different families []. Clans CF, CM, CN, CO, CP and PD contain only one family.Cysteine peptidases are often active at acidic pH and are therefore confined to acidic environments, such as the animal lysosome or plant vacuole. Cysteine peptidases can be endopeptidases, aminopeptidases, carboxypeptidases, dipeptidyl-peptidases or omega-peptidases. They are inhibited by thiol chelators such as iodoacetate, iodoacetic acid, N-ethylmaleimide or p-chloromercuribenzoate.Clan CA includes proteins with a papain-like fold. There is a catalytic triad which occurs in the order: Cys/His/Asn (or Asp). A fourth residue, usually Gln, is important for stabilising the acyl intermediate that forms during catalysis, and this precedes the active site Cys. The fold consists of two subdomains with the active site between them. One subdomain consists of a bundle of helices, with the catalytic Cys at the end of one of them, and the other subdomain is a β-barrel with the active site His and Asn (or Asp). There are over thirty families in the clan, and tertiary structures have been solved for members of most of these. Peptidases in clan CA are usually sensitive to the small molecule inhibitor E64, which is ineffective against peptidases from other clans of cysteine peptidases [].Clan CD includes proteins with a caspase-like fold. Proteins in the clan have an α/β/α sandwich structure. There is a catalytic dyad which occurs in the order His/Cys. The active site His occurs in a His-Gly motif and the active site Cys occurs in an Ala-Cys motif; both motifs are preceded by a block of hydrophobic residues []. Specificity is predominantly directed towards residues that occupy the S1 binding pocket, so that caspases cleave aspartyl bonds, legumains cleave asparaginyl bonds, and gingipains cleave lysyl or arginyl bonds.Clan CE includes proteins with an adenain-like fold. The fold consists of two subdomains with the active site between them. One domain is a bundle of helices, and the other a β-barrell. The subdomains are in the opposite order to those found in peptidases from clan CA, and this is reflected in the order of active site residues: His/Asn/Gln/Cys. This has prompted speculation that proteins in clans CA and CE are related, and that members of one clan are derived from a circular permutation of the structure of the other.Clan CL includes proteins with a sortase B-like fold. Peptidases in the clan hydrolyse and transfer bacterial cell wall peptides. The fold shows a closed β-barrel decorated with helices with the active site at one end of the barrel []. The active site consists of a His/Cys catalytic dyad.Cysteine peptidases with a chymotrypsin-like fold are included in clan PA, which also includes serine peptidases. Cysteine peptidases that are N-terminal nucleophile hydrolases are included in clan PB. Cysteine peptidases with a tertiary structure similar to that of the serine-type aspartyl dipeptidase are included in clan PC. Cysteine peptidases with an intein-like fold are included in clan PD, which also includes asparagine lyases.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The first Kv1 sequence (also known as Shaker) was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shaker channel, now constitute the Kv1 family. The family consists of at least 6 genes (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6) which each play distinct physiological roles. A conserved motif found towards the C terminus of these channels is required for efficient processing and surface expression []. Variations in this motif account for the differences in cell surface expression and localisation between family members. These channels are mostly expressed in the brain, but can also be found in non-excitable cells, such as lymphocytes []. This entry features the tandem inactivation domain found at the N terminus of the Kv1.4 potassium channel. It is composed of two subdomains. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, and is thought to work by occluding the ion pathway, as is the case with a classical ball domain. Inactivation domain 2 (ID2, residues 40-50) is a 2.5 turn helix with a high proportion of hydrophobic residues that probably serves to attach ID1 to the cytoplasmic face of the channel. In this way, it can promote rapid access of ID1 to the receptor site in the open channel. ID1 and ID2 function together to bring about fast inactivation of the Kv1.4 channel, which is important for the role of the channel in short-term plasticity [].
ABC transporters belong to the ATP-Binding Cassette (ABC) superfamily, which uses the hydrolysis of ATP to energise diverse biological systems. ABC transporters minimally consist of two conserved regions: a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD). These can be found on the same protein or on two different ones. Most ABC transporters function as a dimer and therefore are constituted of four domains, two ABC modules and two TMDs.ABC transporters are involved in the export or import of a wide variety of substrates ranging from small ions to macromolecules. The major function of ABC import systems is to provide essential nutrients to bacteria. They are found only in prokaryotes and their four constitutive domains are usually encoded by independent polypeptides (two ABC proteins and two TMD proteins). Prokaryotic importers require additional extracytoplasmic binding proteins (one or more per systems) for function. In contrast, export systems are involved in the extrusion of noxious substances, the export of extracellular toxins and the targeting of membrane components. They are found in all living organisms and in general the TMD is fused to the ABC module in a variety of combinations. Some eukaryotic exporters encode the four domains on the same polypeptide chain [].The ABC module (approximately two hundred amino acid residues) is known to bind and hydrolyse ATP, thereby coupling transport to ATP hydrolysis in a large number of biological processes. The cassette is duplicated in several subfamilies. Its primary sequence is highly conserved, displaying a typical phosphate-binding loop: Walker A, and a magnesium binding site: Walker B. Besides these two regions, three other conserved motifs are present in the ABC cassette: the switch region which contains a histidine loop, postulated to polarise the attaching water molecule for hydrolysis, the signature conserved motif (LSGGQ) specific to the ABC transporter, and the Q-motif (between Walker A and the signature), which interacts with the gamma phosphate through a water bond. The Walker A, Walker B, Q-loop and switch region form the nucleotide binding site [, , ].The 3D structure of a monomeric ABC module adopts a stubby L-shape with two distinct arms. ArmI (mainly β-strand) contains Walker A and Walker B. The important residues for ATP hydrolysis and/or binding are located in the P-loop. The ATP-binding pocket is located at the extremity of armI. The perpendicular armII contains mostly the alpha helical subdomain with the signature motif. It only seems to be required for structural integrity of the ABC module. ArmII is in direct contact with the TMD. The hinge between armI and armII contains both the histidine loop and the Q-loop, making contact with the gamma phosphate of the ATP molecule. ATP hydrolysis leads to a conformational change that could facilitate ADP release. In the dimer the two ABC cassettes contact each other through hydrophobic interactions at the antiparallel β-sheet of armI by a two-fold axis [, , , , , ].The ATP-Binding Cassette (ABC) superfamily forms one of the largest of all protein families with a diversity of physiological functions []. Several studies have shown that there is a correlation between the functional characterisation and the phylogenetic classification of the ABC cassette [, ]. More than 50 subfamilies have been described based on a phylogenetic and functional classification [, , ].This entry represents a small clade of ABC-type transporter ATP-binding protein components encoded as part of a three gene cassette along with a periplasmic substrate-binding protein () and a permease (). The organisms containing this cassette are all Actinobacteria and contain numerous proteins requiring the coenzyme F420. The model in this entry was defined based on five such organisms, four of which are lacking all F420 biosynthetic capability save the final side-chain polyglutamate attachment step (via the gene cofE: ). In Jonesia denitrificans DSM 20603 and marine actinobacterium PHSC20C1 this cassette is in an apparent operon with the cofE gene and, in PHSC20C1, also with a F420-dependent glucose-6-phosphate dehydrogenase (). Based on these observations this ATP-binding protein is predicted to be a component of an F420-0 (that is, F420 lacking only the polyglutamate tail) transporter.
ABC transporters belong to the ATP-Binding Cassette (ABC) superfamily, which uses the hydrolysis of ATP to energise diverse biological systems. ABC transporters minimally consist of two conserved regions: a highly conserved ATP binding cassette (ABC) and a less conserved transmembrane domain (TMD). These can be found on the same protein or on two different ones. Most ABC transporters function as a dimer and therefore are constituted of four domains, two ABC modules and two TMDs.ABC transporters are involved in the export or import of a wide variety of substrates ranging from small ions to macromolecules. The major function of ABC import systems is to provide essential nutrients to bacteria. They are found only in prokaryotes and their four constitutive domains are usually encoded by independent polypeptides (two ABC proteins and two TMD proteins). Prokaryotic importers require additional extracytoplasmic binding proteins (one or more per systems) for function. In contrast, export systems are involved in the extrusion of noxious substances, the export of extracellular toxins and the targeting of membrane components. They are found in all living organisms and in general the TMD is fused to the ABC module in a variety of combinations. Some eukaryotic exporters encode the four domains on the same polypeptide chain [].The ABC module (approximately two hundred amino acid residues) is known to bind and hydrolyse ATP, thereby coupling transport to ATP hydrolysis in a large number of biological processes. The cassette is duplicated in several subfamilies. Its primary sequence is highly conserved, displaying a typical phosphate-binding loop: Walker A, and a magnesium binding site: Walker B. Besides these two regions, three other conserved motifs are present in the ABC cassette: the switch region which contains a histidine loop, postulated to polarise the attaching water molecule for hydrolysis, the signature conserved motif (LSGGQ) specific to the ABC transporter, and the Q-motif (between Walker A and the signature), which interacts with the gamma phosphate through a water bond. The Walker A, Walker B, Q-loop and switch region form the nucleotide binding site [, , ].The 3D structure of a monomeric ABC module adopts a stubby L-shape with two distinct arms. ArmI (mainly β-strand) contains Walker A and Walker B. The important residues for ATP hydrolysis and/or binding are located in the P-loop. The ATP-binding pocket is located at the extremity of armI. The perpendicular armII contains mostly the alpha helical subdomain with the signature motif. It only seems to be required for structural integrity of the ABC module. ArmII is in direct contact with the TMD. The hinge between armI and armII contains both the histidine loop and the Q-loop, making contact with the gamma phosphate of the ATP molecule. ATP hydrolysis leads to a conformational change that could facilitate ADP release. In the dimer the two ABC cassettes contact each other through hydrophobic interactions at the antiparallel β-sheet of armI by a two-fold axis [, , , , , ].The ATP-Binding Cassette (ABC) superfamily forms one of the largest of all protein families with a diversity of physiological functions []. Several studies have shown that there is a correlation between the functional characterisation and the phylogenetic classification of the ABC cassette [, ]. More than 50 subfamilies have been described based on a phylogenetic and functional classification [, , ].This entry represents a small clade of ABC-type transporter periplasmic substrate-binding proteins encoded as part of a three gene cassette along with a permease () and an ATPase (). The organisms containing this cassette are all Actinobacteria and contain numerous proteins requiring the coenzyme F420. The model in this entry was defined based on five such organisms, four of which are lacking all F420 biosynthetic capability save the final side-chain polyglutamate attachment step (via the gene cofE: ). In Jonesia denitrificans DSM 20603 and marine actinobacterium PHSC20C1 this cassette is in an apparent operon with the cofE gene and, in PHSC20C1, also with a F420-dependent glucose-6-phosphate dehydrogenase (). Based on these observations this periplasmic substarte-binding protein is predicted to be a component of an F420-0 (that is, F420 lacking only the polyglutamate tail) transporter.
Aspartic peptidases, also known as aspartyl proteases ([intenz:3.4.23.-]), are widely distributed proteolytic enzymes [, , ]known to exist in vertebrates, fungi, plants, protozoa, bacteria, archaea, retroviruses and some plant viruses. All known aspartic peptidases are endopeptidases. A water molecule, activated by two aspartic acid residues, acts as the nucleophile in catalysis. Aspartic peptidases can be grouped into five clans, each of which shows a unique structural fold [].Peptidases in clan AA are either bilobed (family A1 or the pepsin family) or are a homodimer (all other families in the clan, including retropepsin from HIV-1/AIDS) []. Each lobe consists of a single domain with a closed β-barrel and each lobe contributes one Asp to form the active site. Most peptidases in the clan are inhibited by the naturally occurring small-molecule inhibitor pepstatin [].Clan AC contains the single family A8: the signal peptidase 2 family. Members of the family are found in all bacteria. Signal peptidase 2 processes the premurein precursor, removing the signal peptide. The peptidase has four transmembrane domains and the active site is on the periplasmic side of the cell membrane. Cleavage occurs on the amino side of a cysteine where the thiol group has been substituted by a diacylglyceryl group. Site-directed mutagenesis has identified two essential aspartic acid residues which occur in the motifs GNXXDRX and FNXAD (where X is a hydrophobic residue) []. No tertiary structures have been solved for any member of the family, but because of the intramembrane location, the structure is assumed not to be pepsin-like.Clan AD contains two families of transmembrane endopeptidases: A22 and A24. These are also known as "GXGD peptidases"because of a common GXGD motif which includes one of the pair of catalytic aspartic acid residues. Structures are known for members of both families and show a unique, common fold with up to nine transmembrane regions []. The active site aspartic acids are located within a large cavity in the membrane into which water can gain access [].Clan AE contains two families, A25 and A31. Tertiary structures have been solved for members of both families and show a common fold consisting of an α-β-alpha sandwich, in which the beta sheet is five stranded [, ].Clan AF contains the single family A26. Members of the clan are membrane-proteins with a unique fold. Homologues are known only from bacteria. The structure of omptin (also known as OmpT) shows a cylindrical barrel containing ten beta strands inserted in the membrane with the active site residues on the outer surface [].There are two families of aspartic peptidases for which neither structure nor active site residues are known and these are not assigned to clans. Family A5 includes thermopsin, an endopeptidase found only in thermophilic archaea. Family A36 contains sporulation factor SpoIIGA, which is known to process and activate sigma factor E, one of the transcription factors that controls sporulation in bacteria [].This group of aspartic peptidases belongs to the MEROPS family A26 (clan AF). Members of the family are transmembrane proteins. The type example for the family is omptin (also known as protease VII) from Escherichia coli, the product of the ompTgene. Omptin preferentially cleaves polypeptides between two basically-charged amino acids []. The tertiary structure has been solved and shows a ten-stranded beta barrel, and because the strands are amphipathic, hydrolphilic rsidue point into the barrel with the hydrophobic residue on the outside. The active site residues, two pairs of aspartic acids and a histidine, are on opposite sides of the active site groove and at the periplasmic surface []. Because the enzyme is sensitive to the serine protease inhibitor diisopropylfluoro-phosphate [], omptin was incorrectly identified as a serine peptidase.The family also includes the surface protease Pla from the plague organism Yersinia pestis, which is an important virulence factor. Pla can activate plasminogen and inactivate plasmin inhibitor, which may lead to uncontrolled proteolysis and aid entry of the bacterium into the circulation []. Pla is temperature sensitive, with proteolytic activity changing with temperature. At temperatures below 30 C, Pla acts as a coagulase, but at temperatures above 30 C it is fibrolytic [].This entry represents two well conserved regions in the central part of these proteins.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similarproteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.This entry represents the cysteine rich domain of the connexins.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' groupand the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The first Kv1 sequence (also known as Shaker) was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shaker channel, now constitute the Kv1 family. The family consists of at least 6 genes (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6) which each play distinct physiological roles. A conserved motif found towards the C terminus of these channels is required for efficient processing and surface expression []. Variations in this motif account for the differences in cell surface expression and localisation between family members. These channels are mostly expressed in the brain, but can also be found in non-excitable cells, such as lymphocytes []. Kv1.4 channels are found in similar regions to Kv1.1 channels. They areparticularly targeted to axons and possibly terminals, suggesting apre-synaptic role in synaptic transmission.
The peptidase C45 family includes acyl-coenzyme A:6-aminopenicillanic-acid-acyltransferases from fungi. The active site residue for members of this family and family T1 is C-terminal to the autolytic cleavage site. In Penicillium chrysogenum, A:6-aminopenicillanic-acid-acyltransferase serves as the last enzyme in penicillin biosynthetic pathway, which converts isopenicillin N (IPN) to penicillin G, using phenyl-acetyl-CoA or phenoxyacetyl-CoA as acyl donors []. The active mature form of this enzyme is formed by autoproteolysis of the immature precursor, which leads to the exposure of a flexible pocket that was previously buried []. This entry also includes a number of uncharacterised proteins from bacteria, archaea, animals and plants.A cysteine peptidase is a proteolytic enzyme that hydrolyses a peptide bond using the thiol group of a cysteine residue as a nucleophile. Hydrolysis involves usually a catalytic triad consisting of the thiol group of the cysteine, the imidazolium ring of a histidine, and a third residue, usually asparagine or aspartic acid, to orientate and activate the imidazolium ring. In only one family of cysteine peptidases, is the role of the general base assigned to a residue other than a histidine: in peptidases from family C89 (acid ceramidase) an arginine is the general base. Cysteine peptidases can be grouped into fourteen different clans, with members of each clan possessing a tertiary fold unique to the clan. Four clans of cysteine peptidases share structural similarities with serine and threonine peptidases and asparagine lyases. From sequence similarities, cysteine peptidases can be clustered into over 80 different families []. Clans CF, CM, CN, CO, CP and PD contain only one family.Cysteine peptidases are often active at acidic pH and are therefore confined to acidic environments, such as the animal lysosome or plant vacuole. Cysteine peptidases can be endopeptidases, aminopeptidases, carboxypeptidases, dipeptidyl-peptidases or omega-peptidases. They are inhibited by thiol chelators such as iodoacetate, iodoacetic acid, N-ethylmaleimide or p-chloromercuribenzoate.Clan CA includes proteins with a papain-like fold. There is a catalytic triad which occurs in the order: Cys/His/Asn (or Asp). A fourth residue, usually Gln, is important for stabilising the acyl intermediate that forms during catalysis, and this precedes the active site Cys. The fold consists of twosubdomains with the active site between them. One subdomain consists of a bundle of helices, with the catalytic Cys at the end of one of them, and the other subdomain is a β-barrel with the active site His and Asn (or Asp). There are over thirty families in the clan, and tertiary structures have been solved for members of most of these. Peptidases in clan CA are usually sensitive to the small molecule inhibitor E64, which is ineffective against peptidases from other clans of cysteine peptidases [].Clan CD includes proteins with a caspase-like fold. Proteins in the clan have an α/β/α sandwich structure. There is a catalytic dyad which occurs in the order His/Cys. The active site His occurs in a His-Gly motif and the active site Cys occurs in an Ala-Cys motif; both motifs are preceded by a block of hydrophobic residues []. Specificity is predominantly directed towards residues that occupy the S1 binding pocket, so that caspases cleave aspartyl bonds, legumains cleave asparaginyl bonds, and gingipains cleave lysyl or arginyl bonds.Clan CE includes proteins with an adenain-like fold. The fold consists of two subdomains with the active site between them. One domain is a bundle of helices, and the other a β-barrell. The subdomains are in the opposite order to those found in peptidases from clan CA, and this is reflected in the order of active site residues: His/Asn/Gln/Cys. This has prompted speculation that proteins in clans CA and CE are related, and that members of one clan are derived from a circular permutation of the structure of the other.Clan CL includes proteins with a sortase B-like fold. Peptidases in the clan hydrolyse and transfer bacterial cell wall peptides. The fold shows a closed β-barrel decorated with helices with the active site at one end of the barrel []. The active site consists of a His/Cys catalytic dyad.Cysteine peptidases with a chymotrypsin-like fold are included in clan PA, which also includes serine peptidases. Cysteine peptidases that are N-terminal nucleophile hydrolases are included in clan PB. Cysteine peptidases with a tertiary structure similar to that of the serine-type aspartyl dipeptidase are included in clan PC. Cysteine peptidases with an intein-like fold are included in clan PD, which also includes asparagine lyases.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.A voltage-dependent potassium channel gene designated Shaw was initially isolated from Drosophila melanogaster (Fruit fly). Subsequently, several vetebrate potassium channels with similar amino acid sequences were found and, together with the D. melanogaster channel, now constitute the Kv3 family. These channels are thought to play a role in shortening of action potential durations and modulating pre-synaptic neurotransmitter release. In mammals, the family consists of 4 genes (Kv3.1, Kv3.2, Kv3.3 and Kv3.4). Each gene product has its own subcellular location and function.Kv3.3 channels are evenly distributed over the soma and proximal apical dendrites. They have also been found in the lens epithelium, corneal endothelium, cerebellar cells and the deep cerebellar nuclei. When co-expressed with NADPH oxidase, they function as an oxygen sensor complex in airway chemoreceptors.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.A voltage-dependent potassium channel gene designated Shaw was initially isolated from Drosophila melanogaster (Fruit fly). Subsequently, several vetebrate potassium channels with similar amino acid sequences were found and, together with the D. melanogaster channel, now constitute the Kv3 family. These channels are thought to play a role in shortening of action potential durations and modulating pre-synaptic neurotransmitter release. In mammals, the family consists of 4 genes (Kv3.1, Kv3.2, Kv3.3 and Kv3.4). Each gene product has its own subcellular location and function.Kv3.4 channels are expressed in cells that surround the cerebellar Purkinje cells. In the presence of protein kinase C, rapid inactivation is eliminated, resulting in a non-inactivating delayed rectifying current. The implications of this are seen for signal encoding in the central nervous system.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.A voltage-dependent potassium channel gene designated Shaw was initially isolated from Drosophila melanogaster (Fruit fly). Subsequently, several vetebrate potassium channels with similar amino acid sequences were found and, together with the D. melanogaster channel, now constitute the Kv3 family. These channels are thought to play a role in shortening of action potential durations and modulating pre-synaptic neurotransmitter release. In mammals, the family consists of 4 genes (Kv3.1, Kv3.2, Kv3.3 and Kv3.4). Each gene product has its own subcellular location and function.Kv3.1 channels have slow-rising delayed rectifier-type outward currents that inactivate slowly. They have a tentative role in the fast action potential repolarisation abundant in rapidly firing neurons, such as the auditory brainstem, and hippocampal and cortical interneurons. There are two forms, -a and -b, which differ in expression during development. Kv3.1a appears to be found in the neurons of the adult brain, whereas Kv3.1b is expressed in embryonic and perinatal neurons. In addition, Kv3.1 channels can also be found in T-lymphocytes.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.2P-domain channels influence the resting membrane potential and as a result can control cell excitability. In addition, they pass K+in response to changes in membrane potential, and are also tightly regulated by molecular oxygen, GABA (gamma-aminobutyric acid), noradrenaline and serotonin.The first member of this family (TOK1), cloned from Saccharomyces cerevisiae [], ispredicted to have eight potential transmembrane (TM) helices. However,subsequently-cloned two P-domain family members from Drosophila andmammalian species are predicted to have only four TM segments. They areusually referred to as TWIK-related channels (Tandem of P-domains in a Weakly Inward rectifying K+ channel) [, , , ]. Functional characterisation of these channels has revealed a diversity of properties in that they may show inward or outward rectification, their activity may be modulated in different directions by protein phosphorylation, and their sensitivity to changes in intracellular or extracellular pH varies. Despite these disparate properties, they are all thought to share the same topology offour TM segments, including two P-domains. That TWIK-related K+ channelsall produce instantaneous and non-inactivating K+ currents, which do notdisplay a voltage-dependent activation threshold, suggests that they arebackground (leak) K+ channels involved in the generation and modulation of the resting membrane potential in various cell types. Further studies have revealed that they may be found in many species, including: plants, invertebrates and mammals.TWIK family members (TWIK-1 and TWIK-2) produce constitutive K+ currents of weak amplitude []. They are present in a variety of tissues, including brain and cells of the immune system. Together with their functional properties, theirwide distribution suggests that these channels may be involved in the control of background K+ conductances in many cell types.TWIK-1 was the first two P-domain K+channel subunit cloned from humantissue []. It is widely distributed, being particularly abundant in thebrain and heart. It forms a weak inward rectifer K+channel, and has beenfound to be inhibited by internal acidification; its activity is enhancedby protein kinase C phosphorylation.
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.This entry represents a conserved site of connexins.
Aconitase (aconitate hydratase; ) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function that arises when the cellular environment changes, such as when iron levels drop [, ]. Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal alpha/beta/alpha domains, a linker region, followed by a C-terminal 'swivel' domain with a beta/beta/alpha structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three alpha/beta/alpha domains (HEAT-4-1-2-3) [].Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic formof cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) [, ]. As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism - it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated [].IRP2 is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis []. Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes []. In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica [, ]. In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances the amount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes the synthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.3-isopropylmalate dehydratase (or isopropylmalate isomerase; ) catalyses the stereo-specific isomerisation of 2-isopropylmalate and 3-isopropylmalate, via the formation of 2-isopropylmaleate. This enzyme performs the second step in the biosynthesis of leucine, and is present in most prokaryotes and many fungal species. The prokaryotic enzyme is a heterodimer composed of a large (LeuC) and small (LeuD) subunit, while the fungal form is a monomeric enzyme. Both forms of isopropylmalate are related and are part of the larger aconitase family []. Aconitases are mostly monomeric proteins which share four domains in common and contain a single, labile [4Fe-4S]cluster. Three structural domains (1, 2 and 3) are tightly packed around the iron-sulphur cluster, while a fourth domain (4) forms a deep active-site cleft. The prokaryotic enzyme is encoded by two adjacent genes, leuC and leuD, corresponding to aconitase domains 1-3 and 4 respectively [, ]. LeuC does not bind an iron-sulphur cluster. It is thought that some prokaryotic isopropylamalate dehydrogenases can also function as homoaconitase , converting cis-homoaconitate to homoisocitric acid in lysine biosynthesis []. Homoaconitase has been identified in higher fungi (mitochondria) and several archaea and one thermophilic species of bacteria, Thermus thermophilus []. It is also found in the higher plant Arabidopsis thaliana, where it is targeted to the chloroplast [].This entry represents a region containing 3 domains, each with a 3-layer alpha/beta/alpha topology. This region represents the [4Fe-4S]cluster-binding region found at the N-terminal of eukaryotic mAcn, cAcn/IPR1 and IRP2, and bacterial AcnA, but in the C-terminal of bacterial AcnB. This domain is also found in the large subunit of isopropylmalate dehydratase (LeuC).
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate throughnervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.This domain is found in the N-terminal region of these proteins.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.The first Kv1 sequence (also known as Shaker) was found in Drosophila melanogaster (Fruit fly). Several vertebrate potassium channels with similar amino acid sequences were subsequently found and, together with the D. melanogaster Shaker channel, now constitute the Kv1 family. The family consists of at least 6 genes (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5 and Kv1.6) which each play distinct physiological roles. A conserved motif found towards the C terminus of these channels is required for efficient processing and surface expression []. Variations in this motif account for the differences in cell surface expression and localisation between family members. These channels are mostly expressed in the brain, but can also be found in non-excitable cells, such as lymphocytes []. This entry features the tandem inactivation domain superfamily found at the N terminus of the Kv1.4 potassium channel. It is composed of two subdomains. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, and is thought to work by occluding the ion pathway, as is the case with a classical ball domain. Inactivation domain 2 (ID2, residues 40-50) is a 2.5 turn helix with a high proportion of hydrophobic residues that probably serves to attach ID1 to the cytoplasmic face of the channel. In this way, it can promote rapid access of ID1 to the receptor site in the open channel. ID1 and ID2 function together to bring about fast inactivation of the Kv1.4 channel, which is important for the role of the channel in short-term plasticity [].
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectorsin signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.A voltage-dependent potassium channel gene designated Shaw was initially isolated from Drosophila melanogaster (Fruit fly). Subsequently, several vetebrate potassium channels with similar amino acid sequences were found and, together with the D. melanogaster channel, now constitute the Kv3 family. These channels are thought to play a role in shortening of action potential durations and modulating pre-synaptic neurotransmitter release. In mammals, the family consists of 4 genes (Kv3.1, Kv3.2, Kv3.3 and Kv3.4). Each gene product has its own subcellular location and function.This particular family model is based on Kv3x proteins from invertebrates, such as Caenorhabditis elegans, Drosophila melanogaster (Fruit fly), Anopheles gambiae (African malaria mosquito), and Aplysia californica (California sea hare).
The connexins are a family of integral membrane proteins that oligomerise to form intercellular channels that are clustered at gap junctions. These channels are specialised sites of cell-cell contact that allow the passage of ions, intracellular metabolites and messenger molecules (with molecular weight less than 1-2kDa) from the cytoplasm of one cell to its opposing neighbours. They are found in almost all vertebrate cell types, and somewhat similar proteins have been cloned from plant species. Invertebrates utilise a different family of molecules, innexins, that share a similar predicted secondary structure to the vertebrate connexins, but have no sequence identity to them [].Vertebrate gap junction channels are thought to participate in diverse biological functions. For instance, in the heart they permit the rapid cell-cell transfer of action potentials, ensuring coordinated contraction of the cardiomyocytes. They are also responsible for neurotransmission at specialised 'electrical' synapses. In non-excitable tissues, such as the liver, they may allow metabolic cooperation between cells. In the brain, glial cells are extensively-coupled by gap junctions; this allows waves of intracellular Ca2+to propagate through nervous tissue, and may contribute to their ability to spatially-buffer local changes in extracellular K+concentration [].The connexin protein family is encoded by at least 13 genes in rodents, with many homologues cloned from other species. They show overlapping tissue expression patterns, most tissues expressing more than one connexin type. Their conductances, permeability to different molecules, phosphorylation and voltage-dependence of their gating, have been found to vary. Possible communication diversity is increased further by the fact that gap junctions may be formed by the association of different connexin isoforms from apposing cells. However, in vitro studies have shown that not all possible combinations of connexins produce active channels [, ].Hydropathy analysis predicts that all cloned connexins share a common transmembrane (TM) topology. Each connexin is thought to contain 4 TMdomains, with two extracellular and three cytoplasmic regions. This modelhas been validated for several of the family members by in vitrobiochemicalanalysis. Both N- and C-termini are thought to face the cytoplasm, and thethird TM domain has an amphipathic character, suggesting that it contributesto the lining of the formed-channel. Amino acid sequence identity betweenthe isoforms is ~50-80%, with the TM domains being well conserved. Bothextracellular loops contain characteristically conserved cysteine residues,which likely form intramolecular disulphide bonds. By contrast, the singleputative intracellular loop (between TM domains 2 and 3) and the cytoplasmicC terminus are highly variable among the family members.Six connexins arethought to associate to form a hemi-channel, or connexon. Two connexons theninteract (likely via the extracellular loops of their connexins) to form thecomplete gap junction channel.NH2-*** *** *************-COOH** ** ** **** ** ** ** Cytoplasmic---**----**-----**----**----------------** ** ** ** Membrane** ** ** **---**----**-----**----**----------------** ** ** ** Extracellular** ** ** **** **Two sets of nomenclature have been used to identify the connexins. Thefirst, and most commonly used, classifies the connexin molecules accordingto molecular weight, such as connexin43 (abbreviated to Cx43), indicatinga connexin of molecular weight close to 43kDa. However, studies haverevealed cases where clear functional homologues exist across speciesthat have quite different molecular masses; therefore, an alternativenomenclature was proposed based on evolutionary considerations, whichdivides the family into two major subclasses, alpha and beta, each with anumber of members []. Due to their ubiquity and overlapping tissue distributions, it has proved difficult to elucidate the functions of individual connexin isoforms. To circumvent this problem, particular connexin-encoding genes have been subjected to targeted-disruption in mice, and the phenotype of the resulting animals investigated. Around half the connexin isoforms have been investigated in this manner []. Further insight into the functional roles of connexins has come from the discovery that a number of human diseases are caused by mutations in connexin genes. For instance, mutations in Cx32 give rise to a form of inherited peripheral neuropathy called X-linked dominant Charcot-Marie-Tooth disease []. Similarly, mutations in Cx26 are responsible for both autosomal recessive and dominant forms of nonsyndromic deafness, a disorder characterised by hearing loss, with no apparent effects on other organ systems.Gap junction alpha-1 protein (also called connexin43, or Cx43) is a connexinof 381 amino acid residues (human isoform) that is widely expressed inseveral organs and cell types, and is the principal gap junction protein ofthe heart. Characterisation of genetically-engineered mice that lack Cx43,and also of human patients that have spontaneously-occurring mutations inthe gene encoding it (GJA1), suggest Cx43 is essential for the developmentof normal cardiac architecture and ventricular conduction. Mice lacking Cx43survive to term but die shortly after birth. They have cardiac malformationsthat lead to the obstruction of the pulmonary artery, leading to neonatalcyanosis, and subsequent death. This phenotype is reminiscent of some formsof stenosis of the pulmonary artery. Human subjects with visceroatrialheterotaxia (a heart disorder characterised by arterial defects), have beenfound to have points mutations in the Cx43-encoding gene, as a result of which a potential phosphorylation site within the C terminus is disrupted. Consequently, although these mutant Cx43 molecules still form functional gapjunction channels, their response to protein kinase activation is impaired.This domain is found in the C-terminal region of these proteins.
Aconitase (aconitate hydratase; ) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function that arises when the cellular environment changes, such as when iron levels drop [, ]. Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal alpha/beta/alpha domains, a linker region, followed by a C-terminal 'swivel' domain with a beta/beta/alpha structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three alpha/beta/alpha domains (HEAT-4-1-2-3) [].Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic form of cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) [, ]. As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism - it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated [].IRP2 is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis []. Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes []. In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica [, ]. In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances the amount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes the synthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.This entry represents the N-terminal HEAT-like domain, which is present in bacterial aconitase (AcnB), but not in AcnA or eukaryotic cAcn/IRP2 or mAcn. This domain is multi-helical, forming two curved layers in a right-handed α-α superhelix. HEAT-like domains are usually implicated in protein-protein interactions. The HEAT-like domain and the 'swivel' domain that follows it were shown to be sufficient for dimerisation and for AcnB binding to mRNA. An iron-mediated dimerisation mechanism may be responsible for switching AcnB between its catalytic and regulatory roles, as dimerisation requires iron while mRNA binding is inhibited by iron.
Aconitase (aconitate hydratase; ) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function that arises when the cellular environment changes, such as when iron levels drop [, ]. Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal alpha/beta/alpha domains, a linker region, followed by a C-terminal 'swivel' domain with a beta/beta/alpha structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three alpha/beta/alpha domains (HEAT-4-1-2-3) [].Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic form of cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) [, ]. As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism - it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated [].IRP2 is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis []. Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes []. In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica [, ]. In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances the amount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes the synthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.3-isopropylmalate dehydratase (or isopropylmalate isomerase; ) catalyses the stereo-specific isomerisation of 2-isopropylmalate and 3-isopropylmalate, via the formation of 2-isopropylmaleate. This enzyme performs the second step in the biosynthesis of leucine, and is present in most prokaryotes and many fungal species. The prokaryotic enzyme is a heterodimer composed of a large (LeuC) and small (LeuD) subunit, while the fungal form is a monomeric enzyme. Both forms of isopropylmalate are related and are part of the larger aconitase family []. Aconitases are mostly monomeric proteins which share four domains in common and contain a single, labile [4Fe-4S]cluster. Three structural domains (1, 2 and 3) are tightly packed around the iron-sulphur cluster, while a fourth domain (4) forms a deep active-site cleft. The prokaryotic enzyme is encoded by two adjacent genes, leuC and leuD, corresponding to aconitase domains 1-3 and 4 respectively [, ]. LeuC does not bind an iron-sulphur cluster. It is thought that some prokaryotic isopropylamalate dehydrogenases can also function as homoaconitase , converting cis-homoaconitate to homoisocitric acid in lysine biosynthesis []. Homoaconitase has been identified in higher fungi (mitochondria) and several archaea and one thermophilic species of bacteria, Thermus thermophilus []. It is also found in the higher plant Arabidopsis thaliana, where it is targeted to the chloroplast [].This superfamily represents a domain with an alpha/beta/alpha topology. This structural domain usually occurs in triplicate, with domains 1 and 3 being the most closely related since they share the same pseudo 2-fold symmetry. This entry represents domains 1 and 3. This triple domain region is found at the N-terminal of eukaryotic mAcn, cAcn/IPR1 and IRP2, and bacterial AcnA, but in the C-terminal of bacterial AcnB; in each case, this region binds the [4Fe-4S]-cluster. This triple domain region is also found in the large subunit of isopropylmalate dehydratase (LeuC).
Aconitase (aconitate hydratase; ) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function that ariseswhen the cellular environment changes, such as when iron levels drop [, ]. Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal alpha/beta/alpha domains, a linker region, followed by a C-terminal 'swivel' domain with a beta/beta/alpha structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three alpha/beta/alpha domains (HEAT-4-1-2-3) [].Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic form of cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) [, ]. As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism - it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated [].IRP2 is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis []. Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes []. In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica [, ]. In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances the amount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes the synthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.3-isopropylmalate dehydratase (or isopropylmalate isomerase; ) catalyses the stereo-specific isomerisation of 2-isopropylmalate and 3-isopropylmalate, via the formation of 2-isopropylmaleate. This enzyme performs the second step in the biosynthesis of leucine, and is present in most prokaryotes and many fungal species. The prokaryotic enzyme is a heterodimer composed of a large (LeuC) and small (LeuD) subunit, while the fungal form is a monomeric enzyme. Both forms of isopropylmalate are related and are part of the larger aconitase family []. Aconitases are mostly monomeric proteins which share four domains in common and contain a single, labile [4Fe-4S]cluster. Three structural domains (1, 2 and 3) are tightly packed around the iron-sulphur cluster, while a fourth domain (4) forms a deep active-site cleft. The prokaryotic enzyme is encoded by two adjacent genes, leuC and leuD, corresponding to aconitase domains 1-3 and 4 respectively [, ]. LeuC does not bind an iron-sulphur cluster. It is thought that some prokaryotic isopropylamalate dehydrogenases can also function as homoaconitase , converting cis-homoaconitate to homoisocitric acid in lysine biosynthesis []. Homoaconitase has been identified in higher fungi (mitochondria) and several archaea and one thermophilic species of bacteria, Thermus thermophilus []. It is also found in the higher plant Arabidopsis thaliana, where it is targeted to the chloroplast [].This superfamily represents a domain with an alpha/beta/alpha topology. This structural domain usually occurs in triplicate, with domains 1 and 3 being the most closely related since they share the same pseudo 2-fold symmetry. This entry represents domain 2. This triple domain region is found at the N-terminal of eukaryotic mAcn, cAcn/IPR1 and IRP2, and bacterial AcnA, but in the C-terminal of bacterial AcnB; in each case, this region binds the [4Fe-4S]-cluster.
Aconitase (aconitate hydratase; ) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function that arises when the cellular environment changes, such as when iron levels drop [, ]. Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal alpha/beta/alpha domains, a linker region, followed by a C-terminal 'swivel' domain with a beta/beta/alpha structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three alpha/beta/alpha domains (HEAT-4-1-2-3) [].Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic form of cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) [, ]. As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism - it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated [].IRP2 is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis []. Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes []. In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica [, ]. In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances the amount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes the synthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.3-isopropylmalate dehydratase (or isopropylmalate isomerase; ) catalyses the stereo-specific isomerisation of 2-isopropylmalate and 3-isopropylmalate, via the formation of 2-isopropylmaleate. This enzyme performs the second step in the biosynthesis of leucine, and is present in most prokaryotes and many fungal species. The prokaryotic enzyme is a heterodimer composed of a large (LeuC) and small (LeuD) subunit, while the fungal form is a monomeric enzyme. Both forms of isopropylmalate are related and are part of the larger aconitase family []. Aconitases are mostly monomeric proteins which share four domains in common and contain a single, labile [4Fe-4S]cluster. Three structural domains (1, 2 and 3) are tightly packed around the iron-sulphur cluster, while a fourth domain (4) forms a deep active-site cleft. The prokaryotic enzyme is encoded by two adjacent genes, leuC and leuD, corresponding to aconitase domains 1-3 and 4 respectively [, ]. LeuC does not bind an iron-sulphur cluster. It is thought that some prokaryotic isopropylamalate dehydrogenases can also function as homoaconitase , converting cis-homoaconitate to homoisocitric acid in lysine biosynthesis []. Homoaconitase has been identified in higher fungi (mitochondria) and several archaea and one thermophilic species of bacteria, Thermus thermophilus []. It is also found in the higher plant Arabidopsis thaliana, where it is targeted to the chloroplast [].This superfamily represents the 'swivel' domain found at the C-terminal of eukaryotic mAcn, cAcn/IPR1 and IRP2, and bacterial AcnA, but in the N-terminal region following the HEAT-like domain in bacterial AcnB. This domain has a three layer beta/beta/alpha structure, and in cytosolic Acn is known to rotate between the cAcn and IRP1 forms of the enzyme. This domain is also found in the small subunit of isopropylmalate dehydratase (LeuD).
Neurotransmitter ligand-gatedion channels are transmembrane receptor-ion channel complexes that open transiently upon binding of specific ligands, allowing rapid transmission of signals at chemical synapses [, ]. Five of these ion channel receptor families have been shown to form a sequence-related superfamily:Nicotinic acetylcholine receptor (AchR), an excitatory cation channel in vertebrates and invertebrates; in vertebrate motor endplates it is composed of alpha, beta, gamma and delta/epsilon subunits; in neurons it is composed of alpha and non-alpha (or beta) subunits [].Glycine receptor, an inhibitory chloride ion channel composed of alpha and beta subunits [].Gamma-aminobutyric acid (GABA) receptor, an inhibitory chloride ion channel; at least four types of subunits (alpha, beta, gamma and delta) are known [].Serotonin 5HT3 receptor, of which there are seven major types (5HT3-5HT7) [].Glutamate receptor, an excitatory cation channel of which at least three types have been described (kainate, N-methyl-D-aspartate (NMDA) and quisqualate) [].These receptors possess a pentameric structure (made up of varying subunits), surrounding a central pore. All known sequences of subunits from neurotransmitter-gated ion-channels are structurally related. They are composed of a large extracellular glycosylated N-terminal ligand-binding domain, followed by three hydrophobic transmembrane regions which form the ionic channel, followed by an intracellular region of variable length. A fourth hydrophobic region is found at the C-terminal of the sequence [, ].Gamma-aminobutyric acid type A (GABAA) receptors are members of the neurotransmitter ligand-gated ion channels: they mediate neuronal inhibition on binding GABA. The effects of GABA on GABAA receptors are modulated by a range of therapeutically important drugs, including barbiturates, anaesthetics and benzodiazepines (BZs) []. The BZs are a diverse range of compounds, including widely prescribed drugs, such as librium and valium, and their interaction with GABAA receptors provides the most potent pharmacological means of distinguishing different GABAA receptor subtypes.GABAA receptors are pentameric membrane proteins that operate GABA-gated chloride channels []. Eight types of receptor subunit have been cloned, with multiple subtypes within some classes: alpha 1-6, beta 1-4, gamma 1-4, delta, epsilon, pi, rho 1-3 and theta [, ]. Subunits are typically 50-60kDa in size and comprise a long N-terminal extracellular domain, containing a putative signal peptide and a disulphide-bonded beta structural loop; 4 putative transmembrane (TM) domains; and a large cytoplasmic loop connecting the third and fourth TM domains. Amongst family members, the large cytoplasmic loop displays the most divergence in terms of primary structure, the TM domains showing the highest level of sequence conservation [].Most GABAA receptors contain one type of alpha and beta subunit, and a single gamma polypeptide in a ratio of 2:2:1 [], though in some cases other subunits such as epsilon or delta may replace gamma. The BZ binding site is located at the interface of adjacent alpha and gamma subunits; therefore, the type of alpha and gamma subunits present is instrumental in determining BZ selectivity and sensitivity. Receptors can be categorised into 3 groups based on their alpha subunit content and, hence, sensitivity to BZs: alpha 1-containing receptors have greatest sensitivity towards BZs (type I); alpha 2, 3 and 5-containing receptors have similar but distinguishable properties (type II); and alpha 4- and 6-containing assemblies have very low BZ affinity []. A conserved histidine residue in the alpha subunit of type I and II receptors is believed to be responsible for BZ affinity []. GABAA receptors can be characterised by their sensitivitytowards a selective antagonist, bicuculline. A GABA receptor has been identified that is insensitive to bicuculline and classical GABAA modulators but has an enhanced affinity for GABA. This receptor, unlike most GABAA receptors, is composed principally of rho subunits and was initially termed 'GABAC' in recognition of its altered pharmacology []. Despite these differences, rho subunits are generally considered to be part of the GABAAfamily of receptor proteins due to similarities in sequence and topology.Whilst early studies supported the view that rho subunits assembled to forma homopentamer, it has been shown that a mutant rho 1 protein is able tocoassemble with GABAA gamma 2 subunits as well as the glycine receptor alphasubunit. Rho subunit mRNA occurs prominently in both human and ratretina [], each subunit showing a characteristic pattern of spatial expression. In rat retina, rho 1 mRNA has been detected only in bipolarcells, whereas rho 2 transcripts have been detected in both bipolar andganglion cells. In retinal tissues, expression of rho 3 mRNA is exclusive toganglion cells. Reverse transcriptase PCR (RT-PCR) and in situhybridisation have shown rho transcripts also to be present in other regionsof the brain, specifically those involved in visual signal processing, suchas the superior colliculus and visual cortex.This entry represents Rho 2 subunits.
Neurotransmitter ligand-gated ion channels are transmembrane receptor-ion channel complexes that open transiently upon binding of specific ligands, allowing rapid transmission of signals at chemical synapses [, ]. Five of these ion channel receptor families have been shown to form a sequence-related superfamily:Nicotinic acetylcholine receptor (AchR), an excitatory cation channel in vertebrates and invertebrates; in vertebrate motor endplates it is composed of alpha, beta, gamma and delta/epsilon subunits; in neurons it is composed of alpha and non-alpha (or beta) subunits [].Glycine receptor, an inhibitory chloride ion channel composed of alpha and beta subunits [].Gamma-aminobutyric acid (GABA) receptor, an inhibitory chloride ion channel; at least four types of subunits (alpha, beta, gamma and delta) are known [].Serotonin 5HT3 receptor, of which there are seven major types (5HT3-5HT7) [].Glutamate receptor, an excitatory cation channel of which at least three types have been described (kainate, N-methyl-D-aspartate (NMDA) and quisqualate) [].These receptors possess a pentameric structure (made up of varying subunits), surrounding a central pore. All known sequences of subunits from neurotransmitter-gated ion-channels are structurally related. They are composed of a large extracellular glycosylated N-terminal ligand-binding domain, followed by three hydrophobic transmembrane regions which form the ionic channel, followed by an intracellular region of variable length. A fourth hydrophobic region is found at the C-terminal of the sequence [, ].Gamma-aminobutyric acid type A (GABAA) receptors are members of the neurotransmitter ligand-gated ion channels: they mediate neuronal inhibition on binding GABA. The effects of GABA on GABAA receptors are modulated by a range of therapeutically important drugs, including barbiturates, anaesthetics and benzodiazepines (BZs) []. The BZs are a diverse range of compounds, including widely prescribed drugs, such as librium and valium, and their interaction with GABAA receptorsprovides the most potent pharmacological means of distinguishing different GABAA receptor subtypes.GABAA receptors are pentameric membrane proteins that operate GABA-gated chloride channels []. Eight types of receptor subunit have been cloned, with multiple subtypes within some classes: alpha 1-6, beta 1-4, gamma 1-4, delta, epsilon, pi, rho 1-3 and theta [, ]. Subunits are typically 50-60kDa in size and comprise a long N-terminal extracellular domain, containing a putative signal peptide and a disulphide-bonded beta structural loop; 4 putative transmembrane (TM) domains; and a large cytoplasmic loop connecting the third and fourth TM domains. Amongst family members, the large cytoplasmic loop displays the most divergence in terms of primary structure, the TM domains showing the highest level of sequence conservation [].Most GABAA receptors contain one type of alpha and beta subunit, and a single gamma polypeptide in a ratio of 2:2:1 [], though in some cases other subunits such as epsilon or delta may replace gamma. The BZ binding site is located at the interface of adjacent alpha and gamma subunits; therefore, the type of alpha and gamma subunits present is instrumental in determining BZ selectivity and sensitivity. Receptors can be categorised into 3 groups based on their alpha subunit content and, hence, sensitivity to BZs: alpha 1-containing receptors have greatest sensitivity towards BZs (type I); alpha 2, 3 and 5-containing receptors have similar but distinguishable properties (type II); and alpha 4- and 6-containing assemblies have very low BZ affinity []. A conserved histidine residue in the alpha subunit of type I and II receptors is believed to be responsible for BZ affinity []. GABAA receptors can be characterised by their sensitivitytowards a selective antagonist, bicuculline. A GABA receptor has been identified that is insensitive to bicuculline and classical GABAA modulators but has an enhanced affinity for GABA. This receptor, unlike most GABAA receptors, is composed principally of rho subunits and was initially termed 'GABAC' in recognition of its altered pharmacology []. Despite these differences, rho subunits are generally considered to be part of the GABAAfamily of receptor proteins due to similarities in sequence and topology.Whilst early studies supported the view that rho subunits assembled to forma homopentamer, it has been shown that a mutant rho 1 protein is able tocoassemble with GABAA gamma 2 subunits as well as the glycine receptor alphasubunit. Rho subunit mRNA occurs prominently in both human and ratretina [], each subunit showing a characteristic pattern of spatial expression. In rat retina, rho 1 mRNA has been detected only in bipolarcells, whereas rho 2 transcripts have been detected in both bipolar andganglion cells. In retinal tissues, expression of rho 3 mRNA is exclusive toganglion cells. Reverse transcriptase PCR (RT-PCR) and in situhybridisation have shown rho transcripts also to be present in other regionsof the brain, specifically those involved in visual signal processing, suchas the superior colliculus and visual cortex.This entry represents the GABAA Rho subunits.
Neurotransmitter ligand-gated ion channels are transmembrane receptor-ion channel complexes that open transiently upon binding of specific ligands, allowing rapid transmission of signals at chemical synapses [, ]. Five of these ion channel receptor families have been shown to form a sequence-related superfamily:Nicotinic acetylcholine receptor (AchR), an excitatory cation channel in vertebrates and invertebrates; in vertebrate motor endplates it is composed of alpha, beta, gamma and delta/epsilon subunits; in neurons it is composed of alpha and non-alpha (or beta) subunits [].Glycine receptor, an inhibitory chloride ion channel composed of alpha and beta subunits [].Gamma-aminobutyric acid (GABA) receptor, an inhibitory chloride ion channel; at least four types of subunits (alpha, beta, gamma and delta) are known [].Serotonin 5HT3 receptor, of which there are seven major types (5HT3-5HT7) [].Glutamate receptor, an excitatory cation channel of which at least three types have been described (kainate, N-methyl-D-aspartate (NMDA) and quisqualate) [].These receptors possess a pentameric structure (made up of varying subunits), surrounding a central pore. All known sequences of subunits from neurotransmitter-gated ion-channels are structurally related. They are composed of a large extracellular glycosylated N-terminal ligand-binding domain, followed by three hydrophobic transmembrane regions which form the ionic channel, followed by an intracellular region of variable length. A fourth hydrophobic region is found at the C-terminal of the sequence [, ].Gamma-aminobutyric acid type A (GABAA) receptors are members of the neurotransmitter ligand-gated ion channels: they mediate neuronal inhibition on binding GABA. The effects of GABA on GABAA receptors are modulated by a range of therapeutically important drugs, including barbiturates, anaesthetics and benzodiazepines (BZs) []. The BZs are a diverse range of compounds, including widely prescribed drugs, such as librium and valium, and their interaction with GABAA receptors provides the most potent pharmacological means of distinguishing different GABAA receptor subtypes.GABAA receptors are pentameric membrane proteins that operate GABA-gated chloride channels []. Eight types of receptor subunit have been cloned, with multiple subtypes within some classes: alpha 1-6, beta 1-4, gamma 1-4, delta, epsilon, pi, rho 1-3 and theta [, ]. Subunits are typically 50-60kDa in size and comprise a long N-terminal extracellular domain, containing a putative signal peptide and a disulphide-bonded beta structural loop; 4 putative transmembrane (TM) domains; and a large cytoplasmic loop connecting the third and fourth TM domains. Amongst family members, the large cytoplasmic loop displays the most divergence in terms of primary structure, the TM domains showing the highest level of sequence conservation [].Most GABAA receptors contain one type of alpha and beta subunit, and a single gamma polypeptide in a ratio of 2:2:1 [], though in some cases other subunits such as epsilon or delta may replace gamma. The BZ binding site is located at the interface of adjacent alpha and gamma subunits; therefore, the type of alpha and gamma subunits present is instrumental in determining BZ selectivity and sensitivity. Receptors can be categorised into 3 groups based on their alpha subunit content and, hence, sensitivity to BZs: alpha 1-containing receptors have greatest sensitivity towards BZs (type I); alpha 2, 3 and 5-containing receptors have similar but distinguishable properties (type II); and alpha 4- and 6-containing assemblies have very low BZ affinity []. A conserved histidine residue in the alpha subunit of type I and II receptors is believed to be responsible for BZ affinity []. GABAA receptors can be characterised by their sensitivitytowards a selective antagonist, bicuculline. A GABA receptor has been identified that is insensitive to bicuculline and classical GABAA modulators but has an enhanced affinity for GABA. This receptor, unlike most GABAA receptors, is composed principally of rho subunits and was initially termed 'GABAC' in recognition of its altered pharmacology []. Despite these differences, rho subunitsare generally considered to be part of the GABAAfamily of receptor proteins due to similarities in sequence and topology.Whilst early studies supported the view that rho subunits assembled to forma homopentamer, it has been shown that a mutant rho 1 protein is able tocoassemble with GABAA gamma 2 subunits as well as the glycine receptor alphasubunit. Rho subunit mRNA occurs prominently in both human and ratretina [], each subunit showing a characteristic pattern of spatial expression. In rat retina, rho 1 mRNA has been detected only in bipolarcells, whereas rho 2 transcripts have been detected in both bipolar andganglion cells. In retinal tissues, expression of rho 3 mRNA is exclusive toganglion cells. Reverse transcriptase PCR (RT-PCR) and in situhybridisation have shown rho transcripts also to be present in other regionsof the brain, specifically those involved in visual signal processing, suchas the superior colliculus and visual cortex.This entry represents Rho 1 subunits.
Neurotransmitter ligand-gated ion channels are transmembrane receptor-ion channel complexes that open transiently upon binding of specific ligands, allowing rapid transmission of signals at chemical synapses [, ]. Five of these ion channel receptor families have been shown to form a sequence-related superfamily:Nicotinic acetylcholine receptor (AchR), an excitatory cation channel in vertebrates and invertebrates; in vertebrate motor endplates it is composed of alpha, beta, gamma and delta/epsilon subunits; in neurons it is composed of alpha and non-alpha (or beta) subunits [].Glycine receptor, an inhibitory chloride ion channel composed of alpha and beta subunits [].Gamma-aminobutyric acid (GABA) receptor, an inhibitory chloride ion channel; at least four types of subunits (alpha, beta, gamma and delta) are known [].Serotonin 5HT3 receptor, of which there are seven major types (5HT3-5HT7) [].Glutamate receptor, an excitatory cation channel of which at least three types have been described (kainate, N-methyl-D-aspartate (NMDA) and quisqualate) [].These receptors possess a pentameric structure (made up of varying subunits), surrounding a central pore. All known sequences of subunits from neurotransmitter-gated ion-channels are structurally related. They are composed of a large extracellular glycosylated N-terminal ligand-binding domain, followed by three hydrophobic transmembrane regions which form the ionic channel, followed by an intracellular region of variable length. A fourth hydrophobic region is found at the C-terminal of the sequence [, ].Glycine is a major inhibitory neurotransmitter (NT) in the adult vertebratecentral nervous system (CNS). Glycinergic synapses have a well-establishedrole in the processing of motor and sensory information that controlsmovement, vision and audition []. This action of glycine is mediatedthrough its interaction with the glycine receptor (GlyR): an intrinsicchloride channel is opened in response to agonist binding. The subsequentinflux of anions prevents membrane depolarisation and neuronal firinginduced by excitatory NTs. Strychnine acts as a competitive antagonist ofglycine binding, thereby reducing the activity of inhibitory neurones.Poisoning with strychnine is characterised by over-excitation, muscle spasmsand convulsions. Whilst glycine is the principal physiological agonist atGlyRs, taurine and beta-alanine also behave as agonists []. Compounds thatmodulate GlyR activity include zinc, some alcohols and anaesthetics,picrotoxin, cocaine and some anticonvulsants. GlyRs were thought for sometime to be localised exclusively in the brain stem and spinal cord, but havesince been found to be expressed more widely, including the cochlear nuclei,cerebellar cortex and forebrain [].GlyRs belong to the ligand-gated ion channel family, which also includes theinhibitory gamma-aminobutyric acid type A (GABAA) and excitatory nicotinicacetylcholine (nACh) and serotonin type 3 (5-HT3) receptors [].Affinity-purified GlyR was found to contain two glycosylated membraneproteins of 48kDa and 56kDa, corresponding to alpha and beta subunits,respectively. Four genes encoding alpha subunits have been identified (GLRA1to 4), together with a single beta polypeptide (GLRB). The heterogeneity ofalpha subunits is further increased by alternative exon splicing, yieldingtwo isoforms of GLRA1 to 3 []. The characteristics of different GlyRsubtypes, therefore, can be largely explained by their GLRA content.GlyRs are generally believed to adopt a pentameric structure in vivo: fivesubunits assemble to form a ring structure with a central pore. Typically, astoichiometry of 3:2 (alpha:beta) is observed []. GlyR subunits shareahigh overall level of sequence similarity both with themselves and with thesubunits of the GABAA and nACh receptors. Four highly conserved segmentshave been proposed to correspond to transmembrane (TM) alpha helices (TM1-4), the second of which is thought to contribute to the pore wall []. A long extracellular N-terminal segment precedes TM1 and a long cytoplasmic loop links TM3 and 4. Short cytoplasmic and extracellular loops join TM1-2 andTM2-3, respectively, and a short C-terminal sequence follows TM4. Studiesusing radiolabelled strychnine have shown the alpha subunit to beresponsible for ligand binding, the critical residues for this interaction lying within the N-terminal domain. The beta subunit plays a structuralrole, contributing one of its TM domains to the pore wall as well as playinga putative role in postsynaptic clustering of the receptor.In several mammalian species, defects in glycinergic transmission areassociated with complex motor disorders. Mutations in the gene encodingGLRA1 give rise to hyperplexia, or startle disease []. This ischaracterised by muscular spasms in response to unexpected light or noisestimuli, similar to the symptoms of sublethal doses of strychnine. Themutations result in amino acid substitutions within the TM1-2 and TM3-4loops, suggesting that these regions are involved in the transduction ofligand binding into channel activation.
Neurotransmitter ligand-gated ion channels are transmembrane receptor-ion channel complexes that open transiently upon binding of specific ligands, allowing rapid transmission of signals at chemical synapses [, ]. Five of these ion channel receptor families have been shown to form a sequence-related superfamily:Nicotinic acetylcholine receptor (AchR), an excitatory cation channel in vertebrates and invertebrates; in vertebrate motor endplates it is composed of alpha, beta, gamma and delta/epsilon subunits; in neurons it is composed of alpha and non-alpha (or beta) subunits [].Glycine receptor, an inhibitory chloride ion channel composed of alpha and beta subunits [].Gamma-aminobutyric acid (GABA) receptor, an inhibitory chloride ion channel; at least four types of subunits (alpha, beta, gamma and delta) are known [].Serotonin 5HT3 receptor, of which there are seven major types (5HT3-5HT7) [].Glutamate receptor, an excitatory cation channel of which at least three types have been described (kainate, N-methyl-D-aspartate (NMDA) and quisqualate) [].These receptors possess a pentameric structure (made up of varying subunits), surrounding a central pore. All known sequences of subunits from neurotransmitter-gated ion-channels are structurally related. They are composed of a large extracellular glycosylated N-terminal ligand-binding domain, followed by three hydrophobic transmembrane regions which form the ionic channel, followed by an intracellular region of variable length. A fourth hydrophobic region is found at the C-terminal of the sequence [, ].Glycine is a major inhibitory neurotransmitter (NT) in the adult vertebratecentral nervous system (CNS). Glycinergic synapses have a well-establishedrole in the processing of motor and sensory information that controlsmovement, vision and audition []. This action of glycine is mediatedthrough its interaction with the glycine receptor (GlyR): an intrinsicchloride channel is opened in response to agonist binding. The subsequentinflux of anions prevents membrane depolarisation and neuronal firinginduced by excitatory NTs. Strychnine acts as a competitive antagonist ofglycine binding, thereby reducing the activity of inhibitory neurones.Poisoning with strychnine is characterised by over-excitation, muscle spasmsand convulsions. Whilst glycine is the principal physiological agonist atGlyRs, taurine and beta-alanine also behave as agonists []. Compounds thatmodulate GlyR activity include zinc, some alcohols and anaesthetics,picrotoxin, cocaine and some anticonvulsants. GlyRs were thought for sometime to be localised exclusively in the brain stem and spinal cord, but havesince been found to be expressed more widely, including the cochlear nuclei,cerebellar cortex and forebrain [].GlyRs belong to the ligand-gated ion channel family, which also includes theinhibitory gamma-aminobutyric acid type A (GABAA) and excitatory nicotinicacetylcholine (nACh) and serotonin type 3 (5-HT3) receptors [].Affinity-purified GlyR was found to contain two glycosylated membraneproteins of 48kDa and 56kDa, corresponding to alpha and beta subunits,respectively. Four genes encoding alpha subunits have been identified (GLRA1to 4), together with a single beta polypeptide (GLRB). The heterogeneity ofalpha subunits is further increased by alternative exon splicing, yieldingtwo isoforms of GLRA1 to 3 []. The characteristics of different GlyRsubtypes, therefore, can be largely explained by their GLRA content.GlyRs are generally believed to adopt a pentameric structure in vivo: fivesubunits assemble to form a ring structure with a central pore. Typically, astoichiometry of 3:2 (alpha:beta) is observed []. GlyR subunits share ahigh overall level of sequence similarity both with themselves and with thesubunits of the GABAA and nACh receptors. Four highly conserved segmentshave been proposed to correspond to transmembrane (TM) alpha helices (TM1-4), the second of which is thought to contribute to the pore wall []. A long extracellular N-terminal segment precedes TM1 and a long cytoplasmic loop links TM3 and 4. Short cytoplasmic and extracellular loops join TM1-2 andTM2-3, respectively, and a short C-terminal sequence follows TM4. Studiesusing radiolabelled strychnine have shown the alpha subunit to beresponsible for ligand binding, the critical residues for this interaction lying within the N-terminal domain. The beta subunit plays a structuralrole, contributing one of its TM domains to the pore wall as well as playinga putative role in postsynaptic clustering of the receptor.In several mammalian species, defects in glycinergic transmission areassociated with complex motor disorders. Mutations in the gene encodingGLRA1 give rise to hyperplexia, or startle disease []. This ischaracterised by muscular spasms in response to unexpected light or noisestimuli, similar to the symptoms of sublethal doses of strychnine. Themutations result in amino acid substitutions within the TM1-2 and TM3-4loops, suggesting that these regions are involved in the transduction ofligand binding into channel activation.In humans, the GLRA2 gene is located on chromosome Xp22.2-22.1 []. In situhybridisation studies have shown GLRA2 to be expressed in the hippocampus,cerebral cortex and thalamus. GLRA2 trancripts predominate in the neonataland embyonic CNS, and are replaced postnatally by those of GLRA1 and, to alesser extent, GLRA3.
Neurotransmitter ligand-gated ion channels are transmembrane receptor-ion channel complexes that open transiently upon binding of specific ligands, allowing rapid transmission of signals at chemical synapses [, ]. Five of these ion channel receptor families have been shown to form a sequence-related superfamily:Nicotinic acetylcholine receptor (AchR), an excitatory cation channel in vertebrates and invertebrates; in vertebrate motor endplates it is composed of alpha, beta, gamma and delta/epsilon subunits; in neurons it is composed of alpha and non-alpha (or beta) subunits [].Glycine receptor, an inhibitory chloride ion channel composed of alpha and beta subunits [].Gamma-aminobutyric acid (GABA) receptor, an inhibitory chloride ion channel; at least four types of subunits (alpha, beta, gamma and delta) are known [].Serotonin 5HT3 receptor, of which there are seven major types (5HT3-5HT7) [].Glutamate receptor, an excitatory cation channel of which at least three types have been described (kainate, N-methyl-D-aspartate (NMDA) and quisqualate) [].These receptors possess a pentameric structure (made up of varying subunits), surrounding a central pore. All known sequences of subunits from neurotransmitter-gated ion-channels are structurally related. They are composed of a large extracellular glycosylated N-terminal ligand-binding domain, followed by three hydrophobic transmembrane regions which form the ionic channel, followed by an intracellular region of variable length. A fourth hydrophobic region is found at the C-terminal of the sequence [, ].Glycine is a major inhibitory neurotransmitter (NT) in the adult vertebratecentral nervous system (CNS). Glycinergic synapses have a well-establishedrole in the processing of motor and sensory information that controlsmovement, vision and audition []. This action of glycine is mediatedthrough its interaction with the glycine receptor (GlyR): an intrinsicchloride channel is opened in response to agonist binding. The subsequentinflux of anions prevents membrane depolarisation and neuronal firinginduced by excitatory NTs. Strychnine acts as a competitive antagonist ofglycine binding, thereby reducing the activity of inhibitory neurones.Poisoning with strychnine is characterised by over-excitation, muscle spasmsand convulsions. Whilst glycine is the principal physiological agonist atGlyRs, taurine and beta-alanine also behave as agonists []. Compounds thatmodulate GlyR activity include zinc, some alcohols and anaesthetics,picrotoxin, cocaine and some anticonvulsants. GlyRs were thought for sometime to be localised exclusively in the brain stem and spinal cord, but havesince been found to be expressed more widely, including the cochlear nuclei,cerebellar cortex and forebrain [].GlyRs belong to the ligand-gated ion channel family, which also includes theinhibitory gamma-aminobutyric acid type A (GABAA) and excitatory nicotinicacetylcholine (nACh) and serotonin type 3 (5-HT3) receptors [].Affinity-purified GlyR was found to contain two glycosylated membraneproteins of 48kDa and 56kDa, corresponding to alpha and beta subunits,respectively. Four genes encoding alpha subunits have been identified (GLRA1to 4), together with a single beta polypeptide (GLRB). The heterogeneity ofalpha subunits is further increased by alternative exon splicing, yieldingtwo isoforms of GLRA1 to 3 []. The characteristics of different GlyRsubtypes, therefore, can be largely explained by their GLRA content.GlyRs are generally believed to adopt a pentameric structure in vivo: fivesubunits assemble to form a ring structure with a central pore. Typically, astoichiometry of 3:2 (alpha:beta) is observed []. GlyR subunits share ahigh overall level of sequence similarity both with themselves and with thesubunits of the GABAA and nACh receptors. Four highly conserved segmentshave been proposed to correspond to transmembrane (TM) alpha helices (TM1-4), the second of which is thought to contribute to the pore wall []. A long extracellular N-terminal segment precedes TM1 and a long cytoplasmic loop links TM3 and 4. Short cytoplasmic and extracellular loops join TM1-2 andTM2-3, respectively, and a short C-terminal sequence follows TM4. Studiesusing radiolabelled strychnine have shown the alpha subunit to beresponsible for ligand binding, the critical residues for this interaction lying within the N-terminal domain. The beta subunit plays a structuralrole, contributing one of its TM domains to the pore wall as well as playinga putative role in postsynaptic clustering of the receptor.In several mammalian species, defects in glycinergic transmission areassociated with complex motor disorders. Mutations in the gene encodingGLRA1 give rise to hyperplexia, or startle disease []. This ischaracterised by muscular spasms in response to unexpected light or noisestimuli, similar to the symptoms of sublethal doses of strychnine. Themutations result in amino acid substitutions within the TM1-2 and TM3-4loops,suggesting that these regions are involved in the transduction ofligand binding into channel activation.In humans, the alpha 1 gene is located on chromosome 5p32 []. In situhybridisation studies have shown GLRA1 to be expressed in the spinal cord,brain stem and colliculi. GLRA1 trancripts, together with GLRA3, predominatein the postnatal CNS, replacing GLRA2, which is more abundant in embryonicand neonatal neurones.
Neurotransmitter ligand-gated ion channels are transmembrane receptor-ion channel complexes that open transiently upon binding of specific ligands, allowing rapid transmission of signals at chemical synapses [, ]. Five of these ion channel receptor families have been shown to form a sequence-related superfamily:Nicotinic acetylcholine receptor (AchR), an excitatory cation channel in vertebrates and invertebrates; in vertebrate motor endplates it is composed of alpha, beta, gamma and delta/epsilon subunits; in neurons it is composed of alpha and non-alpha (or beta) subunits [].Glycine receptor, an inhibitory chloride ion channel composed of alpha and beta subunits [].Gamma-aminobutyric acid (GABA) receptor, an inhibitory chloride ion channel; at least four types of subunits (alpha, beta, gamma and delta) are known [].Serotonin 5HT3 receptor, of which there are seven major types (5HT3-5HT7) [].Glutamate receptor, an excitatory cation channel of which at least three types have been described (kainate, N-methyl-D-aspartate (NMDA) and quisqualate) [].These receptors possess a pentameric structure (made up of varying subunits), surrounding a central pore. All known sequences of subunits from neurotransmitter-gated ion-channels are structurally related. They are composed of a large extracellular glycosylated N-terminal ligand-binding domain, followed by three hydrophobic transmembrane regions which form the ionic channel, followed by an intracellular region of variable length. A fourth hydrophobic region is found at the C-terminal of the sequence [, ].Glycine is a major inhibitory neurotransmitter (NT) in the adult vertebratecentral nervous system (CNS). Glycinergic synapses have a well-establishedrole in the processing of motor and sensory information that controlsmovement, vision and audition []. This action of glycine is mediatedthrough its interaction with the glycine receptor (GlyR): an intrinsicchloride channel is opened in response to agonist binding. The subsequentinflux of anions prevents membrane depolarisation and neuronal firinginduced by excitatory NTs. Strychnine acts as a competitive antagonist ofglycine binding, thereby reducing the activity of inhibitory neurones.Poisoning with strychnine is characterised by over-excitation, muscle spasmsand convulsions. Whilst glycine is the principal physiological agonist atGlyRs, taurine and beta-alanine also behave as agonists []. Compounds thatmodulate GlyR activity include zinc, some alcohols and anaesthetics,picrotoxin, cocaine and some anticonvulsants. GlyRs were thought for sometime to be localised exclusively in the brain stem and spinal cord, but havesince been found to be expressed more widely, including the cochlear nuclei,cerebellar cortex and forebrain [].GlyRs belong to the ligand-gated ion channel family, which also includes theinhibitory gamma-aminobutyric acid type A (GABAA) and excitatory nicotinicacetylcholine (nACh) and serotonin type 3 (5-HT3) receptors [].Affinity-purified GlyR was found to contain two glycosylated membraneproteins of 48kDa and 56kDa, corresponding to alpha and beta subunits,respectively. Four genes encoding alpha subunits have been identified (GLRA1to 4), together with a single beta polypeptide (GLRB). The heterogeneity ofalpha subunits is further increased by alternative exon splicing, yieldingtwo isoforms of GLRA1 to 3 []. The characteristics of different GlyRsubtypes, therefore, can be largely explained by their GLRA content.GlyRs are generally believed to adopt a pentameric structure in vivo: fivesubunits assemble to form a ring structure with a central pore. Typically, astoichiometry of 3:2 (alpha:beta) is observed []. GlyR subunits share ahigh overall level of sequence similarity both with themselves and with thesubunits of the GABAA and nACh receptors. Four highly conserved segmentshave been proposed to correspond to transmembrane (TM) alpha helices (TM1-4), the second of which is thought to contribute to the pore wall []. A long extracellular N-terminal segment precedes TM1 and a long cytoplasmic loop links TM3 and 4. Short cytoplasmic and extracellular loops join TM1-2 andTM2-3, respectively, and a short C-terminal sequence follows TM4. Studiesusing radiolabelled strychnine have shown the alpha subunit to beresponsible for ligand binding, the critical residues for this interaction lying within the N-terminal domain. The beta subunit plays a structuralrole, contributing one of its TM domains to the pore wall as well as playinga putative role in postsynaptic clustering of the receptor.In several mammalian species, defects in glycinergic transmission areassociated with complex motor disorders. Mutations in the gene encodingGLRA1 give rise to hyperplexia, or startle disease []. This ischaracterised by muscular spasms in response to unexpected light or noisestimuli, similar to the symptoms of sublethal doses of strychnine. Themutations result in amino acid substitutions within the TM1-2 and TM3-4loops, suggesting that these regions are involved in the transduction ofligand binding into channel activation.GLRA3 is expressed in thecerebellum, olfactory bulb and hippocampus. GLRA3 trancripts, together withGLRA1, predominate in the postnatal CNS, replacing GLRA2, which is moreabundant in embryonic and neonatal neurones.
Potassium channels are the most diverse group of the ion channel family [, ]. They are important in shaping the action potential, and in neuronal excitability and plasticity []. The potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups []: the practically non-inactivating 'delayed' group and the rapidly inactivating 'transient' group.These are all highly similar proteins, with only small amino acid changes causing the diversity of the voltage-dependent gating mechanism, channel conductance and toxin binding properties. Each type of K+channel is activated by different signals and conditions depending on their type of regulation: some open in response to depolarisation of the plasma membrane; others in response to hyperpolarisation or an increase in intracellular calcium concentration; some can be regulated by binding of a transmitter, together with intracellular kinases; while others are regulated by GTP-binding proteins or other second messengers []. In eukaryotic cells, K+channels are involved in neural signalling and generation of the cardiac rhythm, act as effectors in signal transduction pathways involving G protein-coupled receptors (GPCRs) and may have a role in target cell lysis by cytotoxic T-lymphocytes []. In prokaryotic cells, they play a role in the maintenance of ionic homeostasis [].All K+channels discovered so far possess a core of alpha subunits, each comprising either one or two copies of a highly conserved pore loop domain (P-domain). The P-domain contains the sequence (T/SxxTxGxG), which has been termed the K+selectivity sequence. In families that contain one P-domain, four subunits assemble to form a selective pathway for K+across the membrane. However, it remains unclear how the 2 P-domain subunits assemble to form a selective pore. The functional diversity of these families can arise through homo- or hetero-associations of alpha subunits or association with auxiliary cytoplasmic beta subunits. K+channel subunits containing one pore domain can be assigned into one of two superfamilies: those that possess six transmembrane (TM) domains and those that possess only two TM domains. The six TM domain superfamily can be further subdivided into conserved gene families: the voltage-gated (Kv) channels; the KCNQ channels (originally known as KvLQT channels); the EAG-like K+channels; and three types of calcium (Ca)-activated K+channels (BK, IK and SK) []. The 2TM domain family comprises inward-rectifying K+channels. In addition, there are K+channel alpha-subunits that possess two P-domains. These are usually highly regulated K+selective leak channels.The Kv family can be divided into several subfamilies on the basis of sequence similarity and function. Four of these subfamilies, Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw) and Kv4 (Shal), consist of pore-forming alpha subunits that associate with different types of beta subunit. Each alpha subunit comprises six hydrophobic TM domains with a P-domain between the fifth and sixth, which partially resides in the membrane. The fourth TM domain has positively charged residues at every third residue and acts as a voltage sensor, which triggers the conformational change that opens the channel pore in response to a displacement in membrane potential []. More recently, 4 new electrically-silent alpha subunits have been cloned: Kv5 (KCNF), Kv6 (KCNG), Kv8 and Kv9 (KCNS). These subunits do not themselves possess any functional activity, but appear to form heteromeric channels with Kv2 subunits, and thus modulate Shab channel activity []. When highly expressed, they inhibit channel activity, but at lower levels show more specific modulatory actions.A voltage-dependent potassium channel gene designated Shaw was initially isolated from Drosophila melanogaster (Fruit fly). Subsequently, several vetebrate potassium channels with similar amino acid sequences were found and, together with the D. melanogaster channel, now constitute the Kv3 family. These channels are thought to play a role in shortening of action potential durations and modulating pre-synaptic neurotransmitter release. In mammals, the family consists of 4 genes (Kv3.1, Kv3.2, Kv3.3 and Kv3.4). Each gene product has its own subcellular location and function.Fast inactivation of voltage-dependent potassium channels controls membrane excitability and signal propagation in central neurons. This occurs by a 'ball-and-chain'-type mechanism where an N-terminal protein inactivation domain occludes the pore from the cytoplasmic side. In Kv3 channels this process is regulated by protein phosphorylation, where phosphorylation of serine residues leads to a reduction or removal of the fast inactivation [].
Aconitase (aconitate hydratase; ) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function that arises when the cellular environment changes, such as when iron levels drop [, ]. Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal alpha/beta/alpha domains, a linker region, followed by a C-terminal 'swivel' domain with a beta/beta/alpha structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three alpha/beta/alpha domains (HEAT-4-1-2-3) [].Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic form of cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) [, ]. As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism - it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated [].IRP2 is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis []. Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes []. In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica [, ]. In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances the amount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes the synthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.This entry represents mitochondrial aconitase (mAcn), as well as close homologues such as certain bacterial aconitase A (AcnA) enzymes.
Aconitase (aconitate hydratase; ) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function that arises when the cellular environment changes, such as when iron levels drop [, ]. Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal alpha/beta/alpha domains, a linker region, followed by a C-terminal 'swivel' domain with a beta/beta/alpha structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three alpha/beta/alpha domains (HEAT-4-1-2-3) [].Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic form of cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) [, ]. As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism - it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated [].IRP2is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis []. Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes []. In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica [, ]. In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances the amount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes the synthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.This entry represents a small family of proteins homologous and likely functionally equivalent to aconitase 1. Members are found, so far in the anaerobe Clostridium acetobutylicum, in the microaerophilic, early-branching bacterium Aquifex aeolicus, and in the halophilic archaeon Halobacterium sp. NRC-1. No member is experimentally characterised.
