First Author | Brown JP | Year | 1998 |
Journal | J Biol Chem | Volume | 273 |
Issue | 39 | Pages | 25458-65 |
PubMed ID | 9738015 | Mgi Jnum | J:50070 |
Mgi Id | MGI:1289811 | Doi | 10.1074/jbc.273.39.25458 |
Citation | Brown JP, et al. (1998) Cloning and deletion mutagenesis of the alpha2 delta calcium channel subunit from porcine cerebral cortex. Expression of a soluble form of the protein that retains [3H]gabapentin binding activity. J Biol Chem 273(39):25458-65 |
abstractText | The anti-epileptic, anti-hyperalgesic, and anxiolytic agent gabapentin (1-(aminomethyl)-cyclohexane acetic acid or Neurontin) has previously been shown to bind with high affinity to the alpha2delta subunit of voltage-dependent calcium channels (Gee, NS, Brown, JP, Dissanayake, VUK, Offord, J, Thurlow, R, and Woodruff, GN. (1996) J Biol Chem. 271, 5768-5776). We report here the cloning, sequencing, and deletion mutagenesis of the alpha2delta subunit from porcine brain. The deduced protein sequence has a 95.9 and 98.2% identity to the rat and human neuronal alpha2 delta sequences, respectively. [3H]Gabapentin binds with a KD of 37.5 +/- 10.4 nM to membranes prepared from COS-7 cells transfected with wild-type porcine alpha2 delta cDNA. Six deletion mutants (B-G) that lack the delta polypeptide, together with varying amounts of the alpha2 component, failed to bind [3H]gabapentin. C-terminal deletion mutagenesis of the delta polypeptide identified a segment (residues 960-994) required for correct assembly of the [3H]gabapentin binding pocket. Mutant L, which lacks the putative membrane anchor in the delta sequence, was found in both membrane-associated and soluble secreted forms. The soluble form was not proteolytically cleaved into separate alpha2 and delta chains but still retained a high affinity (KD = 30.7 +/- 8.1 nM) for [3H]gabapentin. The production of a soluble alpha2delta mutant supports the single transmembrane model of the alpha2 delta subunit and is an important step toward the large-scale recombinant expression of the protein. |