| First Author | Borre L | Year | 2014 |
| Journal | J Biol Chem | Volume | 289 |
| Issue | 37 | Pages | 25764-73 |
| PubMed ID | 25063810 | Mgi Jnum | J:217226 |
| Mgi Id | MGI:5613419 | Doi | 10.1074/jbc.M114.574269 |
| Citation | Borre L, et al. (2014) The second sodium site in the dopamine transporter controls cation permeation and is regulated by chloride. J Biol Chem 289(37):25764-73 |
| abstractText | The dopamine transporter (DAT) belongs to the family of neurotransmitter:sodium symporters and controls dopamine (DA) homeostasis by mediating Na(+)- and Cl(-)-dependent reuptake of DA. Here we used two-electrode voltage clamp measurements in Xenopus oocytes together with targeted mutagenesis to investigate the mechanistic relationship between DAT ion binding sites and transporter conductances. In Li(+), DAT displayed a cocaine-sensitive cation leak current approximately 10-fold larger than the substrate-induced current in Na(+). Mutation of Na(+) coordinating residues in the first (Na1) and second (Na2) binding sites suggested that the Li(+) leak depends on Li(+) interaction with Na2 rather than Na1. DA caused a marked inhibition of the Li(+) leak, consistent with the ability of the substrate to interact with the Li(+)-occupied state of the transporter. The leak current in Li(+) was also potently inhibited by low millimolar concentrations of Na(+), which according to our mutational data conceivably depended on high affinity binding to Na1. The Li(+) leak was further regulated by Cl(-) that most likely increases Li(+) permeation by allosterically lowering Na2 affinity. Interestingly, mutational lowering of Na2 affinity by substituting Asp-420 with asparagine dramatically increased cation permeability in Na(+) to a level higher than seen in Li(+). In addition to reveal a functional link between the bound Cl(-) and the cation bound in the Na2 site, the data support a key role of Na2 in determining cation permeability of the transporter and thereby possibly in regulating the opening probability of the inner gate. |
