Abstract
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 ~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.
| Original language | English |
|---|---|
| Journal | The Journal of Biological Chemistry |
| Volume | 289 |
| Pages (from-to) | 25764-25773 |
| Number of pages | 10 |
| ISSN | 0021-9258 |
| DOIs | |
| Publication status | Published - 12 Sept 2014 |