The second sodium site in the dopamine transporter controls cation permeability and is regulated by chloride
Research output: Contribution to journal › Journal article › Research › peer-review
The dopamine transporter (DAT) belongs to the family of neurotransmitter:sodium symporters (NSSs) 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 furthermore regulated by Cl(-) that most likely increases Li(+) permeation by allosterically lowering Na2 affinity. Interestingly, mutational lowering of Na2 affinity by substituting Asp420 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 |
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Journal | The Journal of Biological Chemistry |
Volume | 289 |
Pages (from-to) | 25764-25773 |
Number of pages | 10 |
ISSN | 0021-9258 |
DOIs | |
Publication status | Published - 25 Jul 2014 |
ID: 120587517