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The second sodium site in the dopamine transporter controls cation permeability and is regulated by chloride

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  • Lars Borre
  • Thorvald F Andreassen
  • Lei Shi
  • Harel Weinstein
  • Gether, Ulrik

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.

OriginalsprogEngelsk
TidsskriftThe Journal of Biological Chemistry
Vol/bind289
Sider (fra-til)25764-25773
Antal sider10
ISSN0021-9258
DOI
StatusUdgivet - 25 jul. 2014

ID: 120587517