1 Neuropharm and Genetics Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet2 Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet3 Weill Medical College of Cornell University, United States;4 Neuropharm and Genetics Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet5 Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet
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.
Journal of Biological Chemistry, 2014, Vol 289, p. 25764-25773