title: The second sodium site in the dopamine transporter controls cation permeation and is regulated by chloride.
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.
authors: From the Molecular Neuropharmacology Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, The Panum Institute 18.6, University of Copenhagen, 2200 Copenhagen N, Denmark and lbo29917134@gmail.com.
authors: Lars
authors: L
authors: Borre
authors: From the Molecular Neuropharmacology Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, The Panum Institute 18.6, University of Copenhagen, 2200 Copenhagen N, Denmark and.
authors: Thorvald F
authors: TF
authors: Andreassen
authors: the Department of Physiology and Biophysics, Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York 10021.
authors: Lei
authors: L
authors: Shi
authors: the Department of Physiology and Biophysics, Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York 10021.
authors: Harel
authors: H
authors: Weinstein
authors: From the Molecular Neuropharmacology Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, The Panum Institute 18.6, University of Copenhagen, 2200 Copenhagen N, Denmark and gether@sund.ku.dk.
authors: Ulrik
authors: U
authors: Gether
mesh_terms: D000818:Animals; D001665:Binding Sites; D002412:Cations / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry*; D003042:Cocaine / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D008094:Lithium / Q000737:chemistry; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010539:Permeability / Q000187:drug effects; D012964:Sodium / Q000737:chemistry / Q000378:metabolism*; D014982:Xenopus laevis / Q000254:growth & development / Q000378:metabolism
chemical_list: D002412:Cations; D002712:Chlorides; D050483:Dopamine Plasma Membrane Transport Proteins; D008094:Lithium; D012964:Sodium; D003042:Cocaine; D004298:Dopamine
keywords: Dopamine Transporter; Electrophysiology; Membrane Transport; Monoamine Transporter; Neurotransmitter Transport; Site-directed Mutagenesis; Sodium Binding Sites; Structure-Function