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

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The second sodium site in the dopamine transporter controls cation permeability and is regulated by chloride. / Borre, Lars; Andreassen, Thorvald F; Shi, Lei; Weinstein, Harel; Gether, Ulrik.

In: The Journal of Biological Chemistry, Vol. 289, 25.07.2014, p. 25764-25773.

Research output: Contribution to journalJournal articlepeer-review

Harvard

Borre, L, Andreassen, TF, Shi, L, Weinstein, H & Gether, U 2014, 'The second sodium site in the dopamine transporter controls cation permeability and is regulated by chloride', The Journal of Biological Chemistry, vol. 289, pp. 25764-25773. https://doi.org/10.1074/jbc.M114.574269

APA

Borre, L., Andreassen, T. F., Shi, L., Weinstein, H., & Gether, U. (2014). The second sodium site in the dopamine transporter controls cation permeability and is regulated by chloride. The Journal of Biological Chemistry, 289, 25764-25773. https://doi.org/10.1074/jbc.M114.574269

Vancouver

Borre L, Andreassen TF, Shi L, Weinstein H, Gether U. The second sodium site in the dopamine transporter controls cation permeability and is regulated by chloride. The Journal of Biological Chemistry. 2014 Jul 25;289:25764-25773. https://doi.org/10.1074/jbc.M114.574269

Author

Borre, Lars ; Andreassen, Thorvald F ; Shi, Lei ; Weinstein, Harel ; Gether, Ulrik. / The second sodium site in the dopamine transporter controls cation permeability and is regulated by chloride. In: The Journal of Biological Chemistry. 2014 ; Vol. 289. pp. 25764-25773.

Bibtex

@article{faa93e8fce48486c9d5e836761d48241,
title = "The second sodium site in the dopamine transporter controls cation permeability and is regulated by chloride",
abstract = "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.",
author = "Lars Borre and Andreassen, {Thorvald F} and Lei Shi and Harel Weinstein and Ulrik Gether",
note = "Copyright {\textcopyright} 2014, The American Society for Biochemistry and Molecular Biology.",
year = "2014",
month = jul,
day = "25",
doi = "10.1074/jbc.M114.574269",
language = "English",
volume = "289",
pages = "25764--25773",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",

}

RIS

TY - JOUR

T1 - The second sodium site in the dopamine transporter controls cation permeability and is regulated by chloride

AU - Borre, Lars

AU - Andreassen, Thorvald F

AU - Shi, Lei

AU - Weinstein, Harel

AU - Gether, Ulrik

N1 - Copyright © 2014, The American Society for Biochemistry and Molecular Biology.

PY - 2014/7/25

Y1 - 2014/7/25

N2 - 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.

AB - 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.

U2 - 10.1074/jbc.M114.574269

DO - 10.1074/jbc.M114.574269

M3 - Journal article

C2 - 25063810

VL - 289

SP - 25764

EP - 25773

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

ER -

ID: 120587517