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J Biol Chem. 2015 May 29;290(22):13992-4003. doi: 10.1074/jbc.M114.625343. Epub 2015 Apr 13.

Mechanism of the Association between Na+ Binding and Conformations at the Intracellular Gate in Neurotransmitter:Sodium Symporters.

Author information

1
From the Department of Physiology and Biophysics and the Department of Physics, Cornell University, Ithaca, New York, 14850.
2
the Departments of Psychiatry and the Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032.
3
the Centre for Molecular Simulation and Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
4
the Department of Neuroscience and Pharmacology, University of Copenhagen, Copenhagen, Denmark, and.
5
From the Department of Physiology and Biophysics and.
6
the Departments of Psychiatry and the Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York 10032, Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
7
From the Department of Physiology and Biophysics and Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York, 10065, haw2002@med.cornell.edu.
8
From the Department of Physiology and Biophysics and Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York, 10065, the Computational Chemistry and Molecular Biophysics Unit, NIDA, Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224 les2007@med.cornell.edu.

Abstract

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by Na(+)-dependent reuptake of released neurotransmitters. Previous studies suggested that Na(+)-binding reconfigures dynamically coupled structural elements in an allosteric interaction network (AIN) responsible for function-related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. We describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications in both LeuT and human dopamine transporter (hDAT), we apply the novel method to identify the composition and the dynamic properties of their conserved AIN. In LeuT, two different perturbations disrupting Na(+) binding and transport (i.e. replacing Na(+) with Li(+) or the Y268A mutation at the intracellular gate) affect the AIN in strikingly similar ways. In contrast, other mutations that affect the intracellular gate (i.e. R5A and D369A) do not significantly impair Na(+) cooperativity and transport. Our analysis shows these perturbations to have much lesser effects on the AIN, underscoring the sensitivity of this novel method to the mechanistic nature of the perturbation. Notably, this set of observations holds as well for hDAT, where the aligned Y335A, R60A, and D436A mutations also produce different impacts on Na(+) dependence. Thus, the detailed AIN generated from our method is shown to connect Na(+) binding with global conformational changes that are critical for the transport mechanism. That the AIN between the Na(+) binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function.

KEYWORDS:

allosteric regulation; dopamine transporter; metal ion-protein interaction; molecular dynamics; neurotransmitter transport; potential of mean force calculation

PMID:
25869126
PMCID:
PMC4447972
DOI:
10.1074/jbc.M114.625343
[Indexed for MEDLINE]
Free PMC Article

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