Document Detail


Functional defects in the external and internal thin gates of the γ-aminobutyric acid (GABA) transporter GAT-1 can compensate each other.
MedLine Citation:
PMID:  23288838     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The GABA transporter GAT-1 belongs to the neurotransmitter:sodium:symporters which are crucial for synaptic transmission. GAT-1 mediates electrogenic transport of GABA together with sodium and chloride. Structure-function studies indicate that the bacterial homologue LeuT, which possess extra- and intracellular thin gates, is an excellent model for this class of neurotransmitter transporters. We recently showed that a conserved aspartate residue of GAT-1, Asp-451, whose LeuT equivalent participates in its thin extracellular gate, is functionally irreplaceable in GAT-1. Only the D451E mutant exhibited residual transport activity but with an elevated apparent sodium affinity as a consequence of an increased proportion of outward-facing transporters. Because during transport the opening and closing of external and internal gates should be tightly coupled, we have addressed the question of whether mutations of the intracellular thin gate residues Arg-44 and Asp-410 can compensate for the effects of their extracellular counterparts. Mutation of Asp-410 to glutamate resulted in impaired transport activity and a reduced apparent affinity for sodium. However, the transport activity of the double mutant D410E/D451E was increased by approximately 10-fold of that of each of the single mutants. Similar compensatory effects were also seen when other combinations of intra- and extracellular thin gate mutants were analyzed. Moreover, the introduction of D410E into the D451E background resulted in lower apparent sodium affinity than that of D451E alone. Our results indicate that a functional interaction of the external and internal gates of GAT-1 is essential for transport.
Authors:
Assaf Ben-Yona; Baruch I Kanner
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2013-01-03
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  288     ISSN:  1083-351X     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2013 Feb 
Date Detail:
Created Date:  2013-02-18     Completed Date:  2013-04-29     Revised Date:  2014-02-18    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4549-56     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Animals
Biological Transport
Biotinylation
Dose-Response Relationship, Drug
Ethylmaleimide / chemistry
GABA Plasma Membrane Transport Proteins / metabolism*
HeLa Cells
Humans
Kinetics
Models, Molecular
Molecular Conformation
Mutation*
Neurotransmitter Agents / metabolism
Oocytes / metabolism
Protein Transport
Sodium / chemistry
Structure-Activity Relationship
Xenopus laevis
Chemical
Reg. No./Substance:
0/GABA Plasma Membrane Transport Proteins; 0/Neurotransmitter Agents; 9NEZ333N27/Sodium; O3C74ACM9V/Ethylmaleimide
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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