Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Biol Chem
2012 Mar 02;28710:7159-68. doi: 10.1074/jbc.M111.323634.
Show Gene links
Show Anatomy links
An acidic amino acid transmembrane helix 10 residue conserved in the neurotransmitter:sodium:symporters is essential for the formation of the extracellular gate of the γ-aminobutyric acid (GABA) transporter GAT-1.
Ben-Yona A
,
Kanner BI
.
???displayArticle.abstract???
GAT-1 mediates transport of GABA together with sodium and chloride in an electrogenic process enabling efficient GABAergic transmission. Biochemical and modeling studies based on the structure of the bacterial homologue LeuT are consistent with a mechanism whereby the binding pocket is alternately accessible to either side of the membrane and which predicts that the extracellular part of transmembrane domain 10 (TM10) exhibits aqueous accessibility in the outward-facing conformation only. In this study we have engineered cysteine residues in the extracellular half of TM10 of GAT-1 and probed their state-dependent accessibility to sulfhydryl reagents. In three out of four of the accessible cysteine mutants, the inhibition of transport by a membrane impermeant sulfhydryl reagent was diminished under conditions expected to increase the proportion of inward-facing transporters, such as the presence of GABA together with the cotransported ions. A conserved TM10 aspartate residue, whose LeuT counterpart participates in a "thin" extracellular gate, was found to be essential for transport and only the D451E mutant exhibited residual transport activity. D451E exhibited robust sodium-dependent transient currents with a voltage-dependence indicative of an increased apparent affinity for sodium. Moreover the accessibility of an endogenous cysteine to a membrane impermeant sulfhydryl reagent was enhanced by the D451E mutation, suggesting that sodium binding promotes an outward-facing conformation of the transporter. Our results support the idea that TM10 of GAT-1 lines an accessibility pathway from the extracellular space into the binding pocket and plays a role in the opening and closing of the extracellular transporter gate.
Ben-Yona,
A glutamine residue conserved in the neurotransmitter:sodium:symporters is essential for the interaction of chloride with the GABA transporter GAT-1.
2011, Pubmed,
Xenbase
Ben-Yona,
A glutamine residue conserved in the neurotransmitter:sodium:symporters is essential for the interaction of chloride with the GABA transporter GAT-1.
2011,
Pubmed
,
Xenbase
Bennett,
The membrane topology of GAT-1, a (Na+ + Cl-)-coupled gamma-aminobutyric acid transporter from rat brain.
1997,
Pubmed
Bismuth,
Tyrosine 140 of the gamma-aminobutyric acid transporter GAT-1 plays a critical role in neurotransmitter recognition.
1997,
Pubmed
Borre,
Dynamic equilibrium between coupled and uncoupled modes of a neuronal glutamate transporter.
2002,
Pubmed
,
Xenbase
Borre,
Arginine 445 controls the coupling between glutamate and cations in the neuronal transporter EAAC-1.
2004,
Pubmed
Claxton,
Ion/substrate-dependent conformational dynamics of a bacterial homolog of neurotransmitter:sodium symporters.
2010,
Pubmed
Dodd,
Selective amino acid substitutions convert the creatine transporter to a gamma-aminobutyric acid transporter.
2007,
Pubmed
Faham,
The crystal structure of a sodium galactose transporter reveals mechanistic insights into Na+/sugar symport.
2008,
Pubmed
Forrest,
Identification of a chloride ion binding site in Na+/Cl -dependent transporters.
2007,
Pubmed
Forrest,
Mechanism for alternating access in neurotransmitter transporters.
2008,
Pubmed
Forrest,
The rocking bundle: a mechanism for ion-coupled solute flux by symmetrical transporters.
2009,
Pubmed
Fuerst,
Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.
1986,
Pubmed
Golovanevsky,
The reactivity of the gamma-aminobutyric acid transporter GAT-1 toward sulfhydryl reagents is conformationally sensitive. Identification of a major target residue.
1999,
Pubmed
Guastella,
Cloning and expression of a rat brain GABA transporter.
1990,
Pubmed
,
Xenbase
Hilgemann,
GAT1 (GABA:Na+:Cl-) cotransport function. Database reconstruction with an alternating access model.
1999,
Pubmed
,
Xenbase
Kanner,
Sodium-coupled neurotransmitter transporters.
2008,
Pubmed
Kanner,
Transmembrane domain I of the gamma-aminobutyric acid transporter GAT-1 plays a crucial role in the transition between cation leak and transport modes.
2003,
Pubmed
,
Xenbase
Kavanaugh,
Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes.
1992,
Pubmed
,
Xenbase
Keller,
Cysteine-scanning mutagenesis of the fifth external loop of serotonin transporter.
2004,
Pubmed
Keynan,
gamma-Aminobutyric acid transport in reconstituted preparations from rat brain: coupled sodium and chloride fluxes.
1988,
Pubmed
Keynan,
Expression of a cloned gamma-aminobutyric acid transporter in mammalian cells.
1992,
Pubmed
,
Xenbase
Kleinberger-Doron,
Identification of tryptophan residues critical for the function and targeting of the gamma-aminobutyric acid transporter (subtype A).
1994,
Pubmed
Kunkel,
Rapid and efficient site-specific mutagenesis without phenotypic selection.
1987,
Pubmed
Loo,
Relaxation kinetics of the Na+/glucose cotransporter.
1993,
Pubmed
,
Xenbase
Lu,
GAT1 (GABA:Na+:Cl-) cotransport function. Steady state studies in giant Xenopus oocyte membrane patches.
1999,
Pubmed
,
Xenbase
Lu,
GAT1 (GABA:Na+:Cl-) cotransport function. Kinetic studies in giant Xenopus oocyte membrane patches.
1999,
Pubmed
,
Xenbase
Mager,
Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes.
1993,
Pubmed
,
Xenbase
Mager,
Ion binding and permeation at the GABA transporter GAT1.
1996,
Pubmed
,
Xenbase
Mari,
Role of the conserved glutamine 291 in the rat gamma-aminobutyric acid transporter rGAT-1.
2006,
Pubmed
Nelson,
The family of Na+/Cl- neurotransmitter transporters.
1998,
Pubmed
Pantanowitz,
Only one of the charged amino acids located in the transmembrane alpha-helices of the gamma-aminobutyric acid transporter (subtype A) is essential for its activity.
1993,
Pubmed
Radian,
Purification and identification of the functional sodium- and chloride-coupled gamma-aminobutyric acid transport glycoprotein from rat brain.
1986,
Pubmed
Rosenberg,
The substrates of the gamma-aminobutyric acid transporter GAT-1 induce structural rearrangements around the interface of transmembrane domains 1 and 6.
2008,
Pubmed
Shimamura,
Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1.
2010,
Pubmed
Skovstrup,
Homology modelling of the GABA transporter and analysis of tiagabine binding.
2010,
Pubmed
Vandenberg,
Molecular basis for substrate discrimination by glycine transporters.
2007,
Pubmed
,
Xenbase
Weyand,
Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter.
2008,
Pubmed
Yamashita,
Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters.
2005,
Pubmed
Zhang,
The cytoplasmic substrate permeation pathway of serotonin transporter.
2006,
Pubmed
Zhao,
Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue.
2011,
Pubmed
Zhou,
Identification of a lithium interaction site in the gamma-aminobutyric acid (GABA) transporter GAT-1.
2006,
Pubmed
Zomot,
Mechanism of chloride interaction with neurotransmitter:sodium symporters.
2007,
Pubmed