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
2009 Jun 12;28424:16226-16235. doi: 10.1074/jbc.M109.003137.
Show Gene links
Show Anatomy links
Elucidating conformational changes in the gamma-aminobutyric acid transporter-1.
Meinild AK
,
Loo DDF
,
Skovstrup S
,
Gether U
,
MacAulay N
.
???displayArticle.abstract???
The GABA transporter-1 (GAT-1) has three current-generating modes: GABA-coupled current, Li+-induced leak current, and Na+-dependent transient currents. We earlier hypothesized that Li+ is able to substitute for the first Na+ in the transport cycle and thereby induce a distinct conformation in GAT-1 and that the onset of the Li+-induced leak current at membrane potentials more negative than -50 mV was due to a voltage-dependent conformational change of the Li+-bound transporter. In this study, we set out to verify this hypothesis and seek insight into the structural dynamics underlying the leak current, as well as the sodium-dependent transient currents, by applying voltage clamp fluorometry to tetramethylrhodamine 6-maleimide-labeled GAT-1 expressed in Xenopus laevis oocytes. MTSET accessibility studies demonstrated the presence of two distinct conformations of GAT-1 in the presence of Na+ or Li+. The voltage-dependent fluorescence intensity changes obtained in Li+ buffer correlated with the Li+-induced leak currents, i.e. both were highly voltage-dependent and only present at hyperpolarized potentials (<-50 mV). The transient currents correlated directly with the voltage-dependent fluorescence data obtained in sodium buffer and the associated conformational changes were distinct from those associated with the Li+-induced leak current. The inhibitor potency of SKF89976A of the Li+- versus Na+-bound transporter confirmed the cationic dependence of the conformational occupancy. Our observations suggest that the microdomain situated at the external end of transmembrane I is involved in different conformational changes taking place either during the binding and release of sodium or during the initiation of the Li+-induced leak current.
Bennett,
Mutation of arginine 44 of GAT-1, a (Na(+) + Cl(-))-coupled gamma-aminobutyric acid transporter from rat brain, impairs net flux but not exchange.
2000, Pubmed
Bennett,
Mutation of arginine 44 of GAT-1, a (Na(+) + Cl(-))-coupled gamma-aminobutyric acid transporter from rat brain, impairs net flux but not exchange.
2000,
Pubmed
Beuming,
A comprehensive structure-based alignment of prokaryotic and eukaryotic neurotransmitter/Na+ symporters (NSS) aids in the use of the LeuT structure to probe NSS structure and function.
2006,
Pubmed
Bezanilla,
The voltage sensor in voltage-dependent ion channels.
2000,
Pubmed
Bicho,
Rapid substrate-induced charge movements of the GABA transporter GAT1.
2005,
Pubmed
Bismuth,
Tyrosine 140 of the gamma-aminobutyric acid transporter GAT-1 plays a critical role in neurotransmitter recognition.
1997,
Pubmed
Borden,
Tiagabine, SK&F 89976-A, CI-966, and NNC-711 are selective for the cloned GABA transporter GAT-1.
1994,
Pubmed
Borden,
GABA transporter heterogeneity: pharmacology and cellular localization.
1996,
Pubmed
Chen,
External cysteine residues in the serotonin transporter.
1997,
Pubmed
Forrest,
Mechanism for alternating access in neurotransmitter transporters.
2008,
Pubmed
Galli,
Sodium-dependent norepinephrine-induced currents in norepinephrine-transporter-transfected HEK-293 cells blocked by cocaine and antidepressants.
1995,
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
Hazama,
Presteady-state currents of the rabbit Na+/glucose cotransporter (SGLT1).
1997,
Pubmed
,
Xenbase
Hilgemann,
GAT1 (GABA:Na+:Cl-) cotransport function. Database reconstruction with an alternating access model.
1999,
Pubmed
,
Xenbase
Hirayama,
Cation effects on protein conformation and transport in the Na+/glucose cotransporter.
1997,
Pubmed
,
Xenbase
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
Keynan,
gamma-Aminobutyric acid transport in reconstituted preparations from rat brain: coupled sodium and chloride fluxes.
1988,
Pubmed
Larsson,
Fluorometric measurements of conformational changes in glutamate transporters.
2004,
Pubmed
,
Xenbase
Li,
Voltage-dependent transient currents of human and rat 5-HT transporters (SERT) are blocked by HEPES and ion channel ligands.
2002,
Pubmed
,
Xenbase
Li,
An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence.
2000,
Pubmed
,
Xenbase
Lin,
Single-channel currents produced by the serotonin transporter and analysis of a mutation affecting ion permeation.
1996,
Pubmed
,
Xenbase
Liu,
Effect of mutation of glycosylation sites on the Na+ dependence of steady-state and transient currents generated by the neuronal GABA transporter.
1998,
Pubmed
,
Xenbase
Loo,
Relaxation kinetics of the Na+/glucose cotransporter.
1993,
Pubmed
,
Xenbase
Loo,
Conformational changes couple Na+ and glucose transport.
1998,
Pubmed
,
Xenbase
López-Corcuera,
Substrate-induced conformational changes of extracellular loop 1 in the glycine transporter GLYT2.
2001,
Pubmed
MacAulay,
Engineered Zn(2+) switches in the gamma-aminobutyric acid (GABA) transporter-1. Differential effects on GABA uptake and currents.
2001,
Pubmed
,
Xenbase
MacAulay,
Conformational basis for the Li(+)-induced leak current in the rat gamma-aminobutyric acid (GABA) transporter-1.
2002,
Pubmed
,
Xenbase
MacAulay,
Residues in the extracellular loop 4 are critical for maintaining the conformational equilibrium of the gamma-aminobutyric acid transporter-1.
2003,
Pubmed
,
Xenbase
Mager,
Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes.
1993,
Pubmed
,
Xenbase
Mager,
Conducting states of a mammalian serotonin transporter.
1994,
Pubmed
,
Xenbase
Mager,
Ion binding and permeation at the GABA transporter GAT1.
1996,
Pubmed
,
Xenbase
Mannuzzu,
Independence and cooperativity in rearrangements of a potassium channel voltage sensor revealed by single subunit fluorescence.
2000,
Pubmed
,
Xenbase
Meinild,
Fluorescence studies of ligand-induced conformational changes of the Na(+)/glucose cotransporter.
2002,
Pubmed
,
Xenbase
Melamed,
Transmembrane domains I and II of the gamma-aminobutyric acid transporter GAT-4 contain molecular determinants of substrate specificity.
2004,
Pubmed
,
Xenbase
Ni,
A lithium-induced conformational change in serotonin transporter alters cocaine binding, ion conductance, and reactivity of Cys-109.
2001,
Pubmed
,
Xenbase
Radian,
Stoichiometry of sodium- and chloride-coupled gamma-aminobutyric acid transport by synaptic plasma membrane vesicles isolated from rat brain.
1983,
Pubmed
Sali,
Comparative protein modelling by satisfaction of spatial restraints.
1993,
Pubmed
Singh,
Antidepressant binding site in a bacterial homologue of neurotransmitter transporters.
2007,
Pubmed
Sonders,
Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants.
1997,
Pubmed
,
Xenbase
Umbach,
Intestinal Na+/glucose cotransporter expressed in Xenopus oocytes is electrogenic.
1990,
Pubmed
,
Xenbase
Virkki,
Voltage clamp fluorometric measurements on a type II Na+-coupled Pi cotransporter: shedding light on substrate binding order.
2006,
Pubmed
,
Xenbase
Virkki,
Mapping conformational changes of a type IIb Na+/Pi cotransporter by voltage clamp fluorometry.
2006,
Pubmed
,
Xenbase
Wadiche,
Kinetics of a human glutamate transporter.
1995,
Pubmed
,
Xenbase
Yamashita,
Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters.
2005,
Pubmed
Yu,
Topological localization of cysteine 74 in the GABA transporter, GAT1, and its importance in ion binding and permeation.
1998,
Pubmed
,
Xenbase
Zhang,
Identification of acetylcholine receptor channel-lining residues in the M1 segment of the beta-subunit.
1997,
Pubmed
,
Xenbase
Zhou,
The aqueous accessibility in the external half of transmembrane domain I of the GABA transporter GAT-1 Is modulated by its ligands.
2004,
Pubmed
Zhou,
Transporter-associated currents in the gamma-aminobutyric acid transporter GAT-1 are conditionally impaired by mutations of a conserved glycine residue.
2005,
Pubmed
,
Xenbase
Zhou,
Identification of a lithium interaction site in the gamma-aminobutyric acid (GABA) transporter GAT-1.
2006,
Pubmed
Zomot,
The interaction of the gamma-aminobutyric acid transporter GAT-1 with the neurotransmitter is selectively impaired by sulfhydryl modification of a conformationally sensitive cysteine residue engineered into extracellular loop IV.
2003,
Pubmed
,
Xenbase
Zomot,
Proximity of transmembrane domains 1 and 3 of the gamma-aminobutyric acid transporter GAT-1 inferred from paired cysteine mutagenesis.
2005,
Pubmed
