.gamma.-Aminobutyric acid transport in reconstituted preparations from rat brain: coupled sodium and chloride fluxes

Keynan, Shoshi; Kanner, Baruch I.

doi:10.1021/bi00401a003

Cited by 162 publications

(111 citation statements)

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“…3 and 4). GAT-1 catalyzes the electrogenic cotransport of sodium, chloride, and GABA with a stoichiometry of 2:1:1, respectively (5)(6)(7)(8). Although the precise order of events during a transport cycle is not yet established, it is clear that the uptake process is initiated by the binding of at least one sodium ion (6,9,10).…”

mentioning

confidence: 99%

Transmembrane Domain 8 of the γ-Aminobutyric Acid Transporter GAT-1 Lines a Cytoplasmic Accessibility Pathway into Its Binding Pocket

Ben-Yona

Kanner

2009

Journal of Biological Chemistry

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GAT-1 is a sodium-and chloride-coupled ␥-aminobutyric acid (GABA) transporter, which fulfills an essential role in the synaptic transmission by this neurotransmitter. Cysteine-399 is the major site of inhibition of GAT-1 by membrane-permeant sulfhydryl reagents. This cysteine residue was previously thought to reside on a cytoplasmic loop connecting transmembrane domains (TMs) 8 and 9. However, the crystal structure of LeuT, a bacterial homologue of the mammalian neurotransmitter:sodium symporters, revealed that the residue corresponding to Cys-399 is in fact located in the middle of TM 8. This residue is located to the cytoplasmic side of Asp-395 and Ser-396, whose side chains are thought to ligand one of the two cotransported sodium ions. To determine how the sulfhydryl reagents approach cysteine-399, a cysteine scan of all 35 residues of TM 8 was performed. Sulfhydryl reagents inhibited transport when a cysteine residue was present at either of the positions 399, 402, 406, and 410. SKF-89976A and other non-transportable analogues, which are expected to lock the transporter in a conformation facing the extracellular medium, protected against the sulfhydryl modification at positions 399, 402, and 406. Such a protection was not seen by GABA itself, which actually modestly potentiated the modification at positions 399 and 402. Our results point to an ␣-helical stripe on TM8 lining an aqueous access pathway from the cytoplasm into the binding pocket, which gets occluded in the conformation of the transporter where the binding pocket is exposed to the extracellular medium.

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confidence: 99%

Transmembrane Domain 8 of the γ-Aminobutyric Acid Transporter GAT-1 Lines a Cytoplasmic Accessibility Pathway into Its Binding Pocket

Ben-Yona

Kanner

2009

Journal of Biological Chemistry

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“…The GABA transporter cotransports the neurotransmitter with sodium and chloride in an electrogenic fashion (Kanner, 1983;Keynan and Kanner, 1988). The available measurements include tracer fluxes (Keynan and Kanner, 1988) and electrophysiological approaches (Kavanaugh et al 1992;Mager et al 1993).…”

Section: Stoichiometrymentioning

confidence: 99%

“…The available measurements include tracer fluxes (Keynan and Kanner, 1988) and electrophysiological approaches (Kavanaugh et al 1992;Mager et al 1993).…”

Section: Stoichiometrymentioning

confidence: 99%

“…This list includes various subtypes of GABA transporters as well as those for all the above-mentioned neurotransmitters, except glutamate. All of the members of this superfamily are dependent on sodium and chloride and, by analogy with the GABA transporter (Keynan and Kanner, 1988), are likely to cotransport their transmitter with both sodium and chloride. Interestingly, sodium-dependent glutamate transport is not chloride-dependent, but rather sodium and glutamate are countertransported with potassium ( Fig.…”

Section: Introductionmentioning

confidence: 99%

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Ion-coupled neurotransmitter transport

Kanner

1989

Current Opinion in Cell Biology

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SummaryThe removal of neurotransmitters by their transporters -located in the plasma membranes of nerve terminals and glial cells -plays an important role in the termination of synaptic transmission. In the last 3 years, many neurotransmitter transporters have been cloned. Structurally and functionally they can be divided into two groups: glutamate transporters, of which to date three have been cloned, couple the flow of glutamate to that of sodium and potassium. The second group of transporters includes those for GABA, glycine, taurine, norepinephrine, dopamine and serotonin. They are sodium-and chloride-dependent, but do not require potassium for function. One of these, the GABAA transporter, encoded by GAT-1, is perhaps the best characterized. It has been purified and reconstituted and has a molecular mass of around 80 kDa, of which 10-15 kDa is sugar. Amino and carboxyl termini (around 50 amino acids each) are not required for function. The transporter is protected against proteolysis at multiple sites by GABA, provided that the two cosubstrates -sodium and chloride -are present. Several amino acid residues that are critical for function have been identified in the GABA transporter. These include arginine-69 and tryptophan-222 located in the first and fourth putative transmembrane helices, respectively. The first is possibly involved in the binding of chloride. The tryptophan appears to serve as a binding site for the amino group of GABA.

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“…With exception of the transporters for glutamate, the transporters for other neurotransmitters, such as GABA, serotonin, dopamine, norepinephrine, and glycine, are sodiumand chloride-dependent and belong to the NSS family (reviewed in Refs 1 and 2). These eukaryotic NSS transporters couple the flux of neurotransmitters not only to that of sodium but also to that of chloride, and in the case of GABA transporter GAT-1, the stoichiometry of the coupled electrogenic transport is 2Na ϩ :1Cl Ϫ :GABA (3)(4)(5)(6). Therefore, in addition to radioactive GABA uptake, the transport can also be monitored by measuring GABA-induced steady-state currents (5,6).…”

mentioning

confidence: 99%

The Aromatic and Charge Pairs of the Thin Extracellular Gate of the γ-Aminobutyric Acid Transporter GAT-1 Are Differently Impacted by Mutation

Dayan¹,

Ben-Yona²,

Kanner³

2014

Journal of Biological Chemistry

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.gamma.-Aminobutyric acid transport in reconstituted preparations from rat brain: coupled sodium and chloride fluxes

Cited by 162 publications

References 15 publications

Transmembrane Domain 8 of the γ-Aminobutyric Acid Transporter GAT-1 Lines a Cytoplasmic Accessibility Pathway into Its Binding Pocket

Transmembrane Domain 8 of the γ-Aminobutyric Acid Transporter GAT-1 Lines a Cytoplasmic Accessibility Pathway into Its Binding Pocket

Ion-coupled neurotransmitter transport

The Aromatic and Charge Pairs of the Thin Extracellular Gate of the γ-Aminobutyric Acid Transporter GAT-1 Are Differently Impacted by Mutation

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