The Aqueous Accessibility in the External Half of Transmembrane Domain I of the GABA Transporter GAT-1 Is Modulated by Its Ligands

Abstract

The sodium-and chloride-dependent ␥-aminobutyric acid (GABA) transporter GAT-1 is the first identified member of a family of transporters, which maintain low synaptic neurotransmitter levels and thereby enable efficient synaptic transmission. To obtain evidence for the idea that the highly conserved transmembrane domain I (TMD I) participates in the permeation pathway, we have determined the impact of impermeant methanethiosulfonate (MTS) reagents on cysteine residues engineered into this domain. As a background the essentially insensitive but fully active C74A mutant has been used. Transport activity of mutants with a cysteine introduced cytoplasmic to glycine 63 is largely unaffected and is resistant to the impermeant MTS reagents. Conversely, transport activity in mutants extracellular to glycine 63 is strongly impacted. Nevertheless, transport activity could be measured in all but three mutants: G65C, N66C, and R69C. In each of the six active cysteine mutants the activity is highly sensitive to the impermeant MTS reagents. This sensitivity is potentiated by sodium in L64C, F70C, and Y72C, but is protected in V67C and P71C. GABA protects in L64C, W68C, F70C, and P71C. The non-transportable GABA analogue SKF100330A also protects in L64C, W68C, and P71C as well as V67C, but strikingly potentiates inhibition in F70C. Although cysteine substitution in this region may have perturbed the native structure of GAT-1, our observations, taken together with the recently published accessibility study on the related serotonin transporter (Henry, L. K., Adkins, E. M., Han, Q., and Blakely, R. D. (2003) J. Biol. Chem. 278, 37052-37063), suggest that the extracellular part of TMD I is conformationally sensitive, lines the permeation pathway, and forms a more extended structure than expected from a membraneembedded ␣-helix.Neurotransmitter transporters located in the plasma membranes of cells surrounding the synapse are essential for the overall process of synaptic transmission. One of the best examples of the importance of these neurotransmitter transporters comes from studies of dopamine transporter knock-out mice; the decay of extracellular dopamine in brain slices of such mice is about 100 times longer than normal (1). Most neurotransmitters are removed from the synaptic cleft by sodium-and chloride-dependent neurotransmitter transporters and this process is essential to maintain efficient synaptic transmission (for reviews see Refs. 2 and 3). The ␥-aminobutyric acid (GABA) 1 transporter GAT-1 (4, 5) is a particularly well studied member of this family. The transporter catalyzes electrogenic sodium:chloride:GABA cotransport with a stoichiometry of 2:1:1 (6 -9). There is still some dispute on this issue, as it has been proposed that during sodium-coupled GABA transport obligatory chloride out /chloride in exchange takes place (10). GAT-1, as well as the other members of the family, is predicted to have 12 transmembrane domains linked by hydrophilic loops with the amino and carboxyl termini residing inside the cell (5). Stud...

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

“…In GAT-1, GABA reduced the reactivity of cysteines located in the extracellular access pathway (25,31). However, in several cases, the non-transportable analogue SKF100330A did not potentiate but protected (31), presumably by a physical blockade of the sulfhydryl reagent by the bulky blocker. Altogether, the present work and the previous studies suggest a concerted conformational change involving opposite accessibility changes in external and internal pathways.…”

“…Conversely, several residues in the intracellular part of TM 5 of SERT reacted faster in the presence of substrate and slower in the presence of a non-transportable analogue and were proposed to line the intracellular pathway leading to the binding pocket (16). In GAT-1, GABA reduced the reactivity of cysteines located in the extracellular access pathway (25,31). However, in several cases, the non-transportable analogue SKF100330A did not potentiate but protected (31), presumably by a physical blockade of the sulfhydryl reagent by the bulky blocker.…”

Selective Amino Acid Substitutions Convert the Creatine Transporter to a γ-Aminobutyric Acid Transporter

“…Despite only 20 -25% sequence identity between LeuT and its eukaryotic counterparts, there is high conservation in the TMs 1, 3, 6, and 8 that surround the leucine binding site (11). Biochemical studies on mammalian transporters had identified, previously, that residues in TM1 (22,23) and TM3 (12,13,24,25) were important for substrate binding and transporter function.…”

Proximity of Transmembrane Domains 1 and 3 of the γ-AminobutyricAcid Transporter GAT-1 Inferred from Paired CysteineMutagenesis

“…Two TMD 3 residues of SERT, corresponding to leucine 136 and isoleucine 143 of GAT-1, are located one turn of a putative ␣-helix below or above the critical tyrosine 176, respectively (17). The highly conserved TMD 1, which appears to line the permeation pathway and appears to form a more extended structure than expected from a membrane-embedded ␣-helix (18,19), contains several amino acid residues critical for function. They have been implicated in playing an important role in the interaction with the neurotransmitter (20,21), in the determination of the apparent affinity for sodium (21,22), and in the sodium-dependent conversion of the leak mode of the transporter into the coupled mode (21).…”