Topological localization of cysteine 74 in the GABA transporter, GAT1, and its importance in ion binding and permeation

Xenopus oocytes expressing the GABA transporter GAT1 were exposed to membrane-impermeant sulfhydryl reagents, resulting in decreased GABA transport current, decreased capacitive charge movements, and increased Na+ and Li+ leakage currents. Mutation of cysteine 74 to alanine (C74A) eliminated these effects. The W68S and W68L mutations significantly increased and decreased the transporter's sensitivity, respectively, to sulfhydryl reagents. At each of the positions 73 through 76, cysteine residues were accessible to external MTSET. These findings, together with recent evidence placing the HD2-HD3 loop on the extracellular side, suggest that the HD2 region does not traverse the membrane.