GAT1 (GABA:Na⁺:Cl^-) cotransport function: Database reconstruction with an alternating access model

We have developed an alternating access transport model that accounts well for GAT1 (GABA:Na⁺: Cl^-) cotransport function in Xenopus oocyte membranes. To do so, many alternative models were fitted to a database on GAT1 function, and discrepancies were analyzed. The model assumes that GAT1 exists predominantly in two states, E(in), and E(out). In the E(in), state, one chloride and two sodium ions can bind sequentially from the cytoplasmic side. In the E(out) state, one sodium ion is occluded within the transporter, and one chloride, one sodium, and one γ-aminobutyric acid (GABA) molecule can bind from the extracellular side. When E(in) sites are empty, a transition to the E(out) state opens binding sites to the outside and occludes one extracellular sodium ion. This conformational change is the major electrogenic GAT1 reaction, and it rate-limits forward transport (i.e., GABA uptake) at 0 mV. From the E(out) state, one GABA can be translocated with one sodium ion to the cytoplasmic side, thereby forming the *E(in), state. Thereafter, an extracellular chloride ion can be translocated and the occluded sodium ion released to the cytoplasm, which returns the transporter to the E(in), state. GABA-GABA exchange can occur in the absence of extracellular chloride, but a chloride ion must be transported to complete a forward transport cycle. In the reverse transport cycle, one cytoplasmic chloride ion binds first to the E(in), state, followed by two sodium ions. One chloride ion and one sodium ion are occluded together, and thereafter the second sodium ion and GABA are occluded and translocated. The weak voltage dependence of these reactions determines the slopes of outward current- voltage relations. Experimental results that are simulated accurately include (a) all current-voltage relations, (b) all substrate dependencies described to date, (c) cis-cis and cis-trans substrate interactions, (d) charge movements in the absence of transport current, (e) dependencies of charge movement kinetics on substrate concentrations, (f) pre-steady state current transients in the presence of substrates, (g) substrate-induced capacitance changes, (h) GABA-GABA exchange, and (i) the existence of inward transport current and GABA-GABA exchange in the nominal absence of extracellular chloride.