GAT1 (GABA:Na⁺:Cl^-) cotransport function: Kinetic studies in giant Xenopus oocyte membrane patches

To explain cotransport function, the 'alternating access' model requires that conformational changes of the empty transporter allow substrates to bind alternatively on opposite membrane sides. To test this principle for the GAT1 (GABA:Na⁺:Cl^-) cotransporter, we have analyzed how its charge-moving partial reactions depend on substrates on both membrane sides in giant Xenopus oocyte membrane patches. (a) 'Slow' charge movements, which require extracellular Na⁺ and probably reflect occlusion of Na⁺ by GAT1, were defined in three ways with similar results: by application of the high- affinity GAT1 blocker (NO-711), by application of a high concentration (120 mM) of cytoplasmic Cl^-, and by removal of extracellular Na⁺ via pipette perfusion. (b) Three results indicate that cytoplasmic Cl^- and extracellular Na⁺ bind to the transporter in a mutually exclusive fashion: first, cytoplasmic Cl^- (5-140 mM) shifts the voltage dependence of the slow charge movement to more negative potentials, specifically by slowing its 'forward' rate (i.e., extracellular Na⁺ occlusion); second, rapid application of cytoplasmic Cl^- induces an outward current transient that requires extracellular Na⁺, consistent with extracellular Na⁺ being forced out of its binding site; third, fast charge-moving reactions, which can be monitored as a capacitance, are 'immobilized' both by cytoplasmic Cl^- binding and by extracellular Na⁺ occlusion (i.e., by the slow charge movement). (c) In the absence of extracellular Na⁺, three fast (submillisecond) charge movements have been identified, but no slow components. The addition of cytoplasmic Cl^- suppresses two components (τ < 1 ms and 13 μs) and enables a faster component (τ < 1 μs). (d)We failed to identify charge movements of fully loaded GAT1 transporters (i.e., with all substrates on both sides). (e) Under zero-trans conditions, inward (forward) GAT1 current shows pronounced pre- steady state transients, while outward (reverse) GAT1 current does not. (f) Turnover rates for reverse GAT1 transport (33°C), calculated from the ratio of steady state current magnitude to total charge movement magnitude, can exceed 60 s^-1 at positive potentials.