Using lithium to probe sequential cation interactions with GAT1

Li+ interacts with the Na+/Cl−-dependent GABA transporter, GAT1, under two conditions: in the absence of Na+ it induces a voltage-dependent leak current; in the presence of Na+ and GABA, Li+ stimulates GABA-induced steady-state currents. The amino acids directly involved in the interaction with the Na+ and Li+ ions at the so-called “ Na2” binding site have been identified, but how Li+ affects the kinetics of GABA cotransport has not been fully explored. We expressed GAT1 in Xenopus oocytes and applied the two-electrode voltage clamp and 22Na uptake assays to determine coupling ratios and steady-state and presteady-state kinetics under experimental conditions in which extracellular Na+ was partially substituted by Li+. Three novel findings are: 1) Li+ reduced the coupling ratio between Na+ and net charge translocated during GABA cotransport; 2) Li+ increased the apparent Na+ affinity without changing its voltage dependence; 3) Li+ altered the voltage dependence of presteady-state relaxations in the absence of GABA. We propose an ordered binding scheme for cotransport in which either a Na+ or Li+ ion can bind at the putative first cation binding site ( Na2). This is followed by the cooperative binding of the second Na+ ion at the second cation binding site ( Na1) and then binding of GABA. With Li+ bound to Na2, the second Na+ ion binds more readily GAT1, and despite a lower apparent GABA affinity, the translocation rate of the fully loaded carrier is not reduced. Numerical simulations using a nonrapid equilibrium model fully recapitulated our experimental findings.