Using lithium to probe sequential cation interactions with GAT1

摘要

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.