title: Using lithium to probe sequential cation interactions with GAT1.
abstract: 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 (22)Na 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.
authors.affiliation: Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Switzerland. stine.lundby@uzh.ch
authors.forename: Anne-Kristine
authors.initials: AK
authors.lastname: Meinild
authors.forename: Ian C
authors.initials: IC
authors.lastname: Forster
mesh_terms: D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002412:Cations / Q000378:metabolism*; D050485:GABA Plasma Membrane Transport Proteins / Q000378:metabolism*; D008094:Lithium / Q000378:metabolism* / Q000494:pharmacology; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D012964:Sodium / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D002412:Cations; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D008094:Lithium; D012964:Sodium