Using lithium to probe sequential cation interactions with GAT1

Am J Physiol Cell Physiol. 2012 Jun 1;302(11):C1661-75. doi: 10.1152/ajpcell.00446.2011. Epub 2012 Mar 28.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Biological Transport
  • Cations / metabolism*
  • GABA Plasma Membrane Transport Proteins / metabolism*
  • Lithium / metabolism*
  • Lithium / pharmacology
  • Membrane Potentials
  • Membrane Proteins / metabolism
  • Oocytes / metabolism
  • Patch-Clamp Techniques
  • Sodium / metabolism*
  • Xenopus laevis

Substances

  • Cations
  • GABA Plasma Membrane Transport Proteins
  • Membrane Proteins
  • Lithium
  • Sodium