PRESTEADY-STATE AND REVERSE TRANSPORT CURRENTS IN THE GABA TRANSPORTER GAT1

The role of internal substrates on the biophysical properties of GAT1 has been investigated electrophysiologically in Xenopus oocytes heterologously expressing the cotransporter. Increments in the Cl(-) and/or Na(+) concentrations caused by intracellular injections did not produce significant effects on the presteady-state currents, while a positive shift of the Q-V and τ-V curves together with a slowing down of this last quantity at positive potentials were observed following treatments producing cytosolic chloride depletion. Activation of the reverse transport mode by injections of GABA caused a reduction of the amount of displaced charge. In the absence of external chloride a stronger reduction was observed, together with a significant increase of reverse transport current. The complementarity between presteady-state and transport currents, observed for the forward mode, is therefore preserved in the reverse mode. All these findings can be qualitatively reproduced by a kinetic scheme in which, in the forward mode the chloride ion is released first, after the inward charge movement, while the two sodium ions can be released only after binding of external GABA. In the reverse mode, internal GABA must bind first to the empty transporter, followed by internal Na+ and Cl(-).