Pre-steady-state and reverse transport currents in the GABA transporter GAT1

Cherubino F, Bertram S, Bossi E, Peres A. Pre-steady-state and reverse transport currents in the GABA transporter GAT1. Am J Physiol Cell Physiol 302: C1096-C1108, 2012. First published December 14, 2011; doi:10.1152/ajpcell.00268.2011.-The role of internal substrates in the biophysical properties of the GABA transporter GAT1 has been investigated electrophysiologically in Xenopus oocytes heterologously expressing the cotransporter. Increments in Cl- and/or Na+ concentrations caused by intracellular injections did not produce significant effects on the pre-steady-state currents, while a positive shift of the charge-voltage (Q-V) and decay time constant (tau)-voltage (tau-V) curves, together with a slowing of tau at positive potentials, was observed following treatments producing cytosolic Cl- depletion. Activation of the reverse transport mode by injections of GABA caused a reduction in the displaced charge. In the absence of external Cl-, a stronger reduction in the displaced charge, together with a significant increase in reverse transport current, was observed. Therefore, complementarity between pre-steady-state and transport currents, observed in the forward mode, is preserved in the reverse mode. All these findings can be qualitatively reproduced by a kinetic scheme in which, in the forward mode, the Cl- ion is released first, after the inward charge movement, while the two Na+ 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-.