Modulation of GABA flux across rat brain membranes resolved by a rapid quenched incubation technique

The progress and inhibition of [3H]GABA influx in native plasma membrane vesicles from the rat cerebral cortex was studied on a subsecond to minute time scale under different conditions by applying a rapid quenched incubation technique. In the absence of Ca2+ ([Ca2+]free = 10(-8) M), the progress of influx followed by the addition of 10 nM [3H]GABA to the membrane vesicle suspension with time (500 ms to 15 min) can be described by a first-order rate equation giving an overall rate constant, k, of 3.93 +/- 0.48 x 10(-3) s-1 and equilibrium influx value, INFe, of 8.84 +/- 0.41 pmol [3H]GABA/mg protein. In the presence of Ca2+ ([Ca2+]free = 2.4 x 10(-3) M) a significant increase in the INFe value was observed (k = 4.64 +/- 0.41 x 10(-3) s-1 and INFe = 13.9 +/- 0.40 pmol [3H]GABA/mg protein). Multiplicity of GABA transporters was indicated in the time-dependent inhibition of [3H]GABA influx by different uptake blockers. In the absence of Ca2+, depolarization (75 mM KCl) inhibited the influx of [3H]GABA into the vesicles by approximately 70% and initiated the efflux from vesicles loaded with [3H]GABA. Different uptake blockers inhibited the Ca(2+)-independent translocation of [3H]GABA in both directions with similar specificities.