MODULATION OF GABA FLUX ACROSS RAT-BRAIN MEMBRANES RESOLVED BY A RAPID QUENCHED INCUBATION TECHNIQUE

The progress and inhibition of [H-3]GABA influx in native plasma membr ane vesicles from the rat cerebral cortex was studied on a subsecond t o minute time scale under different conditions by applying a rapid que nched incubation technique. In the absence of Ca2+ ([Ca2+](free) = 10( -8) M), the progress of influx followed by the addition of 10 nM [H-3] GABA to the membrane vesicle suspension with time (500 ms to 15 min) c an be described by a first-order rate equation giving an over all rate constant, k, of 3.93 +/- 0.48 x 10(-3) s(-1) and equilibrium influx v alue, INFe, of 8.84 +/- 0.41 pmol [H-3]GABA/mg protein. In the presenc e of Ca2+ ([Ca2+](free) = 2.4 x 10(-3) M) a significant increase in th e INFe value was observed (k = 4.64 +/- 0.41 x 10(-3) s(-1) and INFe = 13.9 +/- 0.40 pmol [H-3]GABA/mg protein). Multiplicity of GABA transp orters was indicated in the time-dependent inhibition of [H-3]GABA inf lux by different uptake blockers. In the absence of Ca2+, depolarizati on (75 mM KCl) inhibited the influx of [H-3]GABA into the vesicles by similar to 70% and initiated the efflux from vesicles loaded with [H-3 ]GABA. Different uptake blockers inhibited the Ca2+-independent transl ocation of [H-3]GABA in both directions with similar specificities.