Efflux of ᵧ-Aminobutyric Acid by Synaptic Plasma Membrane Vesicles Isolated from Rat Brain

Synaptic plasma membrane vesicles isolated from rat brain were “actively” loaded with γ-aminobutyric acid (GABA) by a process driven by a sodium ion as well as a chloride ion gradient (both out > in). Subsequently, dilution-induced efflux from these vesicles was monitored. This efflux was 2-3-fold enhanced by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone and 4-6-fold by externally added GABA. The ability of GABA to stimulate was most pronounced when both sodium and chloride ions were present in the dilution medium. The dependency of efflux of γ-aminobutyric acid on both internal sodium and chloride ions was demonstrated by three independent types of experiments: (a) preloading of the vesicles with sodium and chloride ions markedly stimulated -aminobutyric acid efflux, (b) Conditions presumably enhancing the internal sodium concentration, such as dilution in sodium-containing media in the presence of nigericin, enhanced the efflux about 10-fold. Such stimulation was not observed with vesicles previously loaded with sodium. Efflux into chloride-containing media was only slightly enhanced by triphenyltin chloride; on the other hand, this compound strongly inhibited efflux into chloride-free media, (c) A freeze-thaw technique was used to load GABA passively into the vesicles (thus without the need to introduce the external sodium and chloride required for the active loading). The efflux from such vesicles was dependent on the simultaneous presence of internal sodium and chloride ions. It is concluded that the efflux of γ-aminobutyric acid is in many aspects symmetrical with its influx [Kanner, B. I. (1978) Biochemistry 17, 1207-12.11]. It appears that in order for -aminobutyric acid to interact with the carrier both sodium and chloride have to be present on the same side as the γ-aminobutyric acid. The simplest way to account for these and the previous data is to postulate cotransport of sodium, chloride, and -aminobutyric acid through the carrier.