Ionic requirements for transport and release of [(3)H]GABA by synaptic plasma membrane vesicles

A simple procedure for isolating a fraction enriched in synaptic plasma membrane (SPM) vesicles from sheep brain cortex is described and is used in studying uptake and release of [(3)H]GABA. The crude synaptosomal fraction was subjected to osmotic shock and the lysate was subfractioned in a discontinuous density gradient of dextran T110. The activities of the enzyme markers of SPM, the phospholipid analysis and electron microscopy examination suggest that this fraction of SPM is of a purity similar to that of preparations previously described by more time consuming methods. Superfusion of SPM vesicles with sucrose, ?-aminobutyric acid (GABA), veratridine, increased [K(+)](o), or reduced [Cl(?)](o), induced the release of 4-amino-n-[2,3,(3)H]butyric acid ([(3)H]GABA) previously loaded into these vesicles by a Na(+), Cl(?) and K(+) dependent mechanism. Spontaneous and depolarization induced release of [(3)H]GABA does not require external Na(+) or Cl(?), whereas the release elicited by unlabeled GABA (homoexchange) is stimulated by Na(+) or Cl(?). Thus, both the uptake and homoexchange of [(3)H]GABA are influenced by Na(+) and Cl(?).