On the mechanism of GABA-induced currents in cultured rat cortical neurons

Abstract We applied the perforated-patch-clamp technique to cultured cortical neurons of the rat to characterize the ionic basis of membrane potential changes and membrane currents induced by γ-aminobutyric acid (GABA). Gramicidin was used as the membrane-perforating agent, to allow the recording of whole-cell currents without impairing the intracellular Cl– concentration ([Cl–]i). In current-clamp experiments in the presence of 26 mM HCO3– the application of 50 µM GABA evoked changes in the membrane potential of neurons including depolarizations (19%), hyperpolarizations (38%) and biphasic changes in membrane potential (31%), characterized by a transient hyperpolarization followed by a sustained depolarization. Accordingly, GABA (50–200 µM) induced inward, outward or biphasic current responses under voltage-clamp. Inward and biphasic currents as well as depolarizations and biphasic membrane potential responses, respectively, occurred more frequently in the presence of 26 mM HCO3–. The second phase of the biphasic membrane potential or current responses was markedly reduced when the preparation was bathed in a HCO3–-free saline, indicating a contribution from HCO3–. The reversal potential of the GABA-induced currents (EGABA) determined with the gramicidin-perforated-patch mode and in the nominal absence of HCO3– was –73 mV, while it was shifted to –59 mV in the presence of HCO3–. Combined patch-clamp and microfluorimetric measurements using the Cl–-sensitive dye 6-methoxy-1-(3-sulphonatopropyl)quinolinium (SPQ) showed that GABA evoked an increase of [Cl–]i in 54% (n=13) of the neurons. We conclude that this increase of [Cl–]i in combination with the efflux of HCO3– results in a shift of EGABA above the resting membrane potential that gives rise to GABA-mediated depolarizations.