Activation of calcium-phospholipid-dependent protein kinase enhances benzodiazepine and barbiturate potentiation of the GABAA receptor.

Abstract

The effect of calcium-phospholipid-dependent protein kinase (PKC) on GABAA receptor function was examined in Xenopus oocytes expressing recombinant human GABAA receptor using two-electrode voltage-clamp measurements. Phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC, inhibited GABA-gated chloride currents by approximately 72% in oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs. Phorbol 12-monomyristate (PMM), a negative control analogue of PMA, did not alter GABAA receptor responses. To investigate whether activation of PKC could alter the modulatory responses of the receptor complex the effect of PMA on benzodiazepine and barbiturate potentiation of GABA responses was assessed. In oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs, diazepam (300 nM) potentiated GABA responses by approximately 160%. Following PMA (5-25 nM) treatment, diazepam potentiation was significantly increased to 333%. No effect of the inactive phorbol ester PMM (25 nM) was observed on diazepam potentiation of GABA responses. PMA enhancement of diazepam potentiation of GABA responses was also observed in oocytes expressing alpha 1 beta 1 gamma 2S subunit cDNAs, indicating that the unique PKC site present in the gamma 2L subunit is not required for observing the PMA effect. PMA (5-25 nM) also enhanced pentobarbital potentiation of GABA responses. In oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs, pentobarbital (25 microM) potentiated GABA receptor responses by approximately 97%. Following treatment with PMA (5-25 nM), pentobarbital potentiation of GABA responses increased to approximately 156%. The present results suggest that protein phosphorylation may alter the coupling between the allosteric modulatory sites within the GABAA receptor complex.