Regulation of GABAA receptors by protein kinase C and hypoxia in human NT2-N neurons

GABAA receptors (GABARs) mediate fast inhibitory neurotransmission in the human brain. Since GABARs play a key role in controlling neuronal and network activity, modulating their function will have important consequences for neural excitation and communication. In this dissertation, using human NT2-N cells as a neuronal model system, we evaluated the effects of protein kinase C (PKC) and hypoxia on the regulation of GABAR function. In the first manuscript, we found that PKC, activated by phorbol dibutyrate (PDBu), reduced the apparent affinity of the benzodiazepine, diazepam, to enhance GABAR currents without affecting maximal enhancement; this effect could be blocked by the PKC inhibitor, bisindolylmaleimide. This PKC effect was specific to the benzodiazepine site, as PDBu did not alter potentiation of GABAR currents by the barbiturate, pentobarbital. Reducing the apparent affinity of diazepam at NT2-N GABARs by activation of PKC could be one important mechanism by which GABAR function can be modified by phosphorylation. In the second manuscript, we demonstrated that hypoxic exposure altered GABAR function with a biphasic time-course, accompanied by changes in specific GABAR subunit mRNA levels. We also found that hypoxic exposure induced the accumulation of the oxygen-dependent transcription factor, hypoxia-inducible factor 1alpha (HIF-1alpha), a key regulator of cellular and physiological responses to hypoxic stress. To assess whether hypoxic exposure could directly regulate GABAR gene expression, we studied the effect of two chemical HIF-1alpha inducers, cobalt chloride and deferoxamine. Both of these agents transiently increased HIF-1alpha levels. In contrast to increased GABAR current immediately after hypoxia and decreased current 48 h afterward, exposure to cobalt or deferoxamine resulted in increased maximal GABA currents 48 hours after exposure, with increases in specific GABAR subunit mRNA levels after 24 hours. These data demonstrate that chemical induction of HIF-1alpha alters GABARs differently than HIF-1alpha induction by hypoxia, suggesting that hypoxia triggers additional mechanisms that oppose HIF-1alpha effects on GABAR function. Understanding the mechanisms of GABAR regulation by protein kinses and hypoxia will ultimately improve the treatment of epilepsy and other CNS disorders associated with GABAR dysfunction.