The objective of the proposed research project is to examine how intracellular factors, in particular second messenger-mediated mechanisms, regulate GABAA-receptor function. our interest in this issue is prompted by four recent developments: first, high-frequency stimulation of afferent fibers produces a hyperexcitability in CAl neurons which is accompanied by long-term changes of GABA and NMDA-receptor sensitivity. GABA sensitivity is reduced and NMDA receptor sensitivity is greatly enhanced. Second, functional GABAA receptors require the intracellular presence of phosphorylation factors Mg2+ and ATP: the omission of such factors results in a reversible loss of GABA conductance. Third, elevations of intracellular calcium concentrations (> 10-6 M) result in a run-down of GABAA, responses. Rises in intracellular calcium [Ca2+]i) stimulate a calcium/calmodulin-dependent phosphatase which dephosphorylates the GABAA receptor. Fourth, biochemical studies demonstrate net phosphorylation of the GABAA receptor protein by purified protein kinases A and C. Whole-cell clamp experiments will be carried out in acutely dissociated neurons from the hippocampus of adult guinea-pigs. The recent development of a perfusion system which allows the exchange of the intracellular content during the recording will greatly facilitate intracellular studies. The specific objectives are: 1) To define the intracellular conditions that regulate GABAA receptor function. 2) To investigate the role of known second messenger systems and associated protein kinases in the regulation of GABAA receptor function (e.g. protein kinase A, protein kinase C, calcium/calmodulin-dependent kinase). 3) The role of [Ca2+] i in the regulation of GABAA-receptor function.GABAA receptors play a paramount role in the control of the collective behavior of cortical neuronal populations in the mammalian CNS. Modulation of GABAA-receptor sensitivity via intracellular second messengers provides mechanisms for long-lasting modifications of the state of excitability of neuronal populations.
