GABAA receptor function is regulated by phosphorylation in acutely dissociated guinea‐pig hippocampal neurones.

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Abstract
Current mediated by GABAA receptors was examined in pyramidal cells acutely dissociated from the hippocampus of mature guinea-pigs. Current responses were measured using whole-cell voltage-clamp recordings. An internal perfusion technique was used to change the intracellular contents during recording. Application of GABA (100-300 .mu.M) by short duration pressure pulses produced outward current responses at a holding potential of -10 mV. When recordings were made with intracellular solutions which did not contain Mg-ATP, GABA responses progressively decreased to less than 10% of their initial values after 10 min. This ''run-down'' of the GABA response could not be accounted for by desensitization since the rate of run-down was not dependent upon agonist application. The run-down of the GABAA response was reversed when Mg2+ (4 mM) and ATP (2 mM) were introduced into the intracellular perfusate. In addition to the presence of Mg-ATP, buffering of Ca2+ in the intracellular solution to low levels (.apprx. 10-8 M) was also necessary to stabilize the GABAA response. The role of a phosphorylation process in regulating the GABAA receptor was tested. After the GABA response stabilized, introduction of alkaline phosphatase (100 .mu.g/ml) to the intracellular perfusate caused a complete run-down of the GABA response. Stable GABA responses were obtained when ATP was replaced by ATP-.gamma.-S (adenosine 5''-O-(thiotriphosphate)), an analogue of ATP that donates a thiophosphate group resulting in a product that is more resistant to hydrolysis. Following such treatment GABA responses declined more slowly after the introduction of intracellular alkaline phosphatase. Run-down of GABA responses accelerated when intracellular Ca2+ concentration ([Ca2+]i) was elevated to about 5 .times. 10-4 M. The run-down caused by elevated [Ca2+]i could be stopped and reversed by reducing [Ca2+]i to about 10-8 M. The introduction of ATP-.gamma.-S to the intracellular medium retarded the run-down of GABA responses caused by elevation of [Ca2+]i. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), a calmodulin inhibitor, reduced the rate of run-down induced by elevated [Ca2+]i. These results suggest that the function of the GABAA receptor is maintained by phosphorylation of the receptor or some closely associated regulatory molecule. Elevation of [Ca2+]i destabilizes the function of the GABAA receptor, probably by activating a Ca2+/calmodulin-dependent phosphatase.