GABAA RECEPTOR FUNCTION IS REGULATED BY PHOSPHORYLATION IN ACUTELY DISSOCIATED GUINEA-PIG HIPPOCAMPAL-NEURONS

1. 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. 2. Application of GABA (100‐300 microM) 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. 3. 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 (approximately 10(‐8) M) was also necessary to stabilize the GABAA response. 4. The role of a phosphorylation process in regulating the GABAA receptor was tested. After the GABA response stabilized, introduction of alkaline phosphatase (100 micrograms/ml) to the intracellular perfusate caused a complete run‐down of the GABA response. 5. 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. 6. Run‐down of GABA responses accelerated when intracellular Ca2+ concentration ([Ca2+]i) was elevated to about 5 x 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. 7. The introduction of ATP‐gamma‐S to the intracellular medium retarded the run‐down of GABA responses caused by elevation of [Ca2+]i. 8. N‐(6‐Aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide (W‐7), a calmodulin inhibitor, reduced the rate of run‐down induced by elevated [Ca2+]i. 9. 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. © 1990 The Physiological Society