GABAA receptor function is regulated by phosphorylation in acutely dissociated guinea-pig hippocampal neurones.
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.
Chen
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Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
Stelzer
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A
Kay
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Wong
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RK
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000037:antagonists & inhibitors; D005260:Female; D006168:Guinea Pigs; D006624:Hippocampus / Q000502:physiology*; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D009474:Neurons / Q000502:physiology; D010766:Phosphorylation; D011963:Receptors, GABA-A / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D002147:Calmodulin; D011963:Receptors, GABA-A; D013449:Sulfonamides; D013873:Thionucleotides; D005680:gamma-Aminobutyric Acid; C022570:adenosine 5'-O-(2-thiotriphosphate); C017967:W 7; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D002118:Calcium