GABAA RECEPTOR FUNCTION IS REGULATED BY PHOSPHORYLATION IN ACUTELY DISSOCIATED GUINEA-PIG HIPPOCAMPAL-NEURONS
GABAA RECEPTOR FUNCTION IS REGULATED BY PHOSPHORYLATION IN ACUTELY DISSOCIATED GUINEA-PIG HIPPOCAMPAL-NEURONS
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DOI：
10.1113/jphysiol.1990.sp017908
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发表时间：
1990-01-01
影响因子：
5.5
通讯作者：
WONG, RKS
中科院分区：
文献类型：
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作者：
CHEN, QX;STELZER, A;WONG, RKS
在从成熟豚鼠海马体中急剧分离的锥体细胞中检查了由 GABAA 受体介导的电流。使用全细胞电压钳记录测量电流响应。内部灌注技术用于在记录过程中改变细胞内内容物。通过短持续时间压力脉冲施加 GABA (100-300 μM)，在 -10 mV 的保持电势下产生外向电流响应。当使用不含 Mg-ATP 的细胞内溶液进行记录时，10 分钟后 GABA 响应逐渐降低至初始值的 10% 以下。 GABA 反应的这种“衰退”不能用脱敏来解释，因为衰退速率不依赖于激动剂的应用。当将 Mg2+ (4 mM) 和 ATP (2 mM) 引入细胞内灌流液中时，GABAA 反应的减弱发生逆转。除了 Mg-ATP 的存在之外，还需要将细胞内溶液中的 Ca2+ 缓冲至低水平（约 10-8 M）以稳定 GABAA 反应。测试了磷酸化过程在调节 GABAA 受体中的作用。 GABA反应稳定后，向细胞内灌注液中引入碱性磷酸酶(100μg/ml)导致GABA反应完全减弱。当 ATP 被 ATP-γ-S（腺苷 5''-O-（硫代三磷酸））取代时，获得稳定的 GABA 响应，ATP 的类似物提供硫代磷酸基团，从而产生更耐水解的产物。经过这样的处理，在引入细胞内碱性磷酸酶后，GABA 反应下降得更慢。当细胞内Ca2+浓度([Ca2+]i)升高至约5倍时，GABA反应的衰退加速。 10-4 M。通过将[Ca 2+] i 降低至约10-8 M，可以停止和逆转由升高的[Ca 2+ ] i 引起的衰退。将ATP-γ-S引入细胞内培养基延缓了由[Ca 2+ ] i 升高引起的GABA反应的衰退。 N-(6-氨基己基)-5-氯-1-萘磺酰胺 (W-7) 是一种钙调蛋白抑制剂，可降低 [Ca2+]i 升高引起的衰竭率。这些结果表明GABAA受体的功能是通过受体或一些密切相关的调节分子的磷酸化来维持的。 [Ca2+]i 的升高可能通过激活 Ca2+/钙调蛋白依赖性磷酸酶来破坏 GABAA 受体的功能。
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.