Paradiso K, Brehm P
Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York 11790, USA.
J Neurosci. 1998 Nov 15;18(22):9227-37. doi: 10.1523/JNEUROSCI.18-22-09227.1998.
During prolonged application of transmitter, ligand-gated ion channels enter a nonconducting desensitized state. Studies on Torpedo electroplax nicotinic acetylcholine (ACh) receptors have shown that entry into the desensitized state is accelerated by protein kinase A-dependent (PKA) receptor phosphorylation. To examine the effects of phosphorylation on desensitization of muscle-type ACh receptors, we expressed the frog embryonic receptor type in Xenopus oocytes. Treatment of embryonic muscle ACh receptors with 8-Br cAMP had no measurable effect on the rate of entry into a desensitized state, but it greatly accelerated the recovery from desensitization. Three complementary approaches to reduce the levels of receptor phosphorylation provided additional evidence for a role of PKA-dependent phosphorylation in rescuing receptors from long-term desensitization. Inactivation of the endogenous PKA activity by coexpression of an inhibitor protein, treatment of receptors with phosphatase, and removal of phosphorylation sites by site-specific subunit mutation all resulted in slowed recovery. Our findings point to the existence of two distinct desensitized states: one requiring several seconds for full recovery and a second state from which recovery requires minutes. Receptors lacking PKA phosphorylation sites exhibit a pronounced increase in the slowly recovering component of desensitization, suggesting that receptor phosphorylation speeds overall recovery by reducing the entry into a deep desensitized state. This newly described effect of phosphorylation on ACh receptor function may serve as an important modulator of postsynaptic receptor sensitivity.
在递质长时间作用期间，配体门控离子通道会进入一种非传导性的脱敏状态。对电鳐电器官烟碱型乙酰胆碱（ACh）受体的研究表明，蛋白激酶A依赖性（PKA）受体磷酸化会加速受体进入脱敏状态。为了研究磷酸化对肌肉型ACh受体脱敏的影响，我们在非洲爪蟾卵母细胞中表达了蛙胚胎型受体。用8-溴环磷酸腺苷（8-Br cAMP）处理胚胎肌肉ACh受体，对其进入脱敏状态的速率没有可测量的影响，但极大地加速了从脱敏状态的恢复。三种互补的降低受体磷酸化水平的方法，为PKA依赖性磷酸化在使受体从长期脱敏中恢复的作用提供了额外证据。通过共表达一种抑制蛋白使内源性PKA活性失活、用磷酸酶处理受体以及通过位点特异性亚基突变去除磷酸化位点，均导致恢复减慢。我们的研究结果表明存在两种不同的脱敏状态：一种需要几秒钟才能完全恢复，另一种状态的恢复则需要几分钟。缺乏PKA磷酸化位点的受体在脱敏的缓慢恢复成分上有明显增加，这表明受体磷酸化通过减少进入深度脱敏状态来加速整体恢复。这种新描述的磷酸化对ACh受体功能的影响可能是突触后受体敏感性的重要调节因子。
