Costa M R, Catterall W A
J Biol Chem. 1984 Jul 10;259(13):8210-8.
In purified preparations of voltage-sensitive sodium channels, the alpha subunit is selectively phosphorylated by cAMP-dependent protein kinase (Costa, M. R. C., Casnellie, J. E., and Catterall, W. A. (1982) J. Biol. Chem., 7918-7921). We have developed methods to measure sodium channel phosphorylation in both lysed synaptosomal membranes and intact synaptosomes. Incubation of lysed synaptosomal membranes with exogenously added catalytic subunit of cAMP-dependent kinase and [gamma-32P]ATP resulted in rapid phosphorylation of the alpha subunit as detected by specific immuno-precipitation, sodium dodecyl sulfate-gel electrophoresis, and autoradiography. Analysis of tryptic phosphopeptides revealed five major sites of reaction. The level of phosphorylation of these sites on the sodium channel in intact synaptosomes was monitored using a rephosphorylation method in which those sites not phosphorylated in situ were labeled with [gamma-32P]ATP and exogenously added protein kinase after lysis of the synaptosomes. Incubation of synaptosomes with 8-Br-cAMP completely blocked labeling of the alpha subunit in rephosphorylation indicating marked stimulation of phosphorylation of the sites on the sodium channel in situ. Phosphorylation was complete in 15 s and all four of the tryptic phosphopeptides detected under these conditions could be phosphorylated in situ. These results show that the sodium channel can be rapidly phosphorylated by endogenous cAMP-dependent protein kinase in intact synaptosomes. In addition, since ATP and protein kinase are only available inside the synaptosomes, they also show that the alpha subunit is a transmembrane polypeptide exposed on both sides of the synaptosomal membrane. The functional consequences of 8-Br-cAMP-stimulated phosphorylation were examined using ion flux and neurotoxin-binding methods. Binding of saxitoxin and scorpion toxin were unaffected, but neurotoxin-activated 22Na+ influx mediated by the sodium channel was reduced 16 to 26% (P less than 0.01) under various experimental conditions. The potential physiological significance of this action is considered.
在电压敏感性钠通道的纯化制剂中，α亚基被环磷酸腺苷（cAMP）依赖性蛋白激酶选择性磷酸化（科斯塔，M.R.C.，卡斯内利，J.E.，和卡特拉尔，W.A.（1982年）《生物化学杂志》，7918 - 7921页）。我们已经开发出在裂解的突触体膜和完整突触体中测量钠通道磷酸化的方法。用外源添加的cAMP依赖性激酶催化亚基和[γ - 32P]ATP孵育裂解的突触体膜，通过特异性免疫沉淀、十二烷基硫酸钠 - 凝胶电泳和放射自显影检测到α亚基迅速磷酸化。胰蛋白酶磷酸肽分析揭示了五个主要反应位点。使用再磷酸化方法监测完整突触体中钠通道上这些位点的磷酸化水平，该方法是在突触体裂解后，用[γ - 32P]ATP和外源添加的蛋白激酶标记那些未在原位磷酸化的位点。用8 - 溴 - cAMP孵育突触体完全阻断了再磷酸化中α亚基的标记，表明原位钠通道上的位点磷酸化受到显著刺激。磷酸化在15秒内完成，在这些条件下检测到的所有四个胰蛋白酶磷酸肽都可以在原位被磷酸化。这些结果表明，完整突触体中的钠通道可被内源性cAMP依赖性蛋白激酶迅速磷酸化。此外，由于ATP和蛋白激酶仅在突触体内可用，它们还表明α亚基是一种跨膜多肽，暴露于突触体膜的两侧。使用离子通量和神经毒素结合方法研究了8 - 溴 - cAMP刺激的磷酸化的功能后果。石房蛤毒素和蝎毒素的结合不受影响，但在各种实验条件下，由钠通道介导的神经毒素激活的22Na +内流减少了16%至26%（P小于0.01）。考虑了这种作用的潜在生理意义。
