Murphy B J, Rossie S, De Jongh K S, Catterall W A
Department of Pharmacology, University of Washington, Seattle 98195.
J Biol Chem. 1993 Dec 25;268(36):27355-62.
Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent protein kinase (cA-PK) and protein kinase C. Using synthetic peptides and protein microsequencing, we have determined that the alpha subunit of rat brain Na+ channel is selectively phosphorylated by cA-PK in vitro and in intact cells on 4 serine residues in the intracellular loop connecting homologous domains I and II. Ser-623 was most rapidly and extensively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was most extensively phosphorylated in mammalian cells transfected with the alpha subunit of type IIA Na+ channel in response to an increase in intracellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylated Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to lesser extents. Phosphatase 2A selectively dephosphorylated Ser-610. Together these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent phosphorylation results from selective phosphorylation and dephosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.
电压敏感性脑钠通道受环磷酸腺苷依赖性蛋白激酶（cA-PK）和蛋白激酶C调控。我们使用合成肽和蛋白质微测序技术，确定大鼠脑钠通道的α亚基在体外和完整细胞中，在连接同源结构域I和II的细胞内环中的4个丝氨酸残基上被cA-PK选择性磷酸化。在体外，Ser-623磷酸化最快且程度最高，而Ser-573、Ser-610和Ser-687磷酸化程度较低。相比之下，在转染了IIA型钠通道α亚基的哺乳动物细胞中，响应细胞内cAMP增加，丝氨酸687磷酸化程度最高。纯化的蛋白磷酸酶可选择性地使这些位点去磷酸化。钙调神经磷酸酶能快速且大量地使Ser-623去磷酸化，对Ser-573、Ser-610和Ser-687也有一定程度的去磷酸化作用。蛋白磷酸酶2A选择性地使Ser-610去磷酸化。这些结果共同表明，环磷酸腺苷依赖性磷酸化对神经元钠通道活性进而对神经元兴奋性的调节，是由α亚基连接同源结构域I和II的细胞内环中4个位点的选择性磷酸化和去磷酸化所致。
