Murphy B J, Rogers J, Perdichizzi A P, Colvin A A, Catterall W A
Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280, USA.
J Biol Chem. 1996 Nov 15;271(46):28837-43. doi: 10.1074/jbc.271.46.28837.
The voltage-sensitive Na+ channel is responsible for generating action potentials in the heart which are critical for coordinated cardiac muscle contraction. Cardiac Na+ channels are regulated by cAMP-dependent phosphorylation, but the sites of phosphorylation are not known. Using mammalian cells expressing the rat cardiac Na+ channel (rH1) alpha subunit and site-specific antibodies, we have shown that the alpha subunit of rat heart Na+ channel is phosphorylated selectively by cAMP-dependent protein kinase (PKA) in vitro and in intact cells. Analysis of the sites of phosphorylation by two-dimensional phosphopeptide mapping and site-directed mutagenesis of fusion proteins revealed that the cardiac alpha subunit is phosphorylated selectively in vitro by PKA on Ser526 and Ser529 in the intracellular loop connecting homologous domains I and II (LI-II). These two residues were phosphorylated in intact cells expressing the rH1 alpha subunit when PKA was activated. Our results define a different pattern of phosphorylation of LI-II of cardiac and brain Na+ channels and implicate phosphorylation of Ser526 and Ser529 in the differential regulation of cardiac and brain Na+ channels by PKA.
电压敏感性钠离子通道负责在心脏中产生动作电位，这对于协调心肌收缩至关重要。心脏钠离子通道受cAMP依赖性磷酸化调节，但其磷酸化位点尚不清楚。利用表达大鼠心脏钠离子通道（rH1）α亚基的哺乳动物细胞和位点特异性抗体，我们已表明大鼠心脏钠离子通道的α亚基在体外和完整细胞中可被cAMP依赖性蛋白激酶（PKA）选择性磷酸化。通过二维磷酸肽图谱分析和融合蛋白的定点诱变对磷酸化位点进行分析，结果显示在体外PKA可选择性地使位于连接同源结构域I和II的细胞内环（LI-II）中的Ser526和Ser529位点的心脏α亚基磷酸化。当PKA被激活时，在表达rH1α亚基的完整细胞中这两个残基会发生磷酸化。我们的结果确定了心脏和脑钠离子通道LI-II不同的磷酸化模式，并表明Ser526和Ser529的磷酸化与PKA对心脏和脑钠离子通道的差异调节有关。
