Regulation of surface localization of the small conductance Ca2+-activated potassium channel, Sk2, through direct phosphorylation by cAMP-dependent protein kinase
Regulation of surface localization of the small conductance Ca2+-activated potassium channel, Sk2, through direct phosphorylation by cAMP-dependent protein kinase
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DOI：
10.1074/jbc.m513125200
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发表时间：
2006-04-28
影响因子：
4.8
通讯作者：
Anderson, AE
中科院分区：
文献类型：
--
作者：
Ren, YJ;Barnwell, LF;Anderson, AE
小电导，钙激活的电压非依赖性钾通道（SK通道）广泛表达于不同的组织，然而，很少有人知道的SK通道亚基的分子调控。激酶直接改变离子通道亚基是这些通道功能调节的候选机制。我们发现，环AMP依赖性蛋白激酶（PKA）与毛喉素（50 μ M）的激活引起的表面定位的SK2通道亚基在COS 7细胞中表达的急剧下降，由于直接磷酸化的SK2通道亚基。PKA磷酸化研究使用的SK2通道亚基表达谷胱甘肽S-转移酶融合蛋白构建体的细胞内结构域表明，氨基末端和羧基末端区域的PKA底物在体外。突变分析确定了一个单一的PKA磷酸化位点内的氨基末端的SK2亚基在丝氨酸136。诱变和质谱研究确定了四个PKA磷酸化位点：Ser（465）（次要位点）和羧基末端区域内的三个氨基酸残基Ser（568）、Ser（569）和Ser（570）（主要位点）。突变的SK2通道亚基，三个连续的丝氨酸突变为丙氨酸，以阻止这些位点的磷酸化，PKA刺激后表面表达没有减少。因此，我们的研究结果表明，PKA磷酸化这三个网站是必要的PKA介导的重组SK2表面表达。
Small conductance, Ca2+-activated voltage-independent potassium channels (SK channels) are widely expressed in diverse tissues; however, little is known about the molecular regulation of SK channel subunits. Direct alteration of ion channel subunits by kinases is a candidate mechanism for functional modulation of these channels. We find that activation of cyclic AMP-dependent protein kinase (PKA) with forskolin ( 50 mu M) causes a dramatic decrease in surface localization of the SK2 channel subunit expressed in COS7 cells due to direct phosphorylation of the SK2 channel subunit. PKA phosphorylation studies using the intracellular domains of the SK2 channel subunit expressed as glutathione S-transferase fusion protein constructs showed that both the amino-terminal and carboxyl-terminal regions are PKA substrates in vitro. Mutational analysis identified a single PKA phosphorylation site within the amino-terminal of the SK2 subunit at serine 136. Mutagenesis and mass spectrometry studies identified four PKA phosphorylation sites: Ser(465) ( minor site) and three amino acid residues Ser(568), Ser(569), and Ser(570) ( major sites) within the carboxyl-terminal region. A mutated SK2 channel subunit, with the three contiguous serines mutated to alanines to block phosphorylation at these sites, shows no decrease in surface expression after PKA stimulation. Thus, our findings suggest that PKA phosphorylation of these three sites is necessary for PKA-mediated reorganization of SK2 surface expression.