Martin S W, Butcher A J, Berrow N S, Richards M W, Paddon R E, Turner D J, Dolphin A C, Sihra T S, Fitzgerald E M
Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.
Cell Calcium. 2006 Mar;39(3):275-92. doi: 10.1016/j.ceca.2005.11.002. Epub 2006 Jan 6.
Voltage-dependent calcium channels (VDCCs) in sensory neurones are tonically up-regulated via Ras/extracellular signal regulated kinase (ERK) signalling. The presence of putative ERK consensus sites within the intracellular loop linking domains I and II of neuronal N-type (Ca(v)2.2) calcium channels and all four neuronal calcium channel beta subunits (Ca(v)beta), suggests that Ca(v)2.2 and/or Ca(v)betas may be ERK-phosphorylated. Here we report that GST-Ca(v)2.2 I-II loop, and to a lesser extent Ca(v)beta1b-His(6), are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca(v)2.2 I-II loop significantly reduced phosphorylation. Loss of Ser-447 reduced phosphorylation to a greater extent than mutation of Ser-409. Patch-clamp recordings from wild-type Ca(v)2.2,beta1b,alpha2delta1 versus mutant Ca(v)2.2(S447A) or Ca(v)2.2(S409A) channels revealed that mutation of either site significantly reduced current inhibition by UO126, a MEK (ERK kinase)-specific inhibitor that down-regulates ERK activity. However, no additive effect was observed by mutating both residues together, suggesting some functional redundancy between these sites. Mutation of both Ser-161 and Ser-348 on Ca(v)beta1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca(v)2.2(S447A) with Ca(v)beta1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca(v)beta1b(S161,348A) was co-expressed with Ca(v)2.2(S409A). Thus, Ser-447 on Ca(v)2.2 and Ser-161 and Ser-348 of Ca(v)beta1b appear to be both necessary and sufficient for ERK-dependent modulation of these channels. Together, our data strongly suggest that modulation of neuronal N-type VDCCs by ERK involves phosphorylation of Ca(v)2.2alpha1 and to a lesser extent possibly also Ca(v)beta subunits.
感觉神经元中的电压依赖性钙通道（VDCCs）通过Ras/细胞外信号调节激酶（ERK）信号通路持续上调。在连接神经元N型（Ca(v)2.2）钙通道结构域I和II的细胞内环以及所有四个神经元钙通道β亚基（Ca(v)β）中存在假定的ERK共有位点，这表明Ca(v)2.2和/或Ca(v)β可能被ERK磷酸化。在此我们报告，GST-Ca(v)2.2 I-II环，以及程度较轻的Ca(v)β1b-His(6)，是ERK1/2磷酸化的底物。GST-Ca(v)2.2 I-II环上Ser-409和/或Ser-447突变为丙氨酸显著降低了磷酸化。Ser-447缺失比Ser-409突变更显著地降低了磷酸化。对野生型Ca(v)2.2、β1b、α2δ1与突变型Ca(v)2.2(S447A)或Ca(v)2.2(S409A)通道进行膜片钳记录发现，这两个位点的突变均显著降低了UO126（一种下调ERK活性的MEK（ERK激酶）特异性抑制剂）对电流的抑制作用。然而，同时突变这两个残基未观察到累加效应，表明这些位点之间存在一定的功能冗余。Ca(v)β1b上Ser-161和Ser-348的突变并未显著降低磷酸化，但确实降低了UO126诱导的电流抑制作用。至关重要的是，Ca(v)2.2(S447A)与Ca(v)β1b(S161,348A)共表达具有累加效应，消除了UO126对通道电流的作用，而当Ca(v)β1b(S161,348A)与Ca(v)2.2(S409A)共表达时未观察到这种效应。因此，Ca(v)2.2上的Ser-447以及Ca(v)β1b上的Ser-
