C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation
C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation
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DOI：
10.1074/jbc.m117.787788
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发表时间：
2017-10-20
影响因子：
4.8
通讯作者：
Marionneau, Celine
中科院分区：
文献类型：
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作者：
Burel, Sophie;Coyan, Fabien C.;Marionneau, Celine
电压门控Na+（Na-V）通道是心肌兴奋性的关键调节因子，而Na（V）1.5通道失活的Ca 2 +/钙调蛋白依赖性蛋白激酶II（CaMKII）依赖性改变正在成为心力衰竭心律失常的关键决定因素。然而，与这些致炎性疾病相关的Na（V）1.5亚基的全局天然磷酸化模式和相关的通道调节缺陷仍然未知。在这里，我们进行磷酸化蛋白质组学分析，以确定和定量原位Na（V）1.5蛋白纯化成人WT和失败的CaMKII δ（c）-过表达（CaMKII δ（c）-Tg）小鼠心室中的磷酸化位点。在鉴定的19个天然Na（V）1.5磷酸化位点中，位置1938和1989处的两个C-末端磷酸丝氨酸显示与WT心室相比CaMKII delta（c）-Tg中的磷酸化增加。然后我们检验了这两个位点的磷酸化损害成纤维细胞生长因子13（FGF 13）依赖的Na（V）1.5通道失活调节的假设。HEK 293细胞中的全细胞电压钳分析表明，FGF 13增加Na（V）1.5通道的可用性并降低晚期Na+电流，这两种作用被Na（V）1.5突变体在两个位点模拟磷酸化所消除。另外的免疫共沉淀实验显示，FGF 13增强钙调蛋白与Na（V）1.5的结合，并且两个位点处的拟磷酸化突变降低FGF 13的相互作用，并且因此降低钙调蛋白与Na（V）1.5的相互作用。总之，我们在Na（V）1.5的C末端鉴定了两个新的天然磷酸化位点，其损害了FGF 13依赖的通道失活调节，并可能导致衰竭心脏中CaMKII δ（c）依赖性促炎性疾病。
Voltage-gated Na+ (Na-V) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in Na(V)1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of Na(V)1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the Na(V)1.5 proteins purified from adult WT and failing CaMKII delta(c)-overexpressing (CaMKII delta(c)-Tg) mouse ventricles. Of 19 native Na(V)1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKII delta(c)-Tg compared with the WT ventricles. Wethen tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of Na(V)1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases Na(V)1.5 channel availability and decreases late Na+ current, two effects that were abrogated with Na(V)1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to Na(V)1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with Na(V)1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of Na(V)1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKII delta(c)-dependent arrhythmogenic disorders in failing hearts.