CaMKII Phosphorylation of Na(V)1.5: Novel in Vitro Sites Identified by Mass Spectrometry and Reduced S516 Phosphorylation in Human Heart Failure.
CaMKII Phosphorylation of Na(V)1.5: Novel in Vitro Sites Identified by Mass Spectrometry and Reduced S516 Phosphorylation in Human Heart Failure.
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Na（V）1.5的CAMKII磷酸化：通过质谱法鉴定出的新型体外部位，并降低了人类心力衰竭的S516磷酸化。
DOI：
10.1021/acs.jproteome.5b00107
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发表时间：
2015-05-01
影响因子：
4.4
通讯作者：
Bers DM
中科院分区：
文献类型：
--
作者：
Herren AW;Weber DM;Rigor RR;Margulies KB;Phinney BS;Bers DM
心脏电压门控钠通道NaV1.5驱动心脏动作电位的上升，是肌细胞兴奋性的关键决定因素。最近，钙（Ca 2+）/钙调蛋白（CaM）依赖性蛋白激酶II（CaMKII）已成为一个关键的调节器的NaV1.5功能，通过磷酸化的多个残基，包括S516，T594，和S571，这些磷酸化事件可能是重要的获得性心律失常的发生，发生在心力衰竭。然而，全长人NaV1.5的磷酸化尚未被系统地分析，并且人心力衰竭中的NaV1.5磷酸化不完全理解。在本研究中，我们使用无标记质谱法来评估从HEK 293细胞纯化的人NaV1.5的磷酸化，其磷酸化位点完全覆盖，并鉴定了23个在体外被CaMKII磷酸化的位点。我们通过LC-MS/MS证实了S516和S571的磷酸化，并使用新型磷酸化特异性抗体发现人心力衰竭中S516磷酸化减少。这项工作进一步加深了我们对正常和疾病条件下CaMKII对NaV1.5磷酸化的理解，为功能验证提供了新的CaMKII靶位点，并提供了全长人NaV1.5的第一个磷酸化蛋白质组学图谱。
The cardiac voltage-gated sodium channel, NaV1.5, drives the upstroke of the cardiac action potential and is a critical determinant of myocyte excitability. Recently, calcium (Ca2+)/calmodulin(CaM)-dependent protein kinase II (CaMKII) has emerged as a critical regulator of NaV1.5 function through phosphorylation of multiple residues including S516, T594, and S571, and these phosphorylation events may be important for the genesis of acquired arrhythmias, which occur in heart failure. However, phosphorylation of full-length human NaV1.5 has not been systematically analyzed and NaV1.5 phosphorylation in human heart failure is incompletely understood. In the present study, we used label-free mass spectrometry to assess phosphorylation of human NaV1.5 purified from HEK293 cells with full coverage of phosphorylatable sites and identified 23 sites that were phosphorylated by CaMKII in vitro. We confirmed phosphorylation of S516 and S571 by LC–MS/MS and found a decrease in S516 phosphorylation in human heart failure, using a novel phospho-specific antibody. This work furthers our understanding of the phosphorylation of NaV1.5 by CaMKII under normal and disease conditions, provides novel CaMKII target sites for functional validation, and provides the first phospho-proteomic map of full-length human NaV1.5.