Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex
Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex
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DOI：
10.1016/j.cardiores.2006.10.016
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发表时间：
2007-01-01
影响因子：
10.8
通讯作者：
Groschner, K.
中科院分区：
文献类型：
--
作者：
Eder, P.;Probst, D.;Groschner, K.
目的：经典的瞬时受体电位蛋白（TRPC）家族成员被认为是磷脂酶C（PLC）依赖的Ca 2+信号转导的关键组成部分。先前在HEK 293表达系统中获得的结果表明TRPC 3与心脏型Na+/Ca 2+交换器NCX 1（钠钙交换器1）的物理和功能偶联。本研究旨在检测瞬时受体电位通道3（TRPC 3）在大鼠心肌细胞中的表达和天然TRPC 3/NCX 1信号复合物的存在。通过免疫共沉淀和谷胱甘肽S-转移酶（GST）下拉实验研究蛋白质-蛋白质相互作用。通过表面生物素化测定蛋白质复合物向质膜中的募集。TRPC 3的功能作用进行了研究，采用显性负敲除strategy.Results荧光记录血管紧张素II诱导的钙信号：TRPC 3的免疫反应性观察在表面质膜区域和细胞内膜系统。TRPC 3和NCX 1的共免疫标记表明这两种蛋白质的显著共定位。免疫共沉淀和GST-下拉实验都证明了TRPC 3与NCX 1的关联。PLC刺激被发现触发NCX介导的Ca 2+内流，这依赖于TRPC 3介导的肌细胞Na+负荷。这种NCX介导的Ca 2+信号传导被TRPC 3的显性负性片段的表达显著抑制。结论：PLC依赖性的TPPC 3-NCX 1复合物向细胞膜的募集是调节心肌Ca 2+稳态的重要机制。(c)2006年欧洲心脏病学会。Elsevier B. V.出版，保留所有权利。
Objective: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na+/Ca2+ exchanger, NCX1 (sodium calcium exchanger 1). This study was designed to test for expression of TRPC3 (transient receptor potential channel 3) and for the existence of a native TRPC3/NCX1 signaling complex in rat cardiac myocytes.Methods: Protein expression and cellular distribution were determined by Western blot and immunocytochemistry. Protein-protein interactions were investigated by reciprocal co-immunoprecipitation and glutathione S-transferase (GST)-pulldown experiments. Recruitment of protein complexes into the plasma membrane was assayed by surface biotinylation. The functional role of TRPC3 was investigated by fluorimetric recording of angiotensin II-induced calcium signals employing a dominant negative knockdown strategy.Results: TRPC3 immunoreactivity was observed in surface plasma membrane regions and in an intracellular membrane system. Co-immunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GST-pulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca2+ entry, which was dependent on TRPC3-mediated Na+ loading of myocytes. This NCX-mediated Ca2+ signaling was significantly suppressed by expression of a dominant negative fragment of TRPC3. PLC stimulation was associated with increased membrane presentation of both TRPC3 and NCX1.Conclusion: These results suggest a PLC-dependent recruitment of a TPPC3-NCX1 complex into the plasma membrane as a pivotal mechanism for the control of cardiac Ca2+ homeostasis. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.