Regulation of the transient receptor potential channel TRPM2 by the Ca2+ sensor calmodulin
Regulation of the transient receptor potential channel TRPM2 by the Ca2+ sensor calmodulin
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DOI：
10.1074/jbc.m510422200
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发表时间：
2006-04-07
影响因子：
4.8
通讯作者：
Miller, BA
中科院分区：
文献类型：
--
作者：
Tong, Q;Zhang, WY;Miller, BA
TRPM 2是瞬时受体电位（TRP）超家族的成员，是由氧化应激或肿瘤坏死因子α激活的Ca 2+渗透通道，参与细胞死亡的易感性。TRPM 2的激活取决于细胞内Ca 2+的水平。我们探讨了钙调素（CaM）是否是TRPM 2的Ca 2+传感器。用TRPM 2和野生型CaM或突变型CaM（CaMMUT）转染HEK 293 T细胞，其中所有四个EF手被取代。用H2 O2或肿瘤坏死因子α处理表达TRPM 2的细胞导致细胞内钙（[Ca 2 +]（i））显著增加。这是不受共表达的钙调素，表明内源性钙调素水平是足够的最大响应。CaMMUT与TRPM 2的共转染显著抑制了[Ca 2 +]（i）的增加，证明了TRPM 2活化中对CaM的需要。免疫沉淀证实了钙调素和CaMMUT与TRPM 2的直接相互作用，以及这种关联的钙依赖性。CaM与TRPM 2 N端（氨基酸1-730）强结合，但与C端（氨基酸1060-1503）弱结合。利用凝胶移位、免疫沉淀、生物素化CaM覆盖和下拉测定证明了CaM与TRPM 2 N末端的IQ样基序（氨基酸406-416）的结合。TRPM 2的IQ样基序的取代突变体（TRPM 2-IQ（MUT 1））减少但不消除CaM与TRPM 2的结合，表明存在至少一个其它CaM结合位点。TRPM 2 IQ样基序的功能重要性通过用H2 O2处理TRPM 2-IQ（MUT 1）表达细胞来证明。用野生型TRPM 2观察到的[Ca 2 +]（i）的增加不存在，并且细胞活力得以保留。这些数据证明了TRPM 2激活中对CaM的需求。他们认为，Ca 2+通过TRPM 2进入增强了CaM与TRPM 2在N端IQ样基序处的相互作用，为通道激活提供了至关重要的正反馈。
TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca2+-permeable channel activated by oxidative stress or tumor necrosis factor alpha involved in susceptibility to cell death. TRPM2 activation is dependent on the level of intracellular Ca2+. We explored whether calmodulin (CaM) is the Ca2+ sensor for TRPM2. HEK 293T cells were transfected with TRPM2 and wild type CaM or mutant CaM (CaMMUT) with substitutions of all four EF hands. Treatment of cells expressing TRPM2 with H2O2 or tumor necrosis factor alpha resulted in a significant increase in intracellular calcium ([ Ca2+](i)). This was not affected by coexpression of CaM, suggesting that endogenous CaM levels are sufficient for maximal response. Cotransfection of CaMMUT with TRPM2 dramatically inhibited the increase in [Ca2+](i), demonstrating the requirement for CaM in TRPM2 activation. Immunoprecipitation confirmed direct interaction of CaM and CaMMUT with TRPM2, and the Ca2+ dependence of this association. CaM bound strongly to the TRPM2 N terminus (amino acids 1-730), but weakly to the C terminus (amino acids 1060-1503). CaM binding to an IQ-like motif (amino acids 406-416) in the TRPM2 N terminus was demonstrated utilizing gel shift, immunoprecipitation, biotinylated CaM overlay, and pull-down assays. A substitution mutant of the IQ-like motif of TRPM2 (TRPM2-IQ(MUT1)) reduced but did not eliminate CaM binding to TRPM2, suggesting the presence of at least one other CaM binding site. The functional importance of the TRPM2 IQ-like motif was demonstrated by treatment of TRPM2-IQ(MUT1)-expressing cells with H2O2. The increase in [Ca2+](i) observed with wild type TRPM2 was absent and cell viability was preserved. These data demonstrate the requirement for CaM in TRPM2 activation. They suggest that Ca2+ entering through TRPM2 enhances interaction of CaM with TRPM2 at the IQ-like motif in the N terminus, providing crucial positive feedback for channel activation.