Role of caldesmon in the Ca2+ regulation of smooth muscle thin filaments
Role of caldesmon in the Ca2+ regulation of smooth muscle thin filaments
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DOI：
10.1074/jbc.m706771200
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发表时间：
2008-01-04
影响因子：
4.8
通讯作者：
EL-Mezgueldi, Mohammed
中科院分区：
文献类型：
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作者：
Ansari, Saira;Alahyan, Mustapha;EL-Mezgueldi, Mohammed
平滑肌细丝由肌动蛋白、原肌球蛋白、钙调蛋白和钙结合蛋白组成，它们与肌球蛋白的相互作用受钙调节。我们认为钙调素和钙调素对肌内钙的调节是通过与横纹肌肌钙蛋白-原肌球蛋白同源的协同变构机制来控制肌丝的状态。在目前的工作中，我们已经测试了这个假设。我们直接监测细丝之间的转换ON和OFF状态使用准分子荧光芘碘乙酰胺（PIA）标记的平滑肌α α-原肌球蛋白同源二聚体。在稳态荧光测量中，肌球蛋白亚片段1（S1）协同地将细丝切换到ON状态，并且这表现为受激准分子荧光的增加。相反，钙调素减少受激准分子荧光，表明开关的细丝关闭状态。此外，Ca 2 +-钙调素增加了准分子荧光，表明开关的细丝的ON状态。准分子荧光也被用来监测在停流装置中的ON-OFF转变的动力学。当ATP诱导S1从肌动蛋白-PIA-原肌球蛋白解离时，到OFF状态的转变被延迟，直到所有S1分子都解离为肌动蛋白。与此相反，钙调素开关的细丝在合作的方式关闭状态，并没有滞后显示在钙调素诱导的荧光减少的时间过程。我们还研究了钙调素和钙调素钙调素结合肌动蛋白原肌球蛋白在ON和OFF状态。结果用于讨论肌动蛋白原肌球蛋白的钙调素抑制和Ca ~（2+）-钙调素-钙调素激活。
Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca2+-binding protein and their interaction with myosin is Ca2+-regulated. We suggested that Ca2+ regulation by caldesmon and Ca2+-calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis. We monitored directly the thin filament transition between the ON and OFF state using the excimer fluorescence of pyrene iodoacetamide (PIA)-labeled smooth muscle alpha alpha-tropomyosin homodimers. In steady state fluorescence measurements, myosin subfragment 1 (S1) cooperatively switches the thin filaments to the ON state, and this is exhibited as an increase in the excimer fluorescence. In contrast, caldesmon decreases the excimer fluorescence, indicating a switch of the thin filament to the OFF state. Addition of Ca2+-calmodulin increases the excimer fluorescence, indicating a switch of the thin filament to the ON state. The excimer fluorescence was also used to monitor the kinetics of the ON-OFF transition in a stopped-flow apparatus. When ATP induces S1 dissociation from actin-PIA-tropomyosin, the transition to the OFF state is delayed until all S1 molecules are dissociated actin. In contrast, caldesmon switches the thin filament to the OFF state in a cooperative way, and no lag is displayed in the time course of the caldesmon-induced fluorescence decrease. We have also studied caldesmon and Ca2+-calmodulin-caldesmon binding to actin-tropomyosin in the ON and OFF states. The results are used to discuss both caldesmon inhibition and Ca2+-calmodulin- caldesmon activation of actin-tropomyosin.