Roman Horia Nicolae, Zitouni Nedjma B, Kachmar Linda, Ijpma Gijs, Hilbert Lennart, Matusovsky Oleg, Benedetti Andrea, Sobieszek Apolinary, Lauzon Anne-Marie
Meakins-Christie Laboratories, McGill University, Montréal, Québec, Canada.
Biochim Biophys Acta. 2013 Oct;1830(10):4634-41. doi: 10.1016/j.bbagen.2013.05.042. Epub 2013 Jun 6.
Smooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin.
To measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin.
Funb from F-actin alone (0.12±0.01pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02pN). This enhancement was lost when calponin was phosphorylated (0.12±0.01pN). To further verify that this enhancement of Funb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25mM (0.21±0.02pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01pN).
This study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state.
This study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.
平滑肌具有独特的能力，能够长时间以低能量消耗维持张力。虽然人们普遍认为这种称为闩锁状态的特性是由于肌球蛋白在附着于肌动蛋白时发生去磷酸化所致，但去磷酸化且脱离的肌球蛋白也可附着于肌动蛋白，并可能有助于维持张力。因此，我们研究了钙调蛋白在调节和增强未磷酸化的紧张性肌肉肌球蛋白与肌动蛋白结合力方面的作用。
为了测量钙调蛋白对未磷酸化肌球蛋白与肌动蛋白结合的影响，我们使用激光光镊测定法来量化在不存在和存在钙调蛋白或磷酸化钙调蛋白的情况下的平均解离力（Funb）。
单独F-肌动蛋白的Funb（0.12±0.01pN；平均值±标准误）在存在钙调蛋白时（0.20±0.02pN）显著增加。当钙调蛋白被磷酸化时（0.12±0.01pN），这种增强作用消失。为了进一步验证这种Funb的增强是由于钙调蛋白使肌动蛋白与肌球蛋白交联所致，我们在高离子强度下重复了测量。实际上，在25mM [KCl]时获得的Funb（0.21±0.02pN；平均值±标准误）在150mM [KCl]时（0.13±0.01pN）显著降低。
本研究提供了直接的分子水平证据，即钙调蛋白通过交联未磷酸化的肌球蛋白与肌动蛋白来增强它们之间的结合力，并且这种作用在钙调蛋白磷酸化后会逆转。因此，钙调蛋白可能在闩锁状态中起重要作用。
本研究提出了一种可能导致闩锁状态的新机制，闩锁状态是平滑肌的一种基本且重要但仍未解决的特性。
