Xu Le, Meissner Gerhard
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Biophys J. 2004 Feb;86(2):797-804. doi: 10.1016/S0006-3495(04)74155-7.
The functional effects of calmodulin (CaM) on single cardiac sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors) (RyR2s) were determined in the presence of two endogenous channel effectors, MgATP and reduced glutathione, using the planar lipid bilayer method. Single-channel activities, number of events, and open and close times were determined at varying cytosolic Ca(2+) concentrations. CaM reduced channel open probability at <10 micro M Ca(2+) by decreasing channel events and mean open times and increasing mean close times. At >10 micro M Ca(2+), CaM was less effective in inhibiting RyR2. CaM decreased mean open times but increased channel events, without significantly affecting mean close times. A series of voltage pulses was applied to the bilayer from +50 to -50 mV and from -50 mV to +50 mV to rapidly increase and decrease open channel-mediated sarcoplasmic reticulum lumenal to cytosolic Ca(2+) fluxes. CaM decreased the duration of the open events after the voltage switch from -50 mV to +50 mV. In parallel experiments, a Ca(2+)-insensitive calmodulin mutant was without effect on RyR2 activity. The results are discussed in terms of a possible role of CaM in the termination of cardiac sarcoplasmic reticulum Ca(2+) release.
利用平面脂质双层法，在两种内源性通道效应物（MgATP和还原型谷胱甘肽）存在的情况下，测定了钙调蛋白（CaM）对单个心肌肌浆网Ca(2+)释放通道（兰尼碱受体）（RyR2s）的功能影响。在不同的胞质Ca(2+)浓度下，测定了单通道活性、事件数量以及开放和关闭时间。CaM通过减少通道事件和平均开放时间并增加平均关闭时间，降低了<10微摩尔Ca(2+)时的通道开放概率。在>10微摩尔Ca(2+)时，CaM抑制RyR2的效果较差。CaM降低了平均开放时间，但增加了通道事件，而对平均关闭时间没有显著影响。向双层施加一系列从+50到-50 mV以及从-50 mV到+50 mV的电压脉冲，以快速增加和减少开放通道介导的肌浆网腔到胞质的Ca(2+)通量。CaM缩短了电压从-50 mV切换到+50 mV后开放事件的持续时间。在平行实验中，一种对Ca(2+)不敏感的钙调蛋白突变体对RyR2活性没有影响。根据CaM在心肌肌浆网Ca(2+)释放终止中的可能作用对结果进行了讨论。
