Simms Brett A, Souza Ivana Assis, Zamponi Gerald W
Department of Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Dr, NW, Calgary T2N 4N1, Canada.
Mol Brain. 2014 Apr 28;7:34. doi: 10.1186/1756-6606-7-34.
The L-type calcium channel Cav1.2 is important for brain and heart function. The ubiquitous calcium sensing protein calmodulin (CaM) regulates calcium dependent gating of Cav1.2 channels by reducing calcium influx, a process known as calcium-dependent inactivation (CDI). Dissecting the calcium-dependence of CaM in this process has benefited greatly from the use of mutant CaM molecules which are unable to bind calcium to their low affinity (N-lobe) and high affinity (C-lobe) binding sites. Unlike CDI, it is unknown whether CaM can modulate the activation gating of Cav1.2 channels.
We examined a Cav1.2 point mutant in the N-terminus region of the channel (A39V) that has been previously linked to Brugada syndrome. Using mutant CaM constructs in which the N- and/or C-lobe calcium binding sites were ablated, we were able to show that this Brugada syndrome mutation disrupts N-lobe CDI of the channel. In the course of these experiments, we discovered that all mutant CaM molecules were able to alter the kinetics of channel activation even in the absence of calcium for WT-Cav1.2, but not A39V-Cav1.2 channels. Moreover, CaM mutants differentially shifted the voltage-dependence of activation for WT and A39V-Cav1.2 channels to hyperpolarized potentials. Our data therefore suggest that structural changes in CaM that arise directly from site directed mutagenesis of calcium binding domains alter activation gating of Cav1.2 channels independently of their effects on calcium binding, and that the N-terminus of the channel contributes to this CaM dependent process.
Our data indicate that caution must be exercised when interpreting the effects of CaM mutants on ion channel gating.
L型钙通道Cav1.2对脑和心脏功能至关重要。普遍存在的钙传感蛋白钙调蛋白（CaM）通过减少钙内流来调节Cav1.2通道的钙依赖性门控，这一过程称为钙依赖性失活（CDI）。在这一过程中，通过使用无法将钙结合到其低亲和力（N叶）和高亲和力（C叶）结合位点的突变型CaM分子，极大地有助于剖析CaM的钙依赖性。与CDI不同，CaM是否能调节Cav1.2通道的激活门控尚不清楚。
我们研究了通道N端区域的一个Cav1.2点突变体（A39V），该突变体先前与Brugada综合征有关。使用N叶和/或C叶钙结合位点被切除的突变型CaM构建体，我们能够证明这种Brugada综合征突变破坏了通道的N叶CDI。在这些实验过程中，我们发现所有突变型CaM分子即使在没有钙的情况下也能够改变野生型Cav1.2通道的激活动力学，但对A39V-Cav1.2通道则不然。此外，CaM突变体使野生型和A39V-Cav1.2通道的激活电压依赖性向超极化电位发生不同程度的偏移。因此，我们的数据表明，钙结合域定点诱变直接导致的CaM结构变化独立于其对钙结合的影响而改变Cav1.2通道的激活门控，并且通道的N端有助于这一CaM依赖性过程。
我们的数据表明，在解释CaM突变体对离子通道门控的影响时必须谨慎。
