Roles of voltage sensor, S100A1 and calmodulin in skeletal muscle Ca2+ signaling

描述（由申请人提供）：骨骼肌纤维的激活是所有身体运动和呼吸的先决条件，是由横小管（TTs）的电去极化启动的，导致TT二氢吡啶受体（DHPR）中的膜电压(V)传感器通过邻近的骨骼肌红嘌呤受体(RyR1)/邻近肌浆网膜中的Ca2+释放通道触发Ca2+释放。然而，TT V传感器耦合SR RyR1释放激活的分子机制尚不清楚，各种调节分子（包括S100A1和calmodulin (CaM)）的作用尚不清楚。这些问题很重要，因为任何对Ca2+释放激活过程的病理干扰都可能改变或破坏肌肉功能。在Aim 1中，我们在表达低钾性周期性麻痹（hypoPP） CaV1.1 V传感器电荷突变的转基因小鼠模型中发现了先前完全未被怀疑的肌肉Ca2+释放的显著抑制，并表征了这种肌肉激活缺陷的机制。肌肉Ca2+释放也受多种辅助蛋白的调节。在当前的授权周期中，我们发现Ca2+结合蛋白S100A1与RyR1中先前鉴定的钙调蛋白（CaM）结合域（CaMBD）结合，现在应称为CaM/S100A1结合域，因为这些分子在该位点相互作用以结合。在目的2和3中，我们利用shRNA技术抑制S100A1、CaM或S100A1和CaM的蛋白表达，以研究这些配体的作用以及它们在RyR1的CaMBD以外的位点上的竞争性相互作用。我们将使用含有Ca2+指示剂fluo-4的纤维的高速（<50 us/线）线扫描共聚焦成像来监测Ca2+信号，并计算完整纤维中单个或串联动作电位期间SR的潜在Ca2+释放通量，或在膜片移液溶液中具有高水平EGTA的全细胞电压钳夹纤维的电压钳去极化期间。我们将使用内源性或外源性蛋白质的分子生物学操作表达成人肌肉纤维。平行核磁共振和结合研究将检查S100A1和/或CaM与所鉴定的结合位点对应的肽结合的结构和结合亲和力。该项目将阐明调节骨骼肌Ca2+释放的基本分子机制，以及在低电位电位中突变的电压传感器电荷在肌肉Ca2+释放中的作用。它将表征S100A1和CaM对SR Ca2+释放的调节，这可能在广泛性或特异性肌肉疾病状态或衰老肌肉中受损。因此，该项目对多个学科，以及各种晚期疾病和衰老共同存在的运动和呼吸问题具有很高的影响。

DESCRIPTION (provided by applicant): Activation of skeletal muscle fibers, which is a prerequisite for all bodily movement as well as for respiration, is initiated by electrical depolarization of the transverse tubules (TTs), causing membrane voltage (V) sensors in the TT dihydropyridine receptor (DHPR) to trigger Ca2+ release via the abutting skeletal muscle ryanodine receptor (RyR1)/Ca2+ release channels in the adjacent sarcoplasmic reticulum membrane. However, the molecular mechanisms coupling the TT V sensor to SR RyR1 release activation are poorly understood, and the roles of various modulatory molecules, including S100A1 and calmodulin (CaM) are not clear. These issues are important since any pathologic interference with the Ca2+ release activation process may modify or disrupt muscle function. Here in Aim 1 we identify a previously totally unsuspected marked suppression of muscle Ca2+ release in a transgenic mouse model expressing a hypokalemic periodic paralysis (hypoPP) CaV1.1 V sensor charge mutation, and characterize the mechanism(s) underlying this defect in muscle activation. Muscle Ca2+ release is also modulated by a variety of accessory proteins. During the current grant cycle we have made the novel finding that the Ca2+ binding protein S100A1 binds to the previously identified calmodulin (CaM) binding domain (CaMBD) in RyR1, which should now be referred to as a CaM/S100A1 binding domain since these molecules interact for binding at this site. In Aims 2 and 3 we utilize shRNA techniques to suppress the protein expression of S100A1, CaM or both S100A1 and CaM to investigate the effects of each of these ligands as well as their competitive interaction at sites other than the CaMBD of RyR1. We will use high speed (<50 us/line) line-scan confocal imaging of fibers containing the Ca2+ indicator fluo-4 to monitor Ca2+ signals and calculate the underlying Ca2+ release flux from the SR during single or trains of action potentials in intact fibers, or during voltage clamp depolarization of whole cell voltage clamped fibers with high levels of EGTA in the patch pipette solution. We will use adult muscle fibers with molecular biologically manipulated expression of endogenous or exogenous proteins. Parallel NMR and binding studies will examine the structures and binding affinities of S100A1 and/or CaM binding to peptides corresponding to the identified binding sites. This project will elucidate basic molecular mechanisms regulating Ca2+ release in skeletal muscle and the roles of voltage sensor charges that are mutated in hypoPP in muscle Ca2+ release. It will characterize the modulation of SR Ca2+ release by S100A1 and CaM, which might be compromised in generalized or specific muscle disease states, or in aging muscle. Thus, this project has high impact for multiple disciplines, and for problems of both locomotion and breathing common to a variety of advanced diseased states and aging.