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

描述（申请人提供）：骨骼肌纤维的激活是所有身体运动以及呼吸的先决条件，由横小管（TT）的电去极化启动，导致TT二氢吡啶受体（DHPR）中的膜电压（V）传感器通过邻接的骨骼肌兰尼碱受体（RyR 1）/Ca 2+触发Ca 2+释放在相邻的肌浆网膜上有释放通道。然而，将TT V传感器耦合到SR RyR 1释放激活的分子机制知之甚少，并且包括S100 A1和钙调蛋白（CaM）在内的各种调节分子的作用尚不清楚。这些问题是重要的，因为任何病理干扰的Ca 2+释放激活过程可能会修改或破坏肌肉功能。在目的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鉴定了以前完全未被怀疑的肌肉Ca 2+释放的显著抑制，并表征了肌肉激活中这种缺陷的机制。肌肉Ca 2+释放也受到多种辅助蛋白的调节。在当前的资助周期中，我们已经取得了新的发现，即Ca 2+结合蛋白S100 A1与先前鉴定的RyR 1中的钙调蛋白（CaM）结合结构域（CaMBD）结合，该结构域现在应该被称为CaM/S100 A1结合结构域，因为这些分子在该位点相互作用以结合。在目标2和3中，我们利用shRNA技术抑制S100 A1、CaM或S100 A1和CaM两者的蛋白质表达，以研究这些配体中的每一个的作用以及它们在RyR 1的CaMBD以外的位点的竞争性相互作用。我们将使用含有Ca 2+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Ca 2+信号，并计算在完整纤维中的单个或系列动作电位期间，或在膜片钳溶液中具有高水平EGTA的全细胞电压钳位纤维的电压钳位去极化期间，来自SR的潜在Ca 2+释放通量。我们将使用具有内源性或外源性蛋白质的分子生物学操纵表达的成人肌纤维。并行NMR和结合研究将检查S100 A1和/或CaM结合到对应于所鉴定的结合位点的肽的结构和结合亲和力。本项目将阐明调节骨骼肌中Ca 2+释放的基本分子机制以及hypoPP中突变的电压传感器电荷在肌肉Ca 2+释放中的作用。它将表征S100 A1和CaM对SR Ca 2+释放的调节，这可能在全身性或特定肌肉疾病状态或衰老肌肉中受到损害。因此，该项目对多个学科以及各种晚期疾病状态和衰老常见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.