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

电压传感器、S100A1 和钙调蛋白在骨骼肌 Ca2 信号传导中的作用

• 批准号: 9439143
• 负责人: MARTIN F SCHNEIDER
• 金额: $ 4.18万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9439143
• 关键词: Action Potentials; Adult; Affinity; Aging; Binding; Binding Proteins; Binding Sites; Biological; Breathing; C-terminal; Calcium; Calcium ion; Calmodulin; Cells; Charge; Collaborations; Competitive Binding; Coupled; Coupling; Culture Techniques; Defect; Deterioration; Dihydropyridine Receptors; Discipline; Disease; Egtazic Acid; Electrophysiology (science); Exhibits; Fiber; Fluo 4; Genetically Engineered Mouse; Goals; Grant; Health; Human; Hypokalemic periodic paralysis; Image; Impairment; Investigation; L-Type Calcium Channels; Ligands; Link; Location; Locomotion; Measurement; Mediating; Membrane; Molecular; Molecular Conformation; Monitor; Movement; Mus; Muscle; Muscle Fibers; Muscle function; Muscular Atrophy; Mutate; Mutation; Myopathy; Optics; Pathologic; Peptides; Pharmacology; Preparation; Process; Proteins; Reagent; Respiration; Role; Ryanodine Receptor Calcium Release Channel; S100A1 protein; Sarcoplasmic Reticulum; Scanning; Signal Transduction; Site; Skeletal Muscle; Soleus Muscle; Speed; Structure; Tail; Techniques; Testing; Training; Transgenic Animals; Transgenic Mice; advanced disease; combat; electric field; innovation; mouse model; muscle aging; novel; peptide structure; periodic paralysis; protein expression; protein structure; sensor; small hairpin RNA; small molecule; voltage; voltage clamp

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.

描述（由申请人提供）：骨骼肌纤维的激活是由所有身体运动和呼吸的先决条件，它是通过横向小管（TTS）的电去极化引发的，从而导致TT二甲状腺素受体（dhydydropyridine受体（dhydydropyride）to to to to to try try try try tryy tryy to tryy promighter tt tt dihydropylidine to ryy to try ryy ryy to tt tt dihydropylidine to ryy to tts tts。相邻肌质网膜中的受体（RYR1）/Ca2+释放通道。然而，对TT V传感器与SR RYR1释放激活的分子机制尚不清楚，并且包括S100A1和钙调蛋白（CAM）在内的各种调节分子的作用尚不清楚。这些问题很重要，因为对CA2+释放激活过程的任何病理干扰都可能改变或破坏肌肉功能。在AIM 1中，我们在表达低钾血症周期性瘫痪（HYPOPP）CAV1.1 V传感器电荷突变的转基因小鼠模型中确定了先前完全不引起的明显抑制肌肉Ca2+释放，并表征了肌肉激活中这种缺陷的机制。肌肉Ca2+释放也受各种辅助蛋白的调节。在当前的赠款周期中，我们提出了新发现的发现，即Ca2+结合蛋白S100A1与先前鉴定的钙调蛋白（CAM）结合结构域（CAMBD）结合RyR1中，现在应称为CAM/S100A1结合结构域，因为这些分子在该位点相互作用。在目标2和3中，我们利用SHRNA技术来抑制S100A1，CAM或S100A1和CAM的蛋白质表达，以研究每种配体的效果以及它们在RYR1 CAMBD以外的其他位点的竞争相互作用。我们将使用包含CA2+指示器Fluo-4的纤维的高速（<50 US/线路）线扫描共聚焦成像来监视Ca2+信号，并计算在完整纤维中单个或单个动作电位的基础CA2+释放通量，或者在整个细胞电压隔板的电压夹层中，在整个细胞夹层中均与高级别的高级隔板的较高层的型号的旋转夹层。我们将使用具有分子生物学操纵表达内源或外源蛋白的成年肌肉纤维。平行的NMR和结合研究将检查S100A1和/或CAM结合与对应于鉴定结合位点的肽的结构和结合亲和力。该项目将阐明调节骨骼肌中Ca2+释放的基本分子机制以及在肌肉Ca2+释放中HYPOPP突变的电压传感器电荷的作用。它将表征S100A1和CAM对SR Ca2+释放的调节，这可能在广义或特定的肌肉疾病状态或衰老的肌肉中受到损害。因此，该项目对多个学科以及对各种晚期患病状态和衰老常见的运动和呼吸的问题具有很大的影响。