Analysis of a Novel Regulator of Excitation-Contraction Coupling in Skeletal Musc

骨骼肌兴奋-收缩耦合的新型调节器分析

• 批准号: 8927276
• 负责人: JOHN Y KUWADA
• 金额: $ 1.8万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8927276
• 关键词: Adaptor Signaling Protein; Adaptor Signaling Protein Gene; Affect; Age; Alleles; Anatomy; Behavior; Binding; Biochemistry; Biological Assay; Biology; Birth; Calcium ion; Cell Fractionation; Cell physiology; Complex; Contractile Proteins; Coupled; Coupling; Defect; Dependence; Dihydropyridine Receptors; Embryo; Face; Gene Mutation; Genes; Health; Human; Hybrids; Image; In Vitro; Individual; Knockout Mice; Knowledge; Lead; Life; Mammalian Cell; Mammals; Membrane; Molecular; Motor; Motor Neurons; Muscle; Muscle Contraction; Muscle Fibers; Muscle function; Mutate; Mutation; Myopathy; Native Americans; Organelles; Pharmaceutical Preparations; Phenotype; Physiologic pulse; Physiology; Process; Property; Protein Binding; Proteins; Regulation; Reticulum; Role; Ryanodine Receptor Calcium Release Channel; Site; Skeletal Muscle; Temperature; Therapeutic; Therapeutic Agents; Transgenic Organisms; Triad Acrylic Resin; Western Blotting; Yeasts; Zebrafish; base; congenital myopathy; in vivo imaging; mutant; neurotransmitter release; novel; novel therapeutics; protein complex; protein transport; skeletal; trafficking; voltage

DESCRIPTION (provided by applicant): Contractions of skeletal muscles are regulated by a process called excitation-contraction (EC) coupling and defects in EC coupling are associated with numerous human muscle diseases. Motor neurons activate skeletal muscles by releasing neurotransmitter that causes the voltage across the muscle membrane to change. EC coupling is the process by which the change in muscle voltage is converted to a release of calcium ions from a specialized intracellular organelle called the sarcoplasmic recticulum (SR) in muscles. The increase in calcium ions in turn initiates contraction by activating the contractile proteins. EC coupling occurs at triadic junctions of the transverse tubules that are infoldings of the muscle membrane and outpocketings of the SR. The two main molecular components responsible for EC coupling are the dihydropyridine receptor (DHPR), a voltage dependent protein in the triadic transverse tubule membrane, and the ryanodine receptor (RYR), a calcium ion release channel located in the triadic SR membrane. These two proteins face each other in the triad and are thought to directly interact during EC coupling. The voltage changes across the muscle membrane are detected by DHPRs that in turn directly activate RYRs to release calcium ions from the SR. EC coupling requires a complex of proteins including DHPR and RYR localized to triads. Although much is known about the role of DHPR and RYR, relatively little is known about the identities and functions of other components of the triadic molecular complex. We identified a zebrafish mutation that is deficient in motor behaviors and found that the causative gene encodes a novel muscle adaptor protein that we found is a key regulator of EC coupling. The adaptor protein localizes to triads, binds to the DHPR-RYR1 complex and is required for proper release of calcium ions by the SR and contraction by skeletal muscles. We further found that the gene encoding this adaptor protein in humans is the basis for a debilitating congenital myopathy in which 36% of individuals afflicted die by age 18. Finally our evidence suggests that mutations of this gene lead to a decrease in DHPR in muscle by improper trafficking of DHPR to triads once they are synthesized. We propose to take advantage of the identification of this novel protein as a key regulator of EC coupling and a new causative gene for congenital myopathy to analyze how this protein regulates EC coupling and how a defect in the protein leads to congenital myopathy. For this we will take advantage of the ability to readily generate transgenic zebrafish and the unique ability to examine cellular processes in living zebrafish embryos. We propose to examine how trafficking of DHPRs are affected by mutations in this gene by generating transgenic zebrafish in which DHPRs are tagged with a fluorescent protein. We further found that the adaptor protein binds a subunit of the DHPR so will identify the sequences in the adaptor protein and DHPR subunit required for binding and examine the consequences of a loss of this binding. We also generated adaptor protein gene knockout mice to extend our analysis to mammalian muscles. This knowledge should help us better understand the biology of myopathies and could potentially lead to therapeutic agents for congenital myopathies.

描述(申请人提供)：骨骼肌的收缩由一个称为兴奋-收缩(EC)偶联的过程调节，EC偶联的缺陷与许多人类肌肉疾病有关。运动神经元通过释放神经递质来激活骨骼肌，神经递质导致肌膜电压发生变化。EC偶联是一个过程，通过这个过程，肌肉电压的变化被转化为从肌肉中被称为肌浆网(SR)的特殊细胞内细胞器释放出钙离子。钙离子的增加反过来又通过激活收缩蛋白来启动收缩。EC偶联发生在横管的三叉节，横管是肌肉的内折叠 SR的膜和外袋。负责EC偶联的两个主要分子成分是二氢吡啶受体(DHPR)和兰尼定受体(RyR)。二氢吡啶受体(DHPR)是位于三叉神经横小管膜上的电压依赖性蛋白，兰尼定受体(RyR)是位于三叉神经横管上的钙离子释放通道。这两种蛋白质在三联体中面对面，被认为在EC偶联过程中直接相互作用。肌膜上的电压变化是由DHPR检测到的，DHPR反过来直接激活RYRs，从SR释放钙离子。EC偶联需要一个蛋白质复合体，包括定位于三联体的DHPR和RyR。虽然人们对DHPR和RyR的作用了解很多，但对三元分子复合体的其他成分的身份和功能知之甚少。我们鉴定了一种在运动行为方面缺乏的斑马鱼突变，并发现该致病基因编码一种新的肌肉适配器蛋白，我们发现该蛋白是EC偶联的关键调节因子。接头蛋白定位于三联体，与DHPR-RYR1复合体结合，是SR适当释放钙离子和骨骼肌收缩所必需的。我们进一步发现，在人类中编码这种接头蛋白的基因是一种衰弱的先天性肌病的基础，在这种先天性肌病中，36%的人在18岁前死亡。最后，我们的证据表明，该基因的突变导致肌肉中DHPR的减少，因为一旦DHPR被合成，DHPR就不适当地运输到三联体。我们建议利用这个新的蛋白作为EC偶联的关键调节因子和先天性肌病的新致病基因来分析它是如何调节EC偶联的，以及该蛋白的缺陷是如何导致先天性肌病的。为此，我们将利用容易产生转基因斑马鱼的能力和检测活斑马鱼胚胎细胞过程的独特能力。我们建议通过产生转基因斑马鱼，在斑马鱼中用荧光蛋白标记DHPR，来研究DHPR的贩运如何受到该基因突变的影响。我们进一步发现，适配器蛋白与DHPR的一个亚基结合，因此将识别结合所需的适配器蛋白和DHPR亚单位中的序列，并研究失去这种结合的后果。我们还培育了适配器蛋白基因敲除小鼠，将我们的分析扩展到哺乳动物的肌肉。这些知识应该有助于我们更好地了解肌病的生物学，并可能导致先天性肌病的治疗剂。