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（由申请人提供）：骨骼肌的收缩由称为兴奋-收缩（EC）耦合的过程调节，EC耦合的缺陷与许多人类肌肉疾病相关。运动神经元通过释放神经递质来激活骨骼肌，神经递质会导致肌肉膜上的电压发生变化。 EC 耦合是肌肉电压的变化转化为钙离子从肌肉中称为肌浆网 (SR) 的特殊细胞内细胞器释放的过程。钙离子的增加反过来又通过激活收缩蛋白来启动收缩。 EC耦合发生在肌肉内折的横管的三元连接处 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)（三元横管膜中的电压依赖性蛋白）和兰尼碱受体 (RYR)（位于三元 SR 膜中的钙离子释放通道）。这两种蛋白质在三联体中彼此面对，并且被认为在 EC 偶联过程中直接相互作用。 DHPR 检测到肌肉膜上的电压变化，DHPR 进而直接激活 RYR 以从 SR 中释放钙离子。 EC 偶联需要蛋白质复合物，包括定位于三联体的 DHPR 和 RYR。尽管人们对 DHPR 和 RYR 的作用了解甚多，但对三元分子复合物其他组分的特性和功能知之甚少。我们发现了一种运动行为缺陷的斑马鱼突变，并发现致病基因编码一种新型肌肉适配器蛋白，我们发现该蛋白是 EC 耦合的关键调节因子。接头蛋白定位于三联体，与 DHPR-RYR1 复合物结合，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，编码人类这种接头蛋白的基因是一种使人衰弱的先天性肌病的基础，其中 36% 的患者在 18 岁时死亡。最后，我们的证据表明，该基因的突变导致肌肉中 DHPR 的减少一旦 DHPR 被合成，就会将其不当贩运给三合会。 我们建议利用这种新蛋白作为 EC 偶联的关键调节因子和先天性肌病的新致病基因的鉴定，来分析该蛋白如何调节 EC 偶联以及该蛋白的缺陷如何导致先天性肌病。为此，我们将利用容易产生转基因斑马鱼的能力以及检查活体斑马鱼胚胎中细胞过程的独特能力。我们建议通过生成 DHPR 带有荧光蛋白标记的转基因斑马鱼来研究 DHPR 的运输如何受到该基因突变的影响。我们进一步发现衔接蛋白结合 DHPR 的亚基，因此将识别结合所需的衔接蛋白和 DHPR 亚基中的序列，并检查这种结合丧失的后果。我们还生成了接头蛋白基因敲除小鼠，以将我们的分析扩展到哺乳动物肌肉。这些知识应该有助于我们更好地了解肌病的生物学原理，并有可能开发出治疗先天性肌病的药物。