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）：骨骼肌的收缩受到一种称为激发 - 收缩（EC）耦合的过程，EC耦合中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，从而导致整个肌肉膜的电压发生变化。 EC耦合是将肌肉电压变化转化为从肌肉中称为肌浆肌瘤（SR）的专业细胞内细胞器中释放的钙离子的过程。钙离子的增加反过来通过激活收缩蛋白来启动收缩。 EC耦合发生在横向小管的三合会连接处，是肌肉的插头 SR的膜和溢出。负责EC偶联的两个主要分子成分是二氢吡啶受体（DHPR），三联体横向小管膜中的电压依赖性蛋白，以及位于TriADIC SR膜中的钙离子释放通道的Ryanodine受体（RYR）。这两种蛋白质在三合会中相互面对，并被认为在EC耦合过程中直接相互作用。 DHPR检测到整个肌肉膜之间的电压变化，而DHPR又直接激活Ryrs从SR释放钙离子。 EC耦合需要蛋白质的复合物，包括DHPR和RYR定位于三合会。尽管对DHPR和RYR的作用知之甚少，但对三元分子复合物其他成分的身份和功能的了解相对较少。我们确定了一种缺乏运动行为的斑马鱼突变，发现病因基因编码了一种新型的肌肉适配器蛋白，我们发现它是EC耦合的关键调节剂。衔接蛋白定位于三合会，结合DHPR-RYR1复合物，是通过SR正确释放钙离子并通过骨骼肌收缩所必需的。我们进一步发现，编码人类中这种衔接子蛋白的基因是使先天性肌病的基础，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DHPR的DHPR降低，而不是将DHPR的不当运输dhpr降低到Triad时，一旦它们是合成的。 我们建议利用这种新型蛋白质作为EC偶联的关键调节剂和先天性肌病的新因果基因的关键调节剂，以分析该蛋白如何调节EC耦合以及蛋白质中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斑马鱼胚胎中细胞过程的独特能力。我们建议通过产生转基因斑马鱼在该基因中的突变影响DHPR的运输如何受到DHPR的荧光蛋白标记。我们进一步发现，衔接蛋白结合了DHPR的亚基，因此可以识别衔接蛋白和DHPR亚基中所需的结合所需的序列，并检查这种结合丧失的后果。我们还产生了衔接蛋白基因基因敲除小鼠，以将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，并有可能导致先天性肌病的治疗剂。