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

描述（由申请人提供）：骨骼肌的收缩受称为兴奋-收缩（EC）偶联的过程调节，EC偶联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，神经递质导致肌肉膜上的电压发生变化。EC偶联是将肌肉电压的变化转化为钙离子从肌肉中称为肌浆网（SR）的专门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质来启动收缩。EC偶联发生在横小管的三联连接处，横小管是肌肉的内折 负责EC偶联的两个主要分子组分是二氢吡啶受体（DHPR）（三联横小管膜中的电压依赖性蛋白质）和兰尼碱受体（RYR）（位于三联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偶联期间直接相互作用。跨肌肉膜的电压变化由DHPR检测，DHPR进而直接激活RYR以从SR释放钙离子。EC偶联需要包括定位于三联体的DHPR和RYR的蛋白质复合物。虽然对DHPR和RYR的作用了解很多，但对三元分子复合物的其他组分的身份和功能知之甚少。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的肌肉适配器蛋白，我们发现这种蛋白是EC偶联的关键调节因子。衔接蛋白定位于三联体，结合DHPR-RYR 1复合物，并且是SR适当释放钙离子和骨骼肌收缩所需的。我们进一步发现，人类编码这种衔接蛋白的基因是导致衰弱的先天性肌病的基础，其中36%的患病个体在18岁之前死亡。最后，我们的证据表明，该基因的突变导致肌肉中DHPR的减少，一旦合成，DHPR就被不适当地运输到三个单元中。 我们建议利用这种新的蛋白质作为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这种蛋白质如何调节EC偶联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和在活斑马鱼胚胎中检查细胞过程的独特能力。我们建议研究如何贩运的DHPRs的影响，在这个基因的突变，产生转基因斑马鱼，其中DHPRs标记的荧光蛋白。我们进一步发现衔接蛋白结合DHPR的亚基，因此将鉴定衔接蛋白和DHPR亚基中结合所需的序列，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，并可能导致先天性肌病的治疗药物。

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.