Designer DHPRs, EC coupling and an expanded genetic code in skeletal muscle

描述(申请人提供)：兴奋收缩偶联(EC偶联)是生物功能所必需的真正的电化学信号转导的一个例子，它是通过使二氢吡啶受体(DHPR，一种钙通道)的构象发生短暂的、电压依赖的变化来执行的。然后呢？肌细胞表面的亚基与肌浆网上的兰尼定受体(RyR)相互作用。事实上，电压触发了DHPR(细胞表面)和RyR(细胞内)通道之间的关键蛋白质-蛋白质相互作用，这一事实代表了他们在细胞外研究的一个重大技术障碍。捕捉和表征DHPR和RyR之间的静态和电压驱动的瞬时蛋白质-蛋白质相互作用将需要扩展肌肉中的遗传密码，并随之扩展用于肌肉生物学研究的实验库。我们的长期目标是应用新兴化学生物学领域的进展来检验长期存在的假设，即DHPR和RyR之间的电压驱动蛋白质-蛋白质(机械)偶联促进内部存储器中钙的快速释放。这项拟议研究的目的是在骨骼肌环境中应用一种创新的化学生物学方法-将具有独特光激活交联活性的基因编码的合成氨基酸二苯甲酮-苯丙氨酸(BPA)加入到设计的DHPR中，使用它来共价捕获原本难以捉摸的瞬时蛋白质复合体。几项关键的创新使我们的研究变得可行：使用逆转录病毒和腺病毒来表达正交的tRNA和BPA合成酶基因；大规模的准备工作，涉及以前用来产生Fluo-AM染料的酯化策略，以产生一种高度溶解、无毒和具有生物合成能力的BPA，可以将基因并入肌肉；虽然我们不是目前目标的主题，但我们展示了原理证明的BPA掺入相关的电压门控钠通道，以实现快速(毫秒级)光交联。这里提出的实验将建立在这些进展的基础上-使用各种肌肉细胞模型，包括肌管培养-适应这些方法来分析DHPR-RyR复合体。在目标1中，我们将以现有的a-和b-界面的晶体结构为指导，作为DHPR中遗传编码的光化学的模型系统，在目标2中，我们将识别DHPR中支持电压驱动与RyR相互作用的氨基酸侧链。这项研究的贡献将是研究肌肉中瞬时蛋白质相互作用的新工具(技术、试剂、概念)，以及对DHPR结构的理解，这对于确定到目前为止难以捉摸的RyR与DHPR之间的分子相互作用至关重要。这些贡献将是重大的，因为我们的概念创新和技术突破将为一个以前难以解决的机制提供新的见解，同时在该领域“举起所有的船” 肌肉生物学的研究。

DESCRIPTION (provided by applicant): Excitation contraction coupling (EC-coupling) is an example of bona fide electro-chemical signal transduction essential to biological function, and is executed through short-lived, voltage-dependent changes in conformation that enable the dihydropyridine receptor (DHPR, a calcium channel) ? and ? subunits on the muscle-cell surface to interact with ryanodine receptor (RyR) on the sarcoplasmic reticulum. The fact that voltage triggers the key protein-protein interactions between DHPR (on the cell surface) and RyR (intracellular) channels represents a significant technical barrier to their study outside the cell. The capture and characterization of static and voltage-driven transient protein-protein interactions between DHPR and RyR will require expansion of the genetic code in muscle and, concomitantly, expansion of the experimental arsenal for the study of muscle biology. Our long-term goal is to apply advances in the burgeoning field of chemical biology to testing the long-standing hypothesis that voltage-driven protein-protein (mechanical) coupling between DHPR and RyR facilitates rapid release of Ca2+ from the internal stores. The objective of the proposed research is to apply an innovative chemical biology approach - the incorporation of a genetically encoded synthetic amino acid benzophenone-Phe (Bpa) with a unique photoactivated crosslinking activity into designer DHPR's - in the environment of skeletal muscle, using it to covalently trap otherwise elusive, transient protein complexes. Several key innovations make our study feasible: the use of retro- and adenovirus to express the orthogonal tRNA and Bpa synthetase genes; large-scale preparations involving esterfication strategies used previously to generate Fluo-AM dyes to produce a form of Bpa that is highly soluble, non-toxic and biosynthetically competent for genetic incorporation into muscle; and, while not the subject of our present objective, we show proof-of-principle Bpa incorporation into related voltage-gated sodium channels to enable rapid (millisecond-scale) photo-crosslinking. The experiments proposed here will build on these advances - using a variety of muscle-cell models, including myotube cultures - adapting these approaches to the analysis of DHPR-RyR complexes. In Aim 1 we will be guided by an existing crystal structure of the a- and b-interface as a model system for genetically encoded photochemistry in DHPR's, and in Aim 2 we will identify the amino- acid side chains in the DHPR that support voltage-driven interactions with Ryr. The contributions of this study will be new tools (techniques, reagents, concepts) for the study of transient protein interactions in muscle, and an understanding of DHPR structure that will be essential to identifying the so-far elusive molecular interactions between it and the RyR. These contributions will be significant in that our conceptual innovations and technological breakthroughs will provide fresh insight on a previously intractable mechanism, while 'raising all boats' in the field of muscle biology.