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.
The results of our study will remove a significant technical barrier that has barred a rapid, atomic- level, functional understanding of excitation-contraction coupling and the use of non-natural amino acids to study the study of muscle biology. Success of the proposed study will enable the generation of powerful research tools that will be widely available to the research community.