The long term goal of this research is to understand the mechanism of excitation contraction (E-C) coupling in skeletal muscle. Coupling of electrical excitation to Ca release occurs in the junction between the transverse tubule (TT) and the sarcoplasmic reticulum (SR) membranes. Dihydropyridine receptors (DHPRs) in the TT membrane and ryanodine receptors (RyRs) in the SR membrane are the two main proteins involved in this process. The DHPRs have two functions in skeletal muscle, as the fast E-C coupling voltage sensor and the slow DHP-sensitive Ca channel. It is not known whether the two functions are performed by the same DHPR molecule. To understand the properties of the DHPR, the project has three specific aims: First, the biophysical properties of the DHPR as a functional Ca channel will be characterized in the planar bilayer system, including a newly discovered fast kinetic mode of activation. Second, the regulation and modulation of the DHP-sensitive Ca channels by Ca, Ca antagonists, and protein kinase phosphorylation will be studied. Third, a molecular model, based on the above studies, will be developed to test quantitatively whether it is possible for one DHPR to have both functions. The model will make specific predictions of the kinetics and voltage-dependence of macroscopic Ca current and intramembrane charge movement (voltage sensor), which can be readily tested in the whole cell measurements. The RyRs play two roles in E-C coupling, forming the pore of the Ca release channel and providing the bridging foot that presumably links the release channel with the voltage sensor. By studying the functions of the RyR as a Ca release channel, the proposed research will attempt to address the following three questions: First, what are the differences between a purified RyR and a native release channel? Second, how does one draw conclusions from the reconstitution studies back to the whole cell measurements? Third, how do the multimeric complexes of RyR interact in forming a functional Ca release channel? The functional unit of E-C coupling in skeletal muscle. resides in the T-SR junction. Given the anatomical arrangement and structural features of RyR and DHPR, the signal transduction from DHPR to RyR could occur via an allosteric mechanism. Such reaction requires a physical contact, direct or indirect, between the DHPR and the RyR. This putative interaction will be sought in the reconstitution system, by first incorporating one protein then add the other protein while monitoring channel gating in the bilayer. If the experiments were successful, functional regulation of the release channel by DHPR will be studied, including the voltage dependence of activation and modulation by pharmacological agents. These studies should lead to further insights into the mechanism of E-C coupling in skeletal muscle. Ca signalling and its control are important issues in many other cell types, including the heart and the nervous system, where similar Ca release channel proteins (RyRs or IP3 receptors) have been found in the intracellular organelles (SR or ER). Understanding the mechanism of E-C coupling is of general interest to many physiological functions.
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