The ryanodine receptor (RyR) is an essential component of excitation- contraction (E-C) coupling in striated muscle cells, whereby a depolarization across the transverse tubule (TT) membrane leads to rapid release of Ca from the sarcoplasmic reticulum (SR) membrane. RyR consists of a single polypeptide of about 56O kDa normally arranged in a homotetrameric structure, which contains a C-terminal hydrophobic domain that forms the putative conduction pore of the Ca release channel, and a large cytoplasmic domain that spans the junctional gap between the TT and SRI The long term goal of this research is to understand the function and regulation of the RyR channel, and its role in E-C coupling of skeletal and cardiac muscle cells. The primary sequences of skeletal and cardiac RyRs diverge in three regions referred to as D1, D2 and D3. D 1 resides proximal to the C-terminal transmembrane domain, and may be important for the regulation of the Ca release channel. D2 and D3 are located in the N-terminal cytoplasmic region, which is presumably involved in the contact interaction with the dihydropyridine receptor of the TT membrane. The D3 region coincides with a glutamate-rich region in skeletal RyR, not in cardiac RyR, which may be a potential low-affinity binding site for Ca, and thus responsible for the differential Ca-dependent-inactivation of the skeletal and cardiac RyR channels. The roles of the different regions of RyRs in the function of E-C coupling remain largely unknown. Through heterologous expression of RyR proteins in CHO cells, and construction of chimeras between skeletal and cardiac RyR cDNAs, this project will test the hypotheses that, a) the functional unit of the Ca release channel resides in the C-terminal end of RyR (Aim 1), and b) the divergent regions between skeletal and cardiac RyR are involved in the interaction with the dihydropyridine receptor, and thus may play a role in determining the type of E-C coupling in skeletal and cardiac muscle (Aim 2). The functions of the single Ca release channels will be measured in the lipid bilayer system, and the experiments will be focused on the distinct Ca-dependent inactivation of the-skeletal and cardiac Ca release channels, and the specific interaction between the II-III intracellular loop of dihydropyridine receptor and the various constructs of RyRs. Understanding the structure-function of RyR should provide insights into the molecular mechanism of E-C coupling in striated muscles. The results obtained from these studies 'will, in general, add information about the homeostasis of intracellular Ca in other cell types, such as the smooth muscle and neurons, as the different subtypes of RyRs and their close analogue IP3 receptors, are widely distributed in the intracellular membranes which regulate the movement of Ca across the SR and ER.
Showing the most recent 10 out of 33 publications
