The overall goal of this project is to resolve the molecular mechanism of excitation-contraction-relaxation coupling in normal and diseased skeletal muscles. The whole reaction cycle of E-C-R coupling could be described by three major events occurring in the following sequence: (I) the voltage-dependent interaction between the dihydropyridine receptor (DHPR) and the junctional foot protein (JFP); (II) conformational changes in the JFP to open the SR Ca2+ channel; and (III) release inactivation/recovery processes involving several putative reaction intermediates and modulators. Each of these major events will be investigated utilizing the in vitro E-C coupling assay system (triad) or purified proteins for studying the problems at the molecular level.
In Aim 1 the detailed modes of protein-protein interactions in the signal transmission from the DHPR to the JFP will be resolved. A series of peptides derived from the II-III cytoplasmic loop of the DHPR alpha1 subunit will be screened for the ability to trigger and SR Ca2+ release (Activator) and to block (Blocker) the actions of Activator peptides in the in vitro E-C coupling assay system and peptide binding assays. Appropriate peptides will be used to localize the signal-receiving site(s) in the JFP.
The second aim will explore changes in JFP conformation involved in activation and inactivation of SR Ca2+ release. This will involve investigations of the correlations between the depolarization signal and JFP conformational change, and between the binding of Activator peptide, JFP conformational change and SR Ca2+ release, and of the temporal relation between JFP conformational change and channel opening. The exact location of the site of attachment of conformational probes in the JFP will be investigated to characterize the actual structural changes involved. Local changes and global changes in JFP structure will be monitored by fluorescent reporter groups and circular dichroism, respectively.
The third aim will attempt to elucidate the complete reaction cycle of E-C-R coupling. Efforts will be made to resolve individual reaction intermediates by analyzing five parameters in a parallel. These include: (i) JFP conformational changes; (ii) Ca2+ release rate; (iii) response to a second activation stimulus; (iv) Ca2+ in the SR lumen; and (v) Ca2+ re-uptake rate.
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