One of the most important unsolved questions in muscle physiology is a detailed understanding of the following sequential events: (i) depolarization of the transverse tubular system (T-tubule), (ii) Ca2+ release from sarcoplasmic reticulum (SR) leading to contraction, (iii) reaccumulation of the released Ca2+ by the SR Ca2+ pump leading to relaxation. Our long range goal is to resolve each of these processes at a molecular level in normal and diseased muscles. Our current working hypothesis is as follows. The excitation signal elicited in the T-tubule is transmitted via the dihydropyridine (DHP) receptor, probably with the aid of several other proteins, to the foot protein (FP). This leads to conformational changes in the FP, and in turn activates the Ca2+ channel located in FP. The signal is further transmitted to a protein in the SR lumen calsequestrin (CSQ), dissociating the bound calcium from CSQ. The dissociated Ca2+, which will eventually be released to the cytoplasm through the activated channel, activates the Ca2+ pump leading to a prompt reaccumulation of the released Ca2+.
Our specific aims are to resolve individual steps of the hypothetical chain reactions using the isolated triads capable of physiological excitation-contraction coupling, purified protein components, and the reconstituted system. Various steps involved in the DHP receptor-mediated signal transmission pathway will be resolved by using chemical antagonists and antibodies. Involvement of other proteins in the T-tubule - SR communication will also be examined by using antibodies (and Fab subfragments) directed against them, and by reconstitution experiments. Conformational changes in the FP induced by various effectors of Ca2+ release, especially the conformational changes induced by the T-tubule depolarization, will be investigated using the fluorescent probe incorporated into various domains of the FP moiety in a site-directed fashion. The question regarding the FP - CSQ communication will be investigated by correlating conformational changes of FP and CSQ, and dissociation of the CSQ-bound calcium during Ca2+ release reaction. To test the hypothesis that CSQ controls SR Ca2+ release, the kinetics of the transient increase in the lumenal Ca2+ and the subsequent Ca2+ release from SR will be correlated. Kinetic behavior of the SR Ca2+ pump during Ca2+ release will be followed to examine reactivation of Ca2+ pumping and the postulated role of CSQ in the reactivation process.
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