In striated muscle, cycles of contraction and relaxation are regulated by the cytoplasmic concentration of Ca 2+. Calcium is passively released from the lumen of the sarcoplasmic reticulum (SR) by means of calcium release channel/foot structures causing contraction, while uptake of Ca an active transport process carried out by the SR Ca-ATPase. The mechanism by which depolarization of the plasmalemma and transverse tubules of the myofiber causes the SR calcium channel/foot to open in excitation-contraction coupling is unknown and is perhaps the most significant remaining problem in the molecular biology of muscle contraction. The calcium channel/foot structure is the major component of the triad junction which is located where the transverse tubules and SR membranes are apposed. The triad junction is believed to be the site of excitation-contraction coupling in skeletal muscle. The overall goal of this proposal is to determine the three-dimensional architecture of the triad junction from electron micrographs of purified and reconstituted components. The studies will focus on the calcium channel/foot structure from skeletal muscle, for which we already have determined a 3-D reconstruction from electron micrographs of negatively stained specimens. The channel complex will be locked in its open and closed states by means of channel-specific ligands, electron micrographs will be recorded from frozen-hydrated (non-stained) specimens, and three-dimensional reconstructions will be determined using the image processing methods developed in our laboratory for application to non-crystalline macromolecular specimens. High resolution immunoelectron microscopy will be done on the channel complexes using monospecific antibodies raised against synthetic peptides corresponding to surface-exposed sequences. The mechanism of excitation-contraction coupling differs in skeletal and cardiac muscles, and so comparative studies will be conducted on calcium channel/foot structures isolated from both types of muscle. Two other large, multisubunit ion channels that are believed to function iii E-C coupling, the dihydropyridine receptor and a recently isolated inositol trisphosphate receptor, should also be feasible for structural studies by the same methodology.
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