Dantrolene, a drug which inhibits intracellular Ca/2+ release is the only therapy of malignant hyperthermia, a genetic sensitivity to volatile anesthetics. The drug also shows therapeutic promise in models of neural excitotoxicity and sepsis. However, the molecular mechanism of action of this compound is not known. This application proposes to use a radiolabeled, pharmacologically active, photoaffinity analog of dantrolene, to identify, purify and molecularly characterize the putative dantrolene receptor from skeletal muscle. The results of these experiments will serve as the basis for future experiments leading to an understanding of the role of the dantrolene receptor in regulating intracellular Ca/2+ release in health and disease. Since [3H]dantrolene binding is greatest in the sarcoplasmic reticulum of skeletal muscle, this tissue will be used as a source of putative receptor. We present data that [3H]azidodantrolene, a custom synthesized, photoaffinity analog of dantrolene, specifically photolabels a protein of approximately 140 kDa, as determined by SDS-PAGE, electroblotting to PVDF membranes, and subsequent fluorography. By using this as a method of molecular tagging, we propose to purify the putative dantrolene receptor by standard methods of membrane protein purification, which includes preparative gel electrophoresis, electroelution, detergent solubilization, ion-exchange, hydrophobic interaction and/or affinity chromatography techniques. Once purified, the receptor will be subjected to protein microsequencing. Sequence data will be compared with known sequences from protein databases to determine whether this protein shows homology to any other previously described protein. Polyclonal antibodies to the purified protein or unique synthetic peptides constructed after sequencing will be produced by standard techniques. These antibodies will be used in Western blots to detect the putative dantrolene receptor in tissues other than skeletal muscle since it has been reported that dantrolene is effective in inhibiting Ca/2+ release in a variety of cell types. Immunocytochemistry will be used to characterize the subcellular localization of this protein with respect to other known proteins involved in calcium regulation. The results from these experiments will help in designing future experiments that would help elucidate the role of the dantrolene receptor in excitation- contraction coupling and, ultimately, in Ca/2+ regulation in other tissues.
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