The ryanodine-sensitive Ca2+ channel controls the release of Ca2+ from the lumen of the sarcoplasmic reticulum (SR). The mechanism which links T-tubule depolarization to Ca2+-release from the SR is termed """"""""excitation-contraction"""""""" (E-C) coupling. The long term goal of the research described in this application is the elucidation of the molecular mechanisms by which endogenous compounds regulate the activity of the Ca2+-release channel and the determination of how these modulations contribute to E-C coupling in skeletal muscle. The hypotheses to be tested are 1) the Ca2+-release channel can exist in three or more functional states; 2) Ca2+ binding regulates the interconversions between states and 3) modulators of the channel (both proteins and small molecules) alter activity by stabilizing different functional states.
The specific aims of this proposal are: 1) to identify functional states of the channel and to analyze the effects on modulators (ATP, Ca2+, Mg2+, calmodulin, and adenosine) on the kinetic constants for interconversions between these states, 2) to define the mechanisms by which Ca2+ regulates channel opening and 3) to assess the role of the dihydropyridine binding protein (DHPBP) in controlling the activity of the Ca2+-release channel. The techniques which will be used include: 1) kinetic and equilibrium binding of [3H]-ryanodine to SR membranes, 2) reconstitution of channels into planar lipid bilayers, 3) terbium luminescence to analyze Ca2+ binding sites, 4) immune precipitation, and 5) sucrose gradient sedimentation. A single amino acid change in the sequence of the Ca2+-release channel produces the disease Malignant Hyperthermia in pigs and humans. The genetic defect in malignant hyperthermia facilitates channel opening. A syndrome very similar to malignant hyperthermia is often found to be associated with Central Core Disease (CCD) and Duchenne's Muscular Dystrophy (DMD). Since the defect in DMD does not appear to be in the Ca2+-release channel itself, a secondary effect must alter the regulation of channel activity in these diseases in such a way as to also facilitate channel opening. Therefore, this proposed study of the modulation of functional transitions of the Ca2+-release channel will aid in the interpretation of the events involved in the occurrence of the MH syndrome in these diseases.
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