REGULATION OF MEMBRANE EXCITABILITY

这个项目的长期目标是了解分子

The long-term goal of this project is to understand the molecular principles underlying the function, subcellular localization and biosynthesis of calcium channels and to further the understanding of excitation-contraction coupling in skeletal muscle. During the period of requested funding, normal myotubes and dysgenic myotubes expressing genetically engineered calcium channels will be studied. The specific aims are to: 1. Produce an improved biophysical model of the dihydropyridine receptor (DHPR) functioning as an L-type calcium channel and as a voltage sensor controlling calcium release from the SR. The activation process will be probed by means of comparisons between slowly (skeletal) and rapidly (cardiac) activating L-type calcium channels expressed in dysgenic myotubes. It will be determined whether the strong depolarization causes the skeletal L-type channel to enter a long-lived open state. 2. Identify regions of the DHPR critical for its function as a calcium channel, as a generator of charge movement and as a voltage sensor for E-C coupling. In each repeat, mutations will be made in order to perturb voltage dependence and, thus, identify the critical roles of each repeat in the physiological functions of the DHPR. Chimeras of the skeletal and cardiac DHPRs will be constructed to identify regions important for inactivation. The effects of DHPR mutations causing hypokalemic periodic paralysis will be analyzed biophysically. 3. Probe the nature of the interaction between the DHPR and the SR calcium release process. Construction of skeletal/cardiac DHPR chimeras will be used to identify a minimal region within the II-III loop critical for skeletal-type E-C coupling. A corresponding oligopeptide will be tested to determine whether it acts as an agonist or antagonist of E-C coupling. The stoichiometry of DHPR activation of SR calcium release will be analyzed. 4. Define the morphological arrangement of different kinds of calcium channels exogenously expressed in muscle. The morphological arrangement of different kinds of calcium channels will be characterized using fluorescence microscopy and using electron microscopy of freeze-fractures. 5. Evaluate functional consequences of developmental changes in the skeletal DHPR. The biosynthesis and assembly of DHP receptors in muscle will be characterized. The physiological significance of alternative splices in the skeletal DHPR alpha1 will be addressed by expression of cDNA and functional analysis.