MODULATION OF CA2+ FLUXES IN HEART AND SMOOTH MUSCLE

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The long term goal of our ongoing program is to study cardiac and smooth muscle with focus on the membranes and the molecular machinery which regulate muscle contraction and relaxation. Specifically, we aim to: 1) Define the molecular machinery involved in the modulation of calcium pumping (enables muscle to relax) in heart sarcoplasmic reticulum (SR); 2) Crystalize the calcium binding protein from cardiac SR, which is involved in the storage of calcium in SR (also referred to as calsequestrin), so that its structure can be determined by X-ray crystallography and electron diffraction; 3) Characterize the heart Ca2+ release channel from SR, which is involved in Ca2+ release which triggers muscle contraction. The modulation of Ca2+ release by protein kinases and phosphatases will be studied. The state of modulation might, in part, explain the controversy in the literature regarding IP activation, or lack or it; 4) Obtain the 3- dimensional structure of the heart ryanodine receptor/Ca2+ release channel (no available) and smooth muscle Ca2+ release channels when they become available (see aim 6); 5) Isolate dyads/triads from heart in order to characterize similarities and differences with that from the skeletal muscle. Such studies should help to assess the basis of the observed macroscopic difference in excitation-contraction coupling, i.e., depolarization induced calcium release (DICR) in skeletal muscle vs calcium release (CICR) in heart; 6) Initiate a program to study smooth muscle membranes, with the aim to isolate and characterize the membrane systems and the molecular components involved int eh Ca2+ pumping, storage and release machinery. This program on smooth muscle will parallel that ongoing for heart (Aims 1-5). Smooth muscle SR appears to have two different types of Ca2+ release channels, the ryanodine receptor type and an IP3 receptor. Definition of these two receptors should provide further insight and comparison into channel types operative in heart and skeletal muscle. Our program is multidisciplinary in scope. It relies heavily on subcellular fractionation to prepare defined membranes, and their functional characterization. The dissociation and reconstitution approach is then employed for isolation and characterization of components involved in Ca2+ transport, storage and release, and the nature of their modulation. Methodology includes electron microscopy, enzymology, transport kinetics, binding studies, channel conductance measurement, crystallization of proteins and structural analysis, monoclonal antibody and cloning technology. The basic information of the membrane machinery involved in Ca2+ uptake, storage and release and its regulation for both heart and smooth muscle should provide a better basis for the understanding of heart disease and hypertension and thereby for the development of cardioselective drugs as well as new types of drugs for regulation of blood pressure.