We study the process of intracellular calcium signaling in muscle and heart physiology. In these tissues, large and transient increases in cytosolic calcium are regularly induced by excitatory processes. This calcium then triggers the contractile machinery as required for the heartbeat or for the muscle twitch. In summary, calcium couples the excitatory process at the external membranes with the contractile event inside the cell; namely, “excitation-contraction (EC) coupling.”

   The bulk of calcium that mediates the process of excitation-contraction in muscle and heart originates from an intracellular reticular meshwork of vesicles and tubules (the sarcoplasmic reticulum; SR), which serve as calcium intracellular stores. On the surface of the SR vesicles there are arrays of very large molecules named ryanodine receptors (as they tightly bind the alkaloid ryanodine) or foot structures (as seen by electron microscopy). They are also known as intracellular calcium release channels because they are pores that open and mobilize the bulk of calcium required for the contractile events. The SR membranes also contain large quantities of a second protein, the SR Ca2+ ATP-ase. This is a ATP-driven "calcium pump" that mediates the uptake of Ca2+ from the cytosol back to SR (enabling muscle to relax).

   Our goal is to understand how RyR channels work in cells. It is clear that the magnitude of the calcium released by the RyRs impacts the force of the twitch contraction of the skeletal fibers we use for movement as well as the strength of the heartbeats that pump blood through our body. Mutations in the RyR molecule or anomalies in its physiological modulation result in a variety of heart and skeletal muscle disorders (cardiac failure, arrhythmia, polymorphic ventricular tachicardia, malignant hyperthermia, central core disease, and hypokalemic periodic paralysis). However, we still do not know how RyRs work. Consequently, we have difficulty understanding the genesis of anomalous calcium release found in diseases. Thus, understanding mechanisms that control RyR function represents crucial points at which contractility and intracellular calcium homeostasis can be modulated. Therefore, their study is clinically important as potential sites for therapeutic intervention.

   We study the mechanism of excitation-contraction coupling at cellular levels. Here, we can measure the characteristics of ryanodine receptor-mediated global calcium transients upon stimulation. We also look at the local and elementary events of calcium release (calcium sparks) observed under special conditions in resting cells.    We study SR Ca2+ mobilization at a membrane and molecular level using subcellular membrane preparations from muscle and heart, including terminal cisternae and longitudinal tubules of SR, and triads, i.e., the junctional association of transverse tubules and terminal cisternae. We reconstitute into planar bilayers the intracellular calcium release channels (ryanodine receptors, RyR) both from skeletal (RyR1) and heart muscle (RyR2).

   Numerous reports on RyR behavior describe many aspects of channel modulation. They mostly focus on defining the function of single channels reconstituted into artificial lipid bilayers. However, we found that individual RyR behavior does not correlate with RyR-mediated calcium release in cells. Electron microscopy studies have shown that the RyRs are physically connected in organized arrays at the terminal cisternae (TC) membranes of SR. There, they appear to work as a group (not individuals). That is, SR calcium release events appear to be generated by discrete groups of adjacent channels in the array that open and close their pores in a synchronized manner. Only one study (Marx et al., 1988) showed synchronous behavior of RyR2 reconstituted in artificial bilayers. It was concluded that RyRs do indeed communicate. This process, named "coupled gating," may explain calcium release in muscle cells but we still know very little about it.

   The specific goal of my current studies (funded by the National Institutes of Health) is to test the hypothesis that RyR1 channels communicate in a calcium-dependent manner and to determine the nature of this communication. I believe that this knowledge would be crucial for understanding E-C coupling, both in health and disease.

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