CARDIAC CA CHANNELS--REGULATION OF SLOW GATING BEHAVIOR

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Regulation of the processes that control the opening and closing, or gating, of Ca channels is crucially important to the control of the heartbeat. Classical ideas about how channels are regulated have focused upon changes in the gating behavior of channels that are almost always available to open upon short notice. In contrast, recent studies have hinted at the novel possibility that a predominant mechanism for modulating Ca channels is to shift channels slowly between two radically different modes of gating; (1) an active' mode in which channel openings are probable, and (2) a 'hibernating'mode in which channels are unlikely to open. The overall goal of this project is to use patch clamp techniques to establish a rigorous understanding, at the single channel and molecular level, of the slow transitions of the Ca channel between active and hibernating gating modes in the heart. Unitary "L-type" Ca channel currents will be measured by the patch voltage clamp technique in single mammalian ventricular cells. A new analytic approach, termed "sweep histogram analysis," will be applied to provide quantitative evidence for the genuine existence of distinct active and hibernating gating modes, between which channels cycle slowly. The approach will also enable the identification of explicit kinetic models to explain the transitions between gating modes. The validation of such a model opens the possibility to newly distinguish the specific kinetic steps that are affected by factors that modulate the Ca channel. These factors include regulators of channel phosphorylation (B-adrenergic and cholinergic agonists) or dephosphorylation (okadaic acid), as well as agents that interact with the channel directly (membrane voltage, synthetic Ca channel ligands, and G proteins). Sweep histogram comparison of the effects of these factors to the action of intracellularly applied proteases will provide important clues as to the existence of domains on the channel molecule that may be crucial to slow gating transitions between modes. Clarification of the mechanisms that bias Ca channels toward active or hibernating gating modes promises to provide fundamental insight into the molecular mechanisms by which the Ca channel is regulated.