REGULATION OF CALCIUM-ACTIVATED CHLORIDE CHANNELS

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Ca-activated Cl (CaC) channels play fundamental roles in many physiological processes, including secretion in airway epithelium, repolarization of the cardiac action potential, regulation of vascular tone, and neuronal excitability. These channels are important in several human diseases, including cystic fibrosis and cardiac arrhythmias. Our understanding of the biophysics of CaC channels, however, remains rudimentary, and the mechanisms of CaC channel activation by Ca are enigmatic. This proposal aims to elucidate the mechanism of regulation and gating of CaC channels in Xenopus oocytes and rabbit cardiac myocytes at the single channel level using the patch clamp technique in the cell-attached and excised-patch configurations. These studies will provide important insights into the control and function of this important class of ion channels and will likely have an impact on the understanding of how other types of ion channels are regulated by Ca. Aim 1 addresses whether the apparent diversity in CaC channels and currents is due to molecular diversity in channel types or to complexity of CaC channel regulation. Xenopus oocytes are an ideal system for studying this question because they express three different types of CaC currents. We present evidence that CaC channels exhibit strong voltage-dependent Ca sensitivity and we suggest that at least some of the diversity in CaC currents can be explained by the complexities in channel function that result from this voltage-dependence of Ca regulation. Aim 2. Addresses how this complex regulation comes about, by examining the mechanisms of channel regulation by Ca binding, by CaM binding, and by phosphorylation. Aim 2 then extends these studies to cardiac myocytes. CaC channels play a normal role in repolarization of the cardiac action potential and can also play a pathological role in initiating arrhythmias during Ca overload. The goal of this aim is to characterize CaC channels in cardiac myocytes to understand whether the same CaC channels are responsible for action potential repolarization and the after depolarizations that trigger arrhythmias and to explore the features of channel regulation that permit the CaC channel to become arrhythmogenic.