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Understanding calcium-activated chloride channels in health and disease

F.C. Britton and W.J. Hatton, Department of Biomedical Sciences, School of Dental Medicine, University of Nevada Las Vegas, 1001 Shadow Lane, Las Vegas, NV 89106, USA.

Ca2+-activated Cl (ClCa) channels are small conductance anion channels activated by a rise in intracellular Ca2+ concentration. ClCa channels have multiple important roles in cellular physiology, including epithelial secretion of electrolytes and water, regulation of vascular smooth muscle tone, sensory transduction (pain, smell, temperature), generation of rhythmic slow waves for gut motility, neuronal and cardiac excitability.

My lab is particularly interested in the roles of ClCa channels in cardiac excitability, first described in the early 1990’s in atrial, ventricular and Purkinje cells isolated from several species. During the cardiac action potential ClCa currents are activated in response to Ca2+ entry via voltage-gated Ca2+ channels and Ca2+ release from intracellular sarcoplasmic reticulum (SR) stores. Under physiological conditions, ClCa currents are expected to carry a significant amount of transient outward current and participate in the early repolarization process. During pathological Ca2+-overload, activation of ClCa currents can contribute to the arrhythmogenic transient inward current.

Despite the fact that ClCa channels are implicated in diverse cellular processes, our understanding these channels has been limited by the fact that, for many years, their molecular identity remained unknown. Bestrophins and anoctamins are two families of membrane proteins that function as ClCa channels and are the subject of research activity in my lab. Our group has made strides to provide a complete characterization of the electrical properties of recombinant bestrophin channel genes isolated from heart. We provided evidence that expression of bestrophin channels results in macroscopic chloride currents that are sensitive to physiological levels of intracellular Ca2+ (EC50; 175 nM Ca2+) and that bestrophin ClCa channels are present at the membrane of cardiac myocytes. More recently, biochemical studies identified that bestrophin-3 channel (Best3) physically interacts with histidine rich calcium-binding protein (Hrc), a key regulator of sarcoplasmic reticulum (SR) Ca2+-uptake, storage and release. This direct coupling of Best3 with Hrc suggests that Best3 channels in addition to mediating ClCa currents at the plasma membrane may also function as an intracellular ClCa counterion channel, balancing charge movement across the SR membrane during Ca2+ release and reuptake.

Anoctamin 1 (Ano1) ClCa channel belongs to a family of membrane proteins and has been linked to essential physiological roles in a variety of excitable cells. Our molecular studies in heart identified that Ano1 transcripts undergo complex alternative splicing to create channel diversity. We determined that Ano1 ClCa channels are clustered at the intercalated discs of cardiac myocytes, were they demonstrate strong co-localization with connexin-43 (Cx43) gap junction protein. A direct interaction between Ano1 channels and Cx43 was verified in co-immunoprecipitation studies. These findings indicate that Ano1 in its reported function as a ClCa channel plays a role in cell-cell communication in this excitable membrane domain. In summary, the identification of anoctamins and bestrophins as molecular components of ClCa channels has been fundamental for our understanding of the functions of ClCa channels. Defining the cellular distribution and localization of these channels and unraveling the mechanisms of ClCa channel regulation by Ca2+ are areas that will provide further insight to their physiological functions and pathological roles in human diseases.