Regulation of calcium-activated chloride channels

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DESCRIPTION (provided by applicant): Our long-term goal is to understand the structure and function of a new family of Cl channels, the bestrophins. We have evidence that bestrophins are Ca-activated Cl channels. CaC channels play established roles in many physiological processes, including epithelial secretion. Specific Aim 1 will perform a structure-function analysis of mouse bestrophin 2 (mBest2). Two hypotheses will be tested: (1 A): The pore of the mBest2 Cl channel is formed by several different non-contiguous hydrophobic sequences, including parts of segments B, C, and E. Cysteine-scanning mutagenesis and analysis of bestrophin currents by patch clamp will be employed to understand how the channel selects among ions. Epitope-tagging will be used to establish mBest2 topology. (1B): Native CaC channels are heteromultimers of bestrophin subunits. We will use co-expression of bestrophin subunits, co-immunoprecipitation of interacting subunits, and interference with the expression or function of native bestrophin subunits using siRNA and dominant negative constructs. This aim will (i) provide concepts about the mechanisms of anion selectivity of ion channels, (ii) yield insights into how the channel functions physiologically, and (iii) give additional support for the role of bestrophins as Cl-selective pores. The second specific aim is to understand the physiological and pathophysiological functions of mBest2. Native CaC currents and bestrophin currents are both regulated by extracellular osmolarity as well as cytosolic Ca. Cells in the kidney are subject to widely varying extracellular osmotic environments depending on the animal's hydration state. Two hypotheses will be tested. (2A): CaC and bestrophin currents are modulated by cell membrane tension and play a novel role in cell volume regulation. This hypothesis will be tested by determining the relationships between osmolarity, cell volume, membrane tension, Ca, mBest2 currents, and compensatory cell volume changes. (2B): CaC currents and bestrophins are involved in cyst expansion in autosomal dominant polycystic kidney disease (ADPKD). This hypothesis will be explored using immunocytochemcial, electrophysiological, and pharmacological analysis of Best2 expression in normal and polycystic kidney tissue and cultured epithelial cells from cysts. This aim will establish the role of CaC channels and bestrophins in kidney physiology and disease.