Regulation of calcium-activated chloride channels

描述（由申请人提供）：我们的长期目标是了解一个新的Cl通道家族，即strophins的结构和功能。我们有证据表明，strophins是钙活化的Cl通道。CaC通道在包括上皮分泌在内的许多生理过程中发挥着既定的作用。Specific Aim 1将对小鼠strophin 2 （mBest2）进行结构-功能分析。两种假设将被测试：（1a）： mbest2cl通道的孔隙是由几个不同的不连续疏水序列组成的，包括部分片段B， C和e。半胱氨酸扫描诱变和膜片钳对strophin电流的分析将被用来了解通道如何在离子之间选择。表位标记将用于建立mBest2拓扑结构。（1B）：原生CaC通道是strophin亚基的异聚体。我们将使用双向strophin亚基的共表达，相互作用亚基的共免疫沉淀，以及使用siRNA和显性阴性构建体干扰天然双向strophin亚基的表达或功能。这一目标将(i)提供关于离子通道阴离子选择性机制的概念，（ii）深入了解通道的生理功能，以及（iii）为strophins作为cl选择性孔的作用提供额外的支持。第二个具体目标是了解mBest2的生理和病理生理功能。天然钙离子电流和胞质钙离子电流都受细胞外渗透压和胞质钙离子的调节。肾脏细胞受细胞外渗透环境的影响很大，这取决于动物的水合状态。两个假设将被检验。（2A）： CaC和bestrophin电流受细胞膜张力调节，在细胞体积调节中发挥新的作用。这一假设将通过确定渗透压、细胞体积、膜张力、Ca、mBest2电流和代偿性细胞体积变化之间的关系来验证。（2B）：常染色体显性多囊肾病（ADPKD）的囊肿扩张与CaC电流和嗜中性粒细胞有关。这一假设将通过免疫细胞化学、电生理和药理学分析在正常和多囊肾组织以及培养的囊肿上皮细胞中Best2的表达进行探讨。这一目的将确立CaC通道和嗜中性粒细胞在肾脏生理和疾病中的作用。

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.