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氯离子通道家族--bestrophins的结构和功能。我们有证据表明Bestrophins是钙激活的氯离子通道。CAC通道在许多生理过程中发挥着既定的作用，包括上皮细胞的分泌。特定目标1将对小鼠Bestrophin 2(MBest2)进行结构功能分析。将检验两种假设：(1)A：mBest2氯通道的孔是由几个不同的非连续疏水序列形成的，包括B、C和E段的部分片段；半胱氨酸扫描突变和膜片钳对Bestrophin电流的分析将用于了解该通道是如何在离子之间进行选择的。表位标记将用于建立mBest2拓扑结构。(1B)：天然CAC通道是Bestrophin亚基的异源多聚体。我们将使用Bestrophin亚基的共表达，相互作用的亚基的共免疫沉淀，以及使用siRNA和显性负结构干扰天然Bestrophin亚基的表达或功能。这一目标将(I)提供有关离子通道的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)进一步支持bestrophins作为氯离子选择孔的作用。第二个具体目的是了解mBest2的生理和病理生理功能。天然钙通道电流和Bestrophin电流既受细胞外渗透压调节，也受胞内钙调节。根据动物的水合状态，肾脏中的细胞受到不同的细胞外渗透环境的影响。我们将检验两个假设。(2A)：CAC和Bestrophin电流受细胞膜张力的调节，在细胞体积调节中发挥新的作用。这一假说将通过确定渗透压、细胞体积、膜张力、钙电流、mBest2电流和代偿性细胞体积变化之间的关系来检验。(2B)：CAC电流和Bestrophins参与常染色体显性遗传性多囊肾病(ADPKD)的囊扩张。这一假说将通过免疫细胞化学、电生理和药理学分析Best2在正常和多囊肾组织以及培养的囊变上皮细胞中的表达来探索。这一目标将确立CAC通道和Bestrophins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.