COBRE: UNR: SIGNAL TRANSDUCTION PATHWAYS REGULATED BY CELL VOLUME

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 维持生理细胞体积对细胞的增殖、分化和存活至关重要。阴离子通道，特别是氯离子通道，参与其他重要的生理功能，包括维持心脏的节律周期和血管的肌张力。氯通道ClC-3是肺动脉平滑肌细胞(PASMCs)的主要容量调节因子，但其调节机制尚不明确。本提案的主要目的是通过解决以下特定目标来确定在非等渗细胞环境中调控ClC-3的基本细胞内机制：(1)验证蛋白激酶通过磷酸化调节容量敏感的外向整流阴离子通道(VSOAC)/ClC-3的假说。在PASMCs和NIH3T3细胞的实验中，将利用生化和分子方法来确定ClC-3受其N-末端(NT)和/或C-末端(CT)的磷酸化调控，并在体外和体内鉴定使CLC-3磷酸化的蛋白激酶。(2)验证血清和糖皮质激素依赖激酶(SGK)通过间接磷酸化和/或泛素化来调节VSOAC/ClC-3的假说。这一目标将涉及实验，以测试低渗肿胀介导的SGK激活是否会导致NIH 3T3细胞和犬PASMCs泛素化减少和ClC-3膜表达增加，从而激活一种目前未知的中间蛋白激酶。(3)验证皮质下肌动蛋白和肌动蛋白结合蛋白与VSOAC/ClC-3通道相互作用并参与其激活的假设。这一目标将包括评估细胞内ClC-3末端与肌动蛋白细丝和肌动蛋白结合蛋白的结合能力，识别参与这种结合的ClC-3结构域，并检验抑制这种相互作用干扰ClC-3激活的假设。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Maintenance of physiological cell volume is crucial for cell proliferation, differentiation and survival. Anion channels, and particularly chloride (Cl-) channels, participate in other important physiological functions including maintenance of the rhythmic cycle of the heart and the muscle tone of blood vessels. The Cl- channel ClC-3 is a major volume regulator in pulmonary artery smooth muscle cells (PASMCs), however its regulation is undefined. The major goal of the present proposal is to define the fundamental intracellular mechanisms regulating ClC-3 in anisotonic cell environment, by addressing the following specific aims: (1) Test the hypothesis that protein kinases regulate the volume-sensitive outwardly rectifying anion channel (VSOAC)/ClC-3 by phosphorylation. Biochemical and molecular approaches will be utilized in experiments with PASMCs and NIH 3T3 cells to establish that ClC-3 is regulated by phosphorylation of its N-terminus (NT) and/or C-terminus (CT), and to identify protein kinases that phosphorylate ClC-3 in vitro and in vivo. (2) Test the hypothesis that the serum and glucocorticoid-dependent kinase (SGK) regulates VSOAC/ClC-3 by indirect phosphorylation and/or ubiquitination. This aim will involve experiments to test the possibility that hypotonic swelling-mediated activation of SGK leads to activation of a currently unknown intermediate kinase, or by decreased ubiquitination and increased membrane expression of ClC-3 in NIH 3T3 cells and canine PASMCs. (3) Test the hypothesis that subcortical actin and actin-binding proteins interact with VSOAC/ClC-3 channel and contribute to its activation. This aim will include experiments to assess the binding capacity of the cytosolic ClC-3 termini with actin filaments and actin-binding proteins, to identify ClC-3 domains involved in the association, and to test the hypothesis that inhibition of this interaction interferes with the activation of ClC-3.