COBRE: UNR: SIGNAL TRANSDUCTION PATHWAYS REGULATED BY CELL VOLUME

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 维持生理细胞体积对于细胞增殖、分化和存活至关重要。阴离子通道，特别是氯离子（Cl-）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。Cl-通道ClC-3是肺动脉平滑肌细胞（PASMCs）的主要容量调节因子，但其调节机制尚不清楚。本发明的主要目的是通过解决以下具体目标来确定在非等渗细胞环境中调节ClC-3的基本细胞内机制：（1）检验蛋白激酶通过磷酸化调节体积敏感性向外整流阴离子通道（VSOAC）/ClC-3的假设。生物化学和分子方法将用于PASMCs和NIH 3 T3细胞的实验中，以确定ClC-3受其N-末端（NT）和/或C-末端（CT）磷酸化的调节，并鉴定在体外和体内使ClC-3磷酸化的蛋白激酶。 (2)检验血清和糖皮质激素依赖性激酶（SGK）通过间接磷酸化和/或泛素化调节VSOAC/ClC-3的假设。这一目标将涉及实验来测试低渗溶胀介导的SGK激活导致目前未知的中间激酶激活，或通过NIH 3 T3细胞和犬PASMCs中C1 C-3的泛素化减少和膜表达增加的可能性。 (3)验证皮质下肌动蛋白和肌动蛋白结合蛋白与VSOAC/ClC-3通道相互作用并促进其激活的假设。这一目标将包括实验，以评估的细胞溶质CLC-3终端与肌动蛋白丝和肌动蛋白结合蛋白的结合能力，以确定CLC-3域参与的协会，并测试的假设，这种相互作用的抑制干扰CLC-3的激活。

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.