COBRE: UNR: SIGNAL TRANSDUCTION PATHWAYS REGULATED BY CELL VOLUME

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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.