Differential Regulation of Na-K-Cl Cotransport

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DESCRIPTION (provided by applicant): NKCC1 (Na-K-2Cl co-transport) is an electrolyte transporter localized to the basolateral membrane of lining epithelial cells of the lung and is crucial for optimal mucociliary clearance because it supplies Cl for secretion. Genetically altered NKCC1 leads to abnormal transepithelial fluid secretion. Regulation of NKCC1 is complex, involving in non-mammalian cells, cAMP-dependent PKA and protein phosphatase PP1. The PI discovered that PKC-? is necessary for human airway NKCC1 function and recently found that targeting of PKC-? to NKCC1 involves a novel multiprotein complex that includes NKCC1, PKC-?, actin and NHERF. Very little is known about this multiprotein complex. Binding studies reveal direct association of PKC-? to actin and functional studies indicate a role for PP2A and the state of actin polymerization in NKCC1 function. The hypothesis of this grant is that PKC-? modulates NKCC1 function through the involvement of the actin cytoskeleton. This will be tested in the following specific aims: 1) to test the hypothesis that actin cytoskeleton is necessary for NKCC1 function. Actin organization, dynamics, and function will be manipulated using marine toxins. The functional effects on basal and agonist-stimulated NKCC1 function and turnover will be determined as will the activity of PKC-? and PP1 and PP2A. Mass and activity of PKC-? and PP2A will be downregulated using an antisense approach. 2) To test the hypothesis that PKC-? interacts with NKCC1 through a multiprotein complex. Association of protein pairs will be studied using pulldown, co-immunoprecipitation and binding assays. Specific sites of interaction will be determined using tagged fusion proteins and mutated proteins in competition experiments, Inhibitory peptides will be delivered into cells for functional studies. We will determine, for the first time, whether PP2A is a partner in the multiprotein complex. 3) To test the hypothesis that PKC-? modulates NKCC1 function through a phosphorylation event. We will determine whether PKC-? directly phosphorylates NKCC1 or indirectly through NKCC1-associated protein(s). Sites of phosphorylation by PKC-? and of dephosphorylation by PP2A will be investigated using tagged fusion proteins predicted from the cytoplasmic domains of NKCC1. The results will provide new information on proteins that regulate NKCC1 function and proteins regulated by NKCC1. The long-term outcome is manipulation, at the molecular level, of protein-protein interactions that facilitate NKCC1 function and could lead to improved NKCC1 function in pathophysiological states.