Modulation of renal ion and water excretion by antidiuretic hormone of ADH is a fundamental mechanism regulating total body water content. This competitive renewal application seeks funding to continue the laboratory's effort to define the molecular components of the complex cellular machinery in ADH-responsive cells that regulates apical membrane water permeability (Pf) and thus preserves body homeostasis. Molecular cloning of the ADH elicited water channel called Aquaporin (AQP) 2 has demonstrated that ADH regulates Pf via alterations in AQP2 mRNA and protein content of cells, insertion and removal of AQP2 from the apical membrane in vesicles and perhaps changes in channel permeability. ADH's regulation of Pf is similar to that demonstrated for several membrane transporters in other cell types including the insulin sensitive glucose transporter GLUT-4. Previously, they have demonstrated that ADH water channels are sequestered in a specialized endosomal compartment immediately adjacent to the apical membrane. In recent work, they have purified AQP2 containing endosomes from rat kidney inner medullary collection duct (IMCD) and shown they possess 4 other proteins that modulate AQP2 endosome function. They now propose to define both the structure and function of each of these proteins using a combination of molecular cloning and expression experiments as well as biochemical and physiological studies. The first protein is a Ca2+/polycation receptor protein (CaR) located on the apical membrane of IMCD cells where it alters ADH-elicited IMCD Pf. They hypothesize that this CaR integrates kidney calcium and water metabolism and may be important in the pathogenesis of renal stones. The second protein is cAMP-dependent protein kinase A (PKA) that is present in purified AQP2 endosomes in combination with a PKA anchoring protein or AKAP, protein kinase C and calcineurin. They hypothesize these proteins interact to modulate AQP2 endosome or channel function. The third protein is a homolog of a unique cysteine-rich protein gp55-61 present in the apical endosomes of IMCD as well as other epithelia. They hypothesize a kidney homolog of gp55-61 defines the specialized apical endosomal compartment containing AQP2. The fourth protein is a unique synaptobrevin similar to that present in brain that regulates the fusion of AQP2 They hypothesize this IMCD synaptobrevin participates in the trafficking of AQP2 in a fashion similar to that exhibited by synaptic vesicles in neurons.
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