Na/H exchange activity is ubiquitous and mediates a variety of mammalian cell functions. In the renal proximal tubule, one specific isoform NHE-3 is the principal protein responsible for transepithelial NaCl and NaHCO3 absorption. Regulation of NHE-3 function is pivotal for acid-base and volume homeostasis. A number of hormones that regulate proximal tubule NHE-3 activity also modulate cellular cAMP levels and cAMP-dependent protein kinase activity. A fundamental unresolved question is the mechanism of regulation of NHE-3 by PKA. The present proposal tests the hypothesis that PKA directly phosphorylate NHE-3 leading to inhibition of its transport activity. This study will aim to establish that NHE-3 is a substrate for PKA and perform phosphopeptide mapping using bacterially expressed recombinant NHE-3 proteins in cell-free conditions as well as in mammalian cells transfected with the NHE-3 gene. The exact sites of PKA phosphorylation will then be mapped on NHE-3 and each of these sites will be systematically mutated to non-phosphorylatable residues. Using the wild type and the mutant NHE-3's, the relationship between PKA-induced phosphorylation and transport activity of NHE-3 will be established. This study will establish whether: 1) PKA inhibit NHE-3 function by direct phosphorylation, 2) PKA inhibits NHE-3 function by indirect means through intermediate regulators, or 3) both. This knowledge is of fundamental significance to the understanding of the kidney's role in acid-base and volume homeostasis.
