This proposal will increase understanding of acute regulation of the small intestinal brush border Na+/H+ exchanger NHE3, which accounts for the majority of Na+ absorption in the intestine and contributes to abnormal water and Na+ absorption in most diarrheal diseases. NHE3 transports Na+ and H+ under basal conditions, and in the post-prandial state is first inhibited and then stimulated later in digestion. Studies ae proposed based on our findings during the previous funding period that A) A large, multiprotein complex forms on the NHE3 C-terminus acting as a scaffold between aa 586-605 which is necessary for the post-prandial inhibition of NHE3 activity as is mimicked by elevated Ca2+ and cAMP. Proteins shown to be part of this complex include NHERF 1-4, CaMKII, CK2, PLC?, and probably CaM and the complex is dynamic, changing in composition with conditions which mimick the post-prandial state. B) NHE3 is fixed to the microvillar cytoskeleton under basal conditions, but reversibly dissociates with signaling that mimics the post-prandial state. This proposal will increase understanding of the role of this signaling complex in NHE3 regulation and diarrheal diseases by: A) Testing the hypothesis that the NHE3 C-terminal signaling complex is a physiologic and pathophysiologic regulator of NHE3, which can be affected to stimulate NHE3 activity. The role of CaM KII will be studied, which we have shown is part of the signaling complex and which appears to inhibit basal NHE3 activity and to phosphorylate NHE3. Also, a peptide that mimics the part of the NHE3 C-terminus to which the signaling complex attaches can be loaded into cells, and stimulates basal NHE3 activity and reduces Ca2+ and cAMP-induced NHE3 inhibition, including in an in vivo model of cholera. This peptide will be examined for effects on NHE3 regulation in polarized intestinal cells and intact rodent intestine as well as models of acute diarrhea and used to probe biochemically the nature of the NHE3 signaling complex. This peptide may be useful to develop as a drug to treat diarrhea. B) Testing the hypothesis that acute regulation of NHE3 activity involves two separate events: changes in NHE3 trafficking, which has been studied in the past, and the newly recognized dynamic aspects in the NHE3/microvillar cytoskeletal-NHERF2 association. Advanced imaging/confocal microscopy with polarized epithelial cells will determine which NHE3 regulatory processes are associated with freeing up NHE3 from NHERF2 and the microvillar cytoskeleton and determine which steps in signaling are responsible for this dynamic interaction of NHE3 with the microvillar actin cytoskeleton as well as the consequences on regulation of NHE3 activity of disrupting the dissociation, including in in vivo conditions. These studies will provide insights into how this NHE3 signaling complex helps set NHE3 activity under basal and regulated conditions, including in two in vivo diarrhea models; and also how information about this complex can be used translationally to develop drug therapy of diarrhea by stimulating NHE3.