CAMP-MEDIATED INHIBITION OF THE EPITHELIAL BRUSH-BORDER NA+ H+ EXCHANGER, NHE3, REQUIRES AN ASSOCIATED REGULATORY PROTEIN/

NHE3 is the Na+/H+ exchanger located on the intestinal and renal brush border membrane, where it functions in transepithelial Na+ absorption , Tile brush border Na+ absorptive process is acutely inhibited by act ivation of cAMP-dependent protein kinase, but the molecular mechanism of this inhibitory effect is poorly understood, We have identified two regulatory proteins, E3KARP and NHERF, that interact with NHE3 to ena ble cAMP to inhibit NHE3. The twee regulatory proteins are structurall y related, sharing approximate to 50% identity In amino acid sequences , It has been previously shown that when NHE3 is transfected into PS12 0 fibroblasts or Caco-2 cells, cAMP failed to inhibit NHE3 activity, N orthern blot analysis showed that both PS120 and Caco-2 cells lacked t he expression of both E3KARP and NHERF. In contrast, other cell lines in which cAMP inhibits NHE3, including OK, CHO, and LLC-PK1 cells, exp ressed NHERF-related regulatory proteins, To determine their functions in cAMP-dependent inhibition of NHE3, E3KARP and NHERF were transfect ed into PS120/NHE3 fibroblasts. Transfection in PS120/NHE3 fibroblasts with either NHERF or E3KARP reconstituted cAMP-induced induction of N HE3, resulting in 25-30% inhibition in these cells.