Protein mediated regulation of the NHE1 isoform of the Na+/H+ exchanger in renal cells. A regulatory role of Hsp90 and AKT kinase

Na+/H+ exchanger isoform one (NHE1) is a pH regulatory protein that is present in renal tissues and serves to remove protons from within cells and protect against intracellular acidification. NHE1 has a large 315 amino acid cytosolic regulatory domain that regulates the catalytic membrane domain. We examined protein-mediated regulation of NHE1 through the cytosolic domain. Affinity chromatography with the C-terminus of NHE1 yielded a number of NHE1 binding proteins including 14-3-3 protein, heat shock proteins (Hsp90 and Hsp70) and Na+/K+ ATPase. We confirmed that 14-3-3 and heat shock proteins bind to or regulate NHE1 but could not confirm that Na+/K+ ATPase binds to the intact protein. The Hsp90 inhibitor 17-AAG decreased NHE1 activity and NHE1 phosphorylation in MDCK cells but did not decrease protein levels. Additionally, 17-AAG decreased phospho-AKT levels. Direct inhibition of AKT with MK2206 decreased NHE1 activity, though this effect was not additive with the effect of 17-AAG. The results demonstrate that in renal cells, NHE1 is associated with several regulatory proteins including Hsp90, and that Hsp90 affects its function possibly through altered phosphorylation of the protein via the AKT kinase.