The Calcineurin Homologous Protein (CHP) regulates the Na+/H+ exchanger‐3 (NHE3) traffic by modulation of ezrin phosphorylation

Abstract

NHE3 is essential to the regulation of Na+ absorption in the renal and intestinal epithelium. NHE3 function is regulated in part by changes in its cell surface abundance; however, the upstream molecular signals that control NHE3 surface expression remain elusive. In opossum kidney cells, CHP over‐expression increased NHE3 activity and plasma membrane abundance, while silencing of CHP decreased cell surface NHE3 activity and protein. Consistent with a central role for ezrin dependent NHE3 trafficking, augmented CHP expression increased ezrin phosphorylation at threonine 567 without change in total ezrin. Knockdown of CHP reduces the amount of phosphorylated ezrin with no change in total ezrin. Our findings show that ezrin activation is downstream from CHP. To demonstrate that NHE3 is downstream from ezrin, we showed that over‐expression of wild type (WT) ezrin increased NHE3 activity and surface expression. This effect was enhanced by expression of the constitutively active ezrin mutant T567D, whereas the inactive non‐phosphorylatable mutant T567A did not affect NHE3. To further demonstrate the linear CHP‐ezrin‐NHE3 cascade, we showed that CHP knockdown reversed the ezrin WT mediated NHE3 activation but did not affect NHE3 up‐regulation by T567D ezrin. We conclude that CHP is a critical signal molecule that increases surface NHE3 protein and function by increasing ezrin phosphorylation and NHE3 trafficking.