Regulation of Na+/H+ exchanger 1 allosteric balance by its localization in cholesterol‐ and caveolin‐rich membrane microdomains

Abstract

The Na⁺/H⁺ exchanger 1, which plays an essential role in intracellular pH regulation in most tissues, is also known to be a key actor in both proliferative and apoptotic processes. Its activation by H⁺ is best described by the Monod–Wyman–Changeux model: the dimeric NHE‐1 oscillates between a low and a high affinity conformation, the balance between the two forms being defined by the allosteric constant L₀. In this study, influence of cholesterol‐ and caveolin‐rich microdomains on NHE‐1 activity was examined by using cholesterol depleting agents, including methyl‐β‐cyclodextrin (MBCD). These agents activated NHE‐1 by modulating its L₀ parameter, which was reverted by cholesterol repletion. This activation was associated with NHE‐1 relocation outside microdomains, and was distinct from NHE‐1 mitogenic and hormonal stimulation; indeed MBCD and serum treatments were additive, and serum alone did not change NHE‐1 localization. Besides, MBCD activated a serum‐insensitive, constitutively active mutated NHE‐1 (⁶²⁵KDKEEEIRK⁶³⁵ into KNKQQQIRK). Finally, the membrane‐dependent NHE‐1 regulation occurred independently of Mitogen Activated Protein Kinases, especially Extracellular Regulated Kinase activation, although this kinase was activated by MBCD. In conclusion, localization of NHE‐1 in membrane cholesterol‐ and caveolin‐rich microdomains constitutes a novel physiological negative regulator of NHE‐1 activity. J. Cell. Physiol. 216: 207–220, 2008.