A Slow pH-dependent Conformational Transition Underlies a Novel Mode of Activation of the Epithelial Na+/H+ Exchanger-3 Isoform

Abstract

Allosteric control of Na ؉ /H ؉ exchange by intracellular protons ensures rapid and accurate regulation of the intracellular pH. Although this allosteric effect was heretofore thought to occur almost instantaneously, we report here the occurrence of a slower secondary activation of the epithelial Na ؉ /H ؉ exchanger (NHE)-3 isoform. This slow activation mode developed over the course of minutes and was unique to NHE3 and the closely related isoform NHE5, but was not observed in NHE1 or NHE2. Activation of NHE3 was not due to increased density of exchangers at the cell surface, nor was it accompanied by detectable changes in phosphorylation. The association of NHE3 with the cytoskeleton, assessed by its retention in the detergent-insoluble fraction, was similarly unaffected by acidification. In contrast to the slow progressive activation elicited by acidification, deactivation occurred very rapidly upon restoration of the physiological pH. We propose that NHE3 undergoes a slow pH-dependent transition from a less active to a more active state, likely by changing its conformation or state of association. Na ϩ /H ϩ exchangers (NHEs) 1 catalyze the electroneutral counter-transport of Na ϩ for H ϩ across biological membranes (for review, see Refs. 1-3). To date, seven different NHE isoforms have been cloned, all of which are integral membrane proteins with multiple transmembrane domains and an extensive cytosolic carboxyl-terminal domain. NHE1 and NHE3 are by far the most widely studied isoforms. The former is expressed ubiquitously and, by mediating Na ϩ /H ϩ exchange across the plasma membrane, contributes to the regulation of cytosolic pH and cell volume (2). NHE3 is found in the brush border of epithelial cells in the kidney and gastrointestinal tract and is involved in the transepithelial (re)absorption of NaCl and, indirectly, HCO 3 Ϫ and water (4).Because Na ϩ /H ϩ exchange is electroneutral with a 1:1 stoichiometry, thermodynamic equilibrium is predicted to be attained when [H ϩ where K e is the equilibrium constant and the subscripts i and o refer to intraand extracellular, respectively (5). In almost all cells, a steep inwardly directed Na ϩ gradient is maintained across the plasma membrane due to extrusion of Na ϩ by the Na ϩ /K ϩ -ATPase. Based on the concentrations recorded in most cases ([Na ϩ ] o being at least 10 times higher than [Na ϩ ] i ), it is apparent that NHE could drive the intracellular pH (pH i ) at least 1 unit above the external pH. Nevertheless, pH i rarely exceeds and is usually lower than the extracellular pH because NHE becomes virtually quiescent at pH i Ն7.2. This behavior has been attributed to the existence of an allosteric pH-sensitive site on the cytosolic aspect of NHE. The set point at which quiescence is dictated by this allosteric or "modifier" site is presumably intended to stabilize pH i near the physiological optimum level and to preclude deleterious alkalinization (6, 7).Although little is known about the molecular identity of the modifier site, the pH dependence of acti...

Mitogen-activated Protein Kinase-dependent Activation of the Na+/H+ Exchanger Is Mediated through Phosphorylation of Amino Acids Ser770 and Ser771

“…Our results on stimulation of activity of NHE1 were similar to those of Haworth et al (17). It should be noted that our results differ from those Hayashi et al (22), who did not find that sustained intracellular acidosis stimulates NHE1 activity. However, in their study they did not serum-deprive NHE1 prior to treatment.…”

The Na+/H+exchanger isoform 2 is the predominant NHE isoform in murine colonic crypts and its lack causes NHE3 upregulation

“…When we assume that NHE1-3 in native cells have similar differences in their half-lives [which needs not be the case, as PS120 fibroblasts have been found to lack important PDZ-domain adapter proteins for these transporters (36) which may influence localization of these NHEs in the membrane], this would explain why NHE1 expression levels, which are only one-third to one-fourth of those found for NHE2, are associated with a similar acid-activated transport activity. In addition, activation of NHE3 by pH i has been found to be slower than for NHE1 and NHE2, possibly explaining a relatively minor role in cryptal pH i regulation when the other NHEs are highly active (14). Taking all this into account, there is a surprisingly good correlation between the relative expres- sion levels of the different NHEs and their respective activity in acid-activated Na ϩ /H ϩ exchange in surface and crypts.…”

Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3

“…The initial intracellular pH (pH i) change (first ϳ30 s) was fitted by linear regression using a fitting software (Deltagraph, Nihonporadegital, Japan) (r 2 Ͼ 0.95). In situ calibration was performed at the end of each experiment by the high potassium-nigericin technique, as previously described (22).…”