NHE2 and NHE3 are epithelial specific Na+/H+ exchanger isoforms which are colocalized to the brush border membrane of intestinal and renal epithelial cells. When individually expressed in PS120 cells that are deficient in endogenous NHEs, NHE2 and NHE3 exhibit 150-fold difference in the sensitivity to the amiloride analogue, HOE694 (IC50 is 5 μM vs. 650 μM for NHE2 and NHE3, respectively, at 1 mM NaCl). In the present study, we determined whether NHE2 and NHE3 activity can be separated by HOE694 and whether NHE2 and NHE3 interact when they are co-expressed in PS120 cells, as indicated by co-immunoprecipitation studies. PS120/NHE2 cells were stably transfected with NHE3V (NHE3 tagged with an 11 aminoacid epitope of the vesicular stomatitis virus glycoprotein). PS120/NHE2/NHE3V cells were selected by acid killing in the presence of 300 μM HOE694 and 2 mM Na+. A stable clone, B4, was isolated and was confirmed to express NHE2 and NHE3V by Western blotting. Immunoprecipitation experiments with anti-NHE2 antibody, Ab597, showed that NHE3V did not coimmunoprecipitate with NHE2 and vice versa. Further, B4 exhibited a bi-phasic curve towards HOE694 inhibition (0.01 μM-1 mM) of Na+/H+ exchange (measured as 22Na uptake in the presence of 1 mM Na+) that was intermediate between those of NHE2 and NHE3 espresses alone (both of which were single sigmoidal curves). This curve exhibited a plateau between 30 μM and 100 μM, suggesting that NHE2 could be completely blocked by 30-100 μM HOE694. A similar bi-phasic curve has been also obtained by us with intestinal brush border vesicles. Moreover, the part of total NHE activity located at the left of the plateau (i.e. 0.01μM-30 μM), represinting NHE2, was recalculated and fitted with a single sigmoidal curve, and this was found to have the same sensitivity curve as NHE2 expressed alone. Thus, we conclude: (1) NHE2 and NHE3 do not interact when expressed in the same cell; (2) NHE2 and NHE3 activity can be pharmacologically separated by HOE694.
|Original language||English (US)|
|State||Published - Mar 20 1998|
ASJC Scopus subject areas
- Molecular Biology