The cDNA encoding the Na+/H+ Exchanger (NHE) from red blood cells of Amphiuma tridactylum was cloned, sequenced and found to be highly homologous to the human housekeeping isoform, NHE1, with 79% identity and 89% similarity at the amino acid level. Full length cDNA of Amphiuma NHE (atNHE1) was transfected into a mutant Chinese hamster ovary cell line (AP-1) which lacks endogenous NHE activity, and the role of the exchanger in pH and volume regulation was evaluated. Intracellular pH measurements were conducted by fluorescent spectrophotometry using the pH-sensitive dye BCECF, and Na+ uptake was determined by radioactive flux assays using 22Na. Non-transfected AP-1 cells displayed minor pH and volume regulation in response to acidification or shrinkage, respectively, which could not be inhibited by the NHE1 inhibitor amiloride. Shrinkage of the atNHE1 transfected AP-1 cells resulted in regulatory volume increase manifested by amiloride-sensitive Na+ influx. The atNHE1 transfectants exhibited robust pH recovery in response to an acid load that was Na+-dependent and strongly inhibited by amiloride. However, these transfectants were relatively insensitive to the potent NHE1 inhibitor HOE694, similar to what we have found in intact Amphiuma red blood cells. This was surprising, given that the transmembrane domain, M9, shown to be important in HOE694 inhibition is identical in atNHE1 and human NHE1 (Orlowski, JBC 271, 19922-19927). To further evaluate this phenomenon, an Amphiuma/Human chimera (AHC) was created that consisted of atNHE1 N-terminus, including all putative transmembrane domains, and human NHE1 C-terminus. When transfected into AP-1 cells, the AHC NHE displayed similar drug sensitivities to atNHE1, again showing modest inhibition by HOE694. Thus, it appears that some other portion of the N-terminal transmembrane domain of the exchanger must be involved in the HOE694 interaction. We are currently in the process of producing additional chimeras of Amphiuma and human NHE's to further evaluate this question.
|Original language||English (US)|
|State||Published - Mar 20 1998|
ASJC Scopus subject areas
- Agricultural and Biological Sciences (miscellaneous)
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology