Expression, functional characterization and tissue distribution of a Na⁺/H⁺ exchanger cloned from Xenopus laevis oocytes (XL-NHE)

Abstract

 We examined the functional properties of a Na⁺/H⁺ exchanger cloned from Xenopus laevis oocytes (XL-NHE) upon stable transfection into PS120 fibroblasts which lack endogenous Na⁺/H⁺ exchange. In contrast to untransfected cells, XL-NHE-transfected cells displayed Na⁺-dependent alkalinization upon acidification with nigericin. XL-NHE activity was inhibited by amiloride, ethylisopropylamiloride, HOE694 [(3-methylsulphonyl-4-piperidinobenzoyl)-guanidine methanesulphonate] and HOE642 [4-isopropyl-3-methylsulphonylbenzoyl)-guanidine methanesulphonate], K _i values being calculated at 5 µmol/l, 25 nmol/l, 300 nmol/l and 180 nmol/l, respectively. The Na⁺ dependence of pH_i recovery was compatible with simple Michaelis–Menten kinetics, the K _m for Na⁺ being 22.0±3.2 mmol/l and the Hill coefficient for Na⁺ being approximately 1. XL-NHE was activated by phorbol ester, whereas forskolin exerted no effect, suggesting the involvement of phospholipase C/protein kinase C signalling pathways rather than protein kinase A signalling pathways in XL-NHE stimulation. Using reverse transcription polymerase chain reaction, XL-NHE message could be detected in various Xenopus tissues including heart, brain, skeletal muscle, reticulocytes, A6-kidney cells and oocytes.