Kinetic characterization of a stably expressed novel Na⁺/H⁺ exchanger (NHE-2)

We have recently reported the molecular cloning, sequencing and tissue distribution of a novel Na⁺/H⁺ exchanger (NHE-2). The cDNA for NHE-2 was cloned by screening a rat intestinal cDNA library. This clone was unique due to the fact that it lacks the first two transmembrane domains which are present in the other Na⁺/H⁺ exchanger isoforms (NHE-1, NHE-3, NHE-4). This structural change in the cDNA offered a unique opportunity to study in detail the properties of this stably expressed cDNA in chinese lung fibroblasts that lack the Na⁺/H⁺ exchanger (PS120) cells. Amiloride-sensitive Na⁺ uptake was linear up to 2 min in PS120 cells transfected with the cDNA. Kinetics of the amiloride-sensitive Na⁺ uptake showed a V_max of 24.7 ± 5 nmol/μl ICW per min and a K_m of 33.1 ± 2.0 mM. The inhibitory constant (K_I) for amiloride and its analogue 5-N-_ethyl-N-isopropylamiloride (EIPA) was 0.15 μM and 0.66 μM, respectively. Epidermal growth factor, a known stimulator of NHE-1, also upregulated the expressed NHE-2. These results characterize the kinetic properties of this unique exchanger and suggests that the first two transmembrane domains of the Na⁺/H⁺ exchanger isoforms are not essential for the expression of amiloride-sensitive Na⁺ uptake.