Characterization of a distinct Na⁺-H⁺ exchanger in basolateral membranes of human jejunum

Kinetically distinct Na⁺-H⁺ exchangers exist on the apical and basolateral membranes of rabbit ileal enterocytes. The apical Na⁺-H⁺ exchanger appears to function in electroneutral NaCl transport, whereas the basolateral Na⁺-H⁺ exchanger may function in homeostatic intravesicular pH (pH(i)) regulation and volume regulation. This study is designed to determine the presence and characteristics of the Na⁺-H⁺ exchanger in basolateral membrane vesicles (BLMV) prepared from jejunal tissues of human organ donors. A well-validated Percoll-gradient technique was used to prepare BLMV. An outwardly directed H⁺ gradient [pH(i)/extravesicular pH (pH(o)) = 5.2/7.5] resulted in a Na⁺ uptake overshoot (1.45 ± 0.21 nmol/mg protein) 2.5-fold above equilibrium values (0.59 ± 0.13 nmol/mg protein). Na⁺ uptake at equilibrium represented transport into an osmotically sensitive intravesicular space as validated by an osmolality study. Na⁺ uptake represented an electroneutral process, as shown by studies in which negative membrane potentials were induced by K⁺ and the ionophore valinomycin. Na⁺ uptake was linear for the first 15 s of transport as depicted by y = 0.042x + 0.002, r² = 0.98. Dixon plot analysis of amiloride sensitivity revealed an ID₅₀ value for amiloride of 29 μM (fourfold lower than ID₅₀ for brush- border Na⁺-H⁺ exchanger). Kinetic studies of amiloride-sensitive Na⁺ uptake revealed a maximal velocity = 1.53 ± 0.19 nmol · mg protein^-1 · 5 s^-1 and Michaelis constant = 9.83 ± 3.5 mM. By varying pH(i) a sigmoidal effect of internal H⁺ on Na⁺ uptake was noted consistent with an internal modifier site for protons. To confirm this finding, the effect of pH(i) on Na⁺ efflux and Na⁺-Na⁺ exchange was studied. Na⁺ efflux and Na⁺-Na⁺ exchange were greater with pH(i) of 6.5 compared with 7.4. This study suggests for the first time the presence of a Na⁺-H⁺ exchanger in human jejunal BLMV that differs in its amiloride sensitivity from the Na⁺-H⁺ exchanger found on human jejunal brush-border membranes. This basolateral- membrane Na⁺-H⁺ exchanger does possess an internal modifier site.