Kinetics and stoichiometry of the human red cell Na+/H+ exchanger

Summary We have investigated the kinetic properties of the human red blood cell Na⁺/H⁺ exchanger to provide a tool to study the role of genetic, hormonal and environmental factors in its expression as well as its functional properties in several clinical conditions. The present study reports its stoichiometry and the kinetic effects of internal H⁺ (H_i) and external Na⁺ (Na_o) in red blood cells of normal subjects.Red blood cells with different cell Na⁺ (Na_i) and pH (pH_i) were prepared by nystatin and DIDS treatment of acid-loaded cells. Unidirectional and net Na⁺ influx were measured by varying pH_i (from 5.7 to 7.4), external pH (pH_o), Na_i and Na_o and by incubating the cells in media containing ouabain, bumetanide and methazolamide. Net Na⁺ influx (Na_i<2.0 mmol/liter cell, Na_o= 150mm) increased sigmoidally (Hill coefficient 2.5) when pH_i fell below 7.0 and the external pH_o was 8.0, but increased linearly at pH_o 6.0. The net Na⁺ influx driven by an outward H⁺ gradient was estimated from the difference of Na⁺ influx at the two pH_o levels (pH_o 8 and pH_o 6). The H⁺-driven Na⁺ influx reached saturation between pH_i 5.9 and 6.1. TheV_max had a wide interindividual variation (6 to 63 mmol/liter cell · hr, 31.0±3, mean±sem,n=20). TheK_m for H_i to activate H⁺-driven Na⁺ influx was 347±30nm (n=7). Amiloride (1mm) or DMA (20 m) partially (59±10%) inhibited red cell Na⁺/H⁺ exchange. The stoichiometric ratio between H⁺-driven Na⁺ influx and Na⁺-driven H⁺ efflux was 11. The dependence of Na⁺ influx from Na_o was studied at pH_i 6.0, and Na_i lower than 2 mmol/liter cell at pH_o 6.0 and 8.0. The meanK_m for Na_o of the H⁺-gradient-driven Na⁺ influx was 55±7mm.An increase in Na_i from 2 to 20 mmol/liter cell did not change significantly H⁺-driven net Na⁺ influx as estimated from the difference between unidirectional²²Na influx and efflux. Na⁺/Na⁺ exchange was negligible in acid-loaded, DIDS-treated cells. Na⁺ and H⁺ efflux from acid-loaded cells were inhibited by amiloride analogs in the absence of external Na⁺ indicating that they may represent nonspecific effects of these compounds and/or uncoupled transport modes of the Na⁺/H⁺ exchanger.It is concluded that human red cell Na⁺/H⁺ exchange performs 11 exchange of external Na⁺ for internal protons, which is partially amiloride sensitive. Its kinetic dependence from internal H⁺ and external Na⁺ is similar to other cells, but it displays a larger variability in theV_max between individuals.