Kinetics of the human lymphocyte Na(+)-H+ exchanger

1. A human lymphocyte preparation, obtained by Percoll gradient centrifugation and free of contaminating monocytes and granulocytes, was used to study the kinetics of the Na(+)-H+ exchanger through activation by nigericin-induced acidic loading. The fluorescent probe biscarboxyethylcarboxyfluorescein (acetoxymethyl ester) was used to determine cell pH and buffering capacity and to measure Na(+)-H+ exchange activity as external Na(+)-dependent H+ efflux. 2. At a cell pH of 6.2, H+ efflux was stimulated by external Na+ with a Km of 30 mmol/l (SEM 6, n = 3) and a calculated Vmax. of 0.73 mmol s-1 l-1 of cell water (SEM 0.06, n = 6). External Na(+)-dependent H+ efflux was more than 98% inhibited and half-maximally inhibited by 200 mumol/l and 17 mumol/l amiloride, respectively. The external pH also inhibited Na(+)-H+ exchange, with a Ki of 93 nmol/l. 3. Na(+)-H+ exchange was sigmoidally activated by an internal pH lower than 7.0 with a Hill coefficient of 2.14 (SEM 0.15, n = 6) and a pK of 6.57 (SEM 0.03, n = 6). Cell buffering capacity was also measured as a function of cell pH and found to gradually increase from 14 to 26 mmol l-1 of cell water pH-1 when cell pH fell below 6.6. The maximal transport rate (cell pH 6.0-6.2) was 0.50 mmol (s l of cell water)-1 (SEM 0.08, n = 12) and ranged between 0.25 and 1.10.(ABSTRACT TRUNCATED AT 250 WORDS)