Oxygen-dependent K+ influxes in Mg2+-clamped equine red blood cells

1. Cl--dependent K+ (Rb-86(+)) influxes were measured in oxygenated and deo xygenated equine red blood cells, whose free [Mg2+](i) had been clamped, to examine the effect on O-2 dependency of the K+-Cl- cotransporter. 2. Total [Mg2+](i) was 2.55 +/- 0.07 mM (mean +/- S.E.M., n = 6). Free [Mg2 +](i) was estimated at 0.45 +/- 0.04 and 0.68 +/- 0.03 mM (mean +/- S.E.M., n = 4) in oxygenated and deoxygenated red cells, respectively. 3. K+-Cl- cotransport was minimal in deoxygenated cells but substantial in oxygenated ones. Cl--dependent K+ influx, inhibited by calyculin A, consist ent with mediation via the K+-Cl- cotransporter, was revealed by depleting deoxygenated cells of Mg2+. 4. Decreasing [Mg2+](i) stimulated K+ influx, and increasing [Mg2+](i) inhi bited it, in both oxygenated and deoxygenated red cells. When free [Mg2+](i ) was clamped, Cl--dependent K+ influxes were always greater in oxygenated cells than in deoxygenated ones, and changes in free [Mg2+](i) of the magni tude occurring during oxygenation-deoxygenation cycles had a minimal effect . Physiological fluctuations in free [Mg2+](i) are unlikely to provide the primary link coupling activity of the K+-Cl- cotransporter with O-2 tension . 5. Volume and H+ ion sensitivity of K+ influx in Mg2+-clamped red cells wer e increased in O-2 compared with those in deoxygenated cells at the same fr ee [Mg2+](i), by about 6- and 2-fold, respectively, but again these feature s were not responsible for the higher fluxes in oxygenated cells. 6. Regulation of the K+-Cl- cotransporter by O-2 is very similar in equine, sheep and in normal human (HbA) red cells, but altered in human sickle cel ls. Present results imply that, as in sheep red cells, O-2 dependence of K-Cl- cotransport in equine red cells is not mediated via changes in free [M g2+](i) and that cotransport in Mg2+-clamped red cells is still stimulated by O-2. This behaviour is contrary to that reported for human sickle (HbS) cells.