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

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Abstract
1 Cl−-dependent K+ (86Rb+) influxes were measured in oxygenated and deoxygenated equine red blood cells, whose free [Mg2+]i had been clamped, to examine the effect on O2 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, consistent 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 inhibited 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 magnitude 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 O2 tension. 5 Volume and H+ ion sensitivity of K+ influx in Mg2+-clamped red cells were increased in O2 compared with those in deoxygenated cells at the same free [Mg2+]i, by about 6- and 2-fold, respectively, but again these features were not responsible for the higher fluxes in oxygenated cells. 6 Regulation of the K+-Cl− cotransporter by O2 is very similar in equine, sheep and in normal human (HbA) red cells, but altered in human sickle cells. Present results imply that, as in sheep red cells, O2 dependence of K+-Cl− cotransport in equine red cells is not mediated via changes in free [Mg2+]i and that cotransport in Mg2+-clamped red cells is still stimulated by O2. This behaviour is contrary to that reported for human sickle (HbS) cells.