Oxygen dependence of K+–Cl− cotransport in human red cell ghosts and sickle cells

KCC activity in normal human red cells (containing haemoglobin A, HbA, and termed HbA cells) is O2-dependent, being active in oxygenated cells but inactive in deoxygenated ones. The mechanism for O2 dependence is unknown but a role for Hb has been suggested. In this paper, we address two main questions. First, do membrane ghosts prepared from HbA cells retain an O2-sensitive KCC activity? Second, how is the response of KCC to changes in O2 tension altered in sickle cell patients heterozygous for HbS and HbC? We found that substantial Cl−-dependent K+ influx, indicative of KCC activity, was present in both pink (5–10% normal Hb complement) and white (no measurable Hb) ghosts when equilibrated with air. KCC responded to deoxygenation in pink ghosts only (86±10% inhibition, mean±S.E.M., n=3), whilst KCC activity in white ghosts remained high (23±8% inhibition). Results indicate that pink ghosts retain an O2-dependent KCC activity but that this is lost in white ghosts. Second, HbSC-containing red cells showed sickling (88±3%) when deoxygenated, together with activation of the deoxygenation-induced cation pathway (Psickle) and the Gardos channel. KCC activity, however, was elevated in oxygenated HbSC cells, but inhibited by deoxygenation. Thus Hb polymerisation and sickling could be dissociated from the abnormal response of KCC to deoxygenation observed in HbS-containing red cells. These preparations provide a useful system with which to study the components involved in O2-sensitive membrane transport and why it is perturbed in certain pathological conditions (such as sickle cell disease and oxidant toxicity).