Effect of intracellular magnesium and oxygen tension on K ⁺ -Cl ^- cotransport in normal and sickle human red cells

In red cells from normal individuals (HbA cells), the K ⁺ -Cl ^- cotransporter (KCC) is inactivated by low O ₂ tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg ²⁺ ] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg ²⁺ depletion and inhibited by Mg ²⁺ loading but the effect of O ₂ was independent of Mg ²⁺ . At all [Mg ²⁺ ] _i s, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O ₂ was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg ²⁺ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, P _sickle and KCC. Results indicate a role for protein phosphorylation in O ₂ dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb polymerisation, but not on [Mg ²⁺ ] _i .