Effect of phenazine methosulphate on K ⁺ transport in human red cells

The effect of phenazine methosulphate (PMS; 1 mM) on ( ⁸⁶ Rb ⁺ ) K ⁺ transport in human red cells was investigated to ascertain its action on the K ⁺ -Cl ^- cotransporter (KCC; defined as the Cl ^- dependent component of K ⁺ flux measured in the presence of ouabain and bumetanide) and the Ca ²⁺ -activated K ⁺ channel (Gardos channel; defined as the clotrimazole, 5 μM, -sensitive K ⁺ flux). In the presence of Ca ²⁺ , both transport pathways were stimulated but effects were markedly greater under deoxygenated conditions (5-fold for KCC; 20-fold for the Gardos channel). KCC activation was inhibited by prior treatment with calyculin A (100nM), implying action via protein dephosphorylation. Activation of the Gardos channel correlated with 28 ± 3% inhibition of the plasma membrane Ca ²⁺ pump, with maximal activity reduced from 7.7 ± 1.1 to 2. 7 ± 0.7 μmol (l cells.h) ^-1 (all means ± S.E.M. for n = 3), and a 3-fold increase in sensitivity of the channel to Ca ²⁺ (EC ₅₀ reduced from 437 ± 156 to 152 ± 57 nM). Increased availability of NADH in deoxygenated conditions, resulting in increased free radical generation by PMS, may be responsible. We speculate that the similarity of the K ⁺ transport phenotype produced by PMS to that seen in deoxygenated sickle cells is relevant to the pathophysiology of sickle cell disease.