Effect of Phenazine Methosulphate on K+ Transport in Human red Cells

Abstract

The effect of phenazine methosulphate (PMS; 1 mM) on (⁸⁶Rb⁺) 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 Ca²⁺-activated K⁺ channel (Gardos channel; defined as the clotrimazole, 5 µM, -sensitive K⁺ flux). In the presence of Ca²⁺, 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 Ca²⁺ 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 Ca²⁺ (EC₅₀ 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.