Influence of Haemoglobin Conformation, Nitrite and Eicosanoids on K+ Transport Across the Carp Red Blood Cell Membrane

The regulation of K⁺ transport across the red blood cell (RBC) membrane by haemoglobin (Hb) conformation was studied in carp, and the K⁺ transport mechanisms were identified. When a large proportion of Hb in the R quaternary structure was secured by oxygenation of blood at pH8.14, a net RBC K⁺ efflux was induced, which was accompanied by RBC shrinkage. This K⁺ efflux was resistant to ouabain and inhibited by furosemide and DIDS and by substitution of NO₃⁻ for Cl⁻, showing it to result from a K⁺/Cl⁻ cotransport mechanism. Deoxygenation of the RBCs (Hb in T structure) eliminated the Cl⁻-dependent K⁺ efflux and resulted in a net K⁺ uptake via the Na⁺/K⁺ pump. These changes were fully reversible. Nitrite-induced methaemoglobin formation in deoxygenated blood, which converts a large fraction of the T structure Hb into an R-like conformation, shifted the K⁺ uptake to a Cl⁻-dependent K⁺ efflux similar to that seen in oxygenated cells. When the allosteric equilibrium between the R and T structures of Hb was gradually shifted towards the T state by decreases in pH, the Cl⁻-dependent K⁺ efflux from oxygenated cells decreased. At pH7.52, where the Root effect caused a potent stabilisation of the T state, the K⁺ efflux was reversed to a net K⁺ uptake. A similar change was induced in methaemoglobin-containing deoxygenated blood, since low pH also favours a T-like conformation of metHb. The variable K⁺ fluxes could not be related to changes in membrane potential or pH but were always directly related to the experimental modulation of the relative proportions of R- and T-structure Hb. It is proposed that Hb conformation governs K⁺ movements via a different binding of T and R structures to integral membrane proteins, and that a large fraction of R-structure Hb triggers the Cl⁻dependent K⁺ efflux mechanism. Application of inhibitors and a substrate of prostaglandin and leukotriene synthesis did not influence the K⁺ efflux from oxygenated erythrocytes. However, a fraction of the K⁺ efflux from nitrite-treated deoxygenated cells was inhibited by nordihydroguaiaretic acid, suggesting that a slightly larger K⁺ efflux from these RBCs than from oxygenated RBCs was related to leukotriene production caused by nitrite entry. A much larger influx of nitrite to deoxygenated than to oxygenated RBCs was positively correlated with the distribution ratio of H⁺ and the membrane potential, supporting the view that nitrite primarily enters the cells via conductive transport. The physiological implications of the results are discussed.