Kinetic comparison of ouabain-resistant K:Cl fluxes (K:Cl [Co]-transport) stimulated in sheep erythrocytes by membrane thiol oxidation and alkylation

Abstract

The stimulatory effects of two thiol (SH) group oxidants, methylmethane thiosulfonate (MMTS) and diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide), on the kinetics of ouabain-resistant (OR) K:Cl [co]-transport in low K (LK) sheep red blood cells were compared with the effects of alkylating agents, notably N-ethylmaleimide (NEM). At low concentrations, both MMTS and diamide stimulated K:Cl [co]-transport, and with a latency period, as measured by OR zero-trans K efflux and OR uptake of external Rb, Rbo, as K congener in Cl and NO3 media. At high concentrations the effect of diamide saturated, and that of MMTS disappeared. The stimulatory effect of MMTS was partially reversed by the reducing agent dithiothreitol (DTT) known to fully restore the diamide-activated K flux (Lauf, J. Memb. Biol. 101:179-188, 1988). In diamide preequilibrated LK sheep red cells, the Km of K:Cl [co]-transport for external Cl, Clo, was 84.3 mM, and 18.7 mM for Rbo, with nearly identical Vmax values around 4 mmol Rb/L cells x h for K (Rb) fluxes in Cl and after correction for the small Cl-independent component. Zero net K (Rb) flux existed at Kc (cell K)/Rbo concentration ratios, [K]c/[Rb]c, of 0.8 i.e. when the electrochemical driving forces across the membrane were about equal. The measured K efflux/Rb influx ratios were almost twice those predicted from [K]c/[Rb]o and the Cl equilibrium potential suggesting that the diamide-stimulated K (Rb) flux may occur through non-diffusional, carrier-mediated transport.(ABSTRACT TRUNCATED AT 250 WORDS)