Functional evidence for a pH sensor of erythrocyte K-Cl cotransport through inhibition by internal protons and diethylpyrocarbonate

The sidedness of proton modulation of K-Cl cotransport (K-Cl COT) was studied in low K sheep red blood cells stripped of cellular Mg, Mg,, at alkaline medium pH, pH(o), by the divalent ionophore A23187 and a chelator. This procedure activates K-CI GOT. presumably, by inhibition of MgATP-dependent kinases. Ouabain-resistant K efflux and Rb influx were measured in Cl or NO3 either at variable pH(i) and fixed pH(o), or vice versa. in erythrocytes pH- and volume-clamped with the anion exchange inhibitor 4,4'-diisothiocyanato-2,2'-disulfonic stilbene (DIDS) and sucrose. Between pH(i) 9 and 6, and at constant pH(o) 9, K effluxes decreased hyperbolically in Cl and linearly in NO3 whereas Rb influxes fell almost linearly in Cl and asymptotically in NO3. Thus, saturation of outward and inward K-Cl COT, the calculated difference of the fluxes in Cl and NO3, occurred at slightly different pH(i) values. Hill plots revealed pK(a) values of 6.5 and 7.0, and Hill coefficients of > 1 for outward and inward K-Cl COT. respectively. Raising pH(o), from 6 to 9 at fixed pH(i) slightly increased K and Rb fluxes in both Cl or NO3, but not K-Cl COT. The histidine reagent diethylpyrocarbonate (DEPC) inhibited low Mg-i-activated K-Cl COT at similar to 4 mM, an effect partially reversible by subsequent treatment with hydroxylamine. It is concluded that protons inhibit erythrocyte K-Cl COT through internal histidine(s) which may be part of a pH sensor.