Direct estimate of 1:1 stoichiometry of K⁺-Cl⁻ cotransport in rabbit erythrocytes

This work was undertaken to obtain a direct measure of the stoichiometry of Na(+)-independent K(+)-Cl(-) cotransport (KCC), with rabbit red blood cells as a model system. To determine whether (86)Rb(+) can be used quantitatively as a tracer for KCC, (86)Rb(+) and K(+) effluxes were measured in parallel after activation of KCC with N-ethylmaleimide (NEM). The rate constant for NEM-stimulated K(+) efflux into isosmotic NaCl was smaller than that for (86)Rb(+) by a factor of 0.68 +/- 0.11 (SD, n = 5). This correction factor was used in all other experiments to calculate the K(+) efflux from the measured (86)Rb(+) efflux. To minimize interference from the anion exchanger, extracellular Cl(-) was replaced with SO, and 4,4'-diisothiocyanothiocyanatodihydrostilbene-2,2'-disulfonic acid was present in the flux media. The membrane potential was clamped near 0 mV with the protonophore 2,4-dinitrophenol. The Cl(-) efflux at 25 degrees C under these conditions is approximately 100,000-fold smaller than the uninhibited Cl(-)/Cl(-) exchange flux and is stimulated approximately 2-fold by NEM. The NEM-stimulated (36)Cl(-) flux is inhibited by okadaic acid and calyculin A, as expected for KCC. The ratio of the NEM-stimulated K(+) to Cl(-) efflux is 1.12 +/- 0.26 (SD, n = 5). We conclude that K(+)-Cl(-) cotransport in rabbit red blood cells has a stoichiometry of 1:1.