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Am J Physiol Cell Physiol 281: C825-C832, 2001;
0363-6143/01 $5.00
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Vol. 281, Issue 3, C825-C832, September 2001

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

Michael L. Jennings and Mark F. Adame

Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

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 86Rb+ can be used quantitatively as a tracer for KCC, 86Rb+ 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 86Rb+ 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 86Rb+ efflux. To minimize interference from the anion exchanger, extracellular Cl- was replaced with SO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, 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°C under these conditions is ~100,000-fold smaller than the uninhibited Cl-/Cl- exchange flux and is stimulated ~2-fold by NEM. The NEM-stimulated 36Cl- 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.

red blood cells; N-ethylmaleimide; ion regulation


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