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.
Jennings
M L
ML
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA. JenningsMichaelL@uams.edu
Adame
M F
MF
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000097:blood* / Q000187:drug effects; D002712:Chlorides / Q000097:blood*; D004910:Erythrocyte Membrane / Q000502:physiology*; D004912:Erythrocytes / Q000187:drug effects / Q000502:physiology*; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D011188:Potassium / Q000097:blood*; D011817:Rabbits; D012413:Rubidium / Q000097:blood; D027981:Symporters; D000096922:K Cl- Cotransporters
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002352:Carrier Proteins; D002712:Chlorides; D005033:Ethylmaleimide; D011188:Potassium; D012413:Rubidium; D027981:Symporters; D000096922:K Cl- Cotransporters