Flux ratio of valinomycin-mediated K+ fluxes across the human red cell membrane in the presence of the protonophore CCCP

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Abstract
The ratio of valinomycin-mediated unidirectional K+ fluxes across the human red cell membrane, has been determined in the presence of the protonophore carbonylcyanidem-chlorophenylhydrazone, CCCP, using the K+ net efflux and42K influx. The driving force for the net efflux (V m −E K +) has been calculated from the membrane potential, estimated by the CCCP-mediated proton distribution and the Nernst potential for potassium ions across the membrane. An apparent driving potential for the K+ net efflux has been calculated from the K+ flux ratio, determined in experiments where the valinomycin and CCCP concentrations were varied systematically. This apparent driving force, in conjunction with the actual driving force calculated on basis of the CCCP estimated membrane potential, is used to calculate a flux ratio exponent, which represents an estimate of the deviation of valinomycin-mediated K+ transport from unrestricted electrodiffusion, when protonophore is present. In the present work, the flux ratio exponent is found to be 0.90 when the CCCP concentration is 5.0 μm and above, while the exponent decreases to about 0.50 when no CCCP is present. The influence of CCCP upon the rate constants in the valinomycin transport cycle is discussed. The significance of this result is that red cell membrane potentials are overestimated, when calculated from valinomycin-mediated potassium isotope fluxes, using a constant field equation.