Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Abstract

Current-voltage curves for DIDS-insensitive Cl- conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous C1- conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K+ and Cl- were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at approximately 65% above 1 microM DIDS at both 25 degrees C and 37 degrees C. The nonlinear current-voltage curves for DIDS-insensitive net Cl- effluxes, induced by valinomycin or gramicidin at varied [K+] o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K+]o. Under our experimental conditions (pH 7.5, 23 degrees C, 1-3 microM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio PK/PCl is 74 +/- 9 with 10 microM DIDS, corresponding to 73% inhibition of PCl. Fitting the constant field current-voltage equation to the measured Cl- currents yields PCl = 0.13 h-1 with DIDS, compared to 0.49 h-1 without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl- current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 +/- 0.005 S/g Hb, half the channels are open at -27 +/- 2 mV, and the equivalent gating charge is -1.2 +/- 0.3.