ELECTRODIFFUSION, BARRIER, AND GATING ANALYSIS OF DIDS-INSENSITIVE CHLORIDE CONDUCTANCE IN HUMAN RED-BLOOD-CELLS TREATED WITH VALINOMYCIN OR GRAMICIDIN

Current-voltage curves for DIDS-insensitive Cl- conductance have been determined in human red blood cells from five donors. Currents were es timated from the rate of cell shrinkage using flow cytometry and dif f erential laser light scattering. Membrane potentials were estimated fr om the extracellular pH of unbuffered suspensions using the proton ion ophore FCCP. The width of the Gaussian distribution of cell volumes re mained invariant during cell shrinkage, indicating a homogeneous Cl- c onductance among the cells. After pretreatment for 30 min with DIDS, n et effluxes of K+ and Cl- were induced by valinomycin and were measure d in the continued presence of DIDS; inhibition was maximal at similar to 65% above 1 mu M DIDS at both 25 degrees C and 37 degrees C. The n onlinear current-voltage curves for DIDS-insensitive net Cl- effluxes, induced by valinomycin or gramicidin at varied [K+],, were compared w ith predictions based on (1) the theory of electrodiffusion, (2) a sin gle barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes th e relationship between the measured transmembrane voltage and [K+](o). Under our experimental conditions (pH 7.5, 23 degrees C, 1-3 mu M val inomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P-K/P-Cl is 74 +/- 9 with 10 mu M DIDS, correspondi ng to 73% inhibition of P-Cl. Fitting the constant field current-volta ge equation to the measured Cl- currents yields P-Cl = 0.13 h(-1) with DIDS, compared to 0.49 h(-1) without DIDS, in good agreement with mos t previous studies. The inward rectifying DIDS-insensitive Cl- current , however, is inconsistent with electrodiffusion and with certain sing le-occupancy multiple barrier models. The data are well de scribed eit her by a single barrier located near the center of the transmembrane e lectric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 +/- 0.005 S/g H-b , half the channels are open at -27 +/- 2 mV, and the equivalent gatin g charge is -1.2 +/- 0.3.