Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin
| Autor: | Terri S. Novak, Jeffrey C. Freedman |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Erythrocytes
Physiology Ionophore Analytical chemistry membrane potentials Gating 4 4'-Diisothiocyanostilbene-2 2'-Disulfonic Acid In Vitro Techniques Article ion transport Diffusion 03 medical and health sciences Valinomycin chemistry.chemical_compound 0302 clinical medicine Humans 030304 developmental biology band 3 protein Membrane potential 0303 health sciences Erythrocyte Membrane Gramicidin Temperature Conductance Flow Cytometry Anti-Bacterial Agents Electrophysiology chloride channels chemistry DIDS Potassium Chloride channel Ion Channel Gating 030217 neurology & neurosurgery |
| Zdroj: | The Journal of General Physiology |
| ISSN: | 1540-7748 0022-1295 |
| DOI: | 10.1085/jgp.109.2.201 |
| Popis: | Current-voltage curves for DIDS-insensitive Cl 2 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 dif- ferential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered sus- pensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained in- variant during cell shrinkage, indicating a homogeneous Cl 2 conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K 1 and Cl 2 were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at z 65% above 1 m M DIDS at both 25 8 C and 37 8 C. The nonlinear cur- rent-voltage curves for DIDS-insensitive net Cl 2 effluxes, induced by valinomycin or gramicidin at varied (K 1 ) 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 1 ) o . Under our experimental conditions (pH 7.5, 23 8 C, 1-3 m M valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P K /P Cl is 74 6 9 with 10 m M DIDS, corresponding to 73% inhibition of P Cl . Fitting the constant field current-volt- age equation to the measured Cl 2 currents yields P Cl 5 0.13 h 2 1 with DIDS, compared to 0.49 h 2 1 without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl 2 current, however, is in- consistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well de- scribed 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 6 0.005 S/g Hb, half the channels are open at 2 27 6 2 mV, and the equivalent gating charge is 2 1.2 6 0.3. |
| Databáze: | OpenAIRE |
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