CHLORIDE-BICARBONATE EXCHANGE THROUGH THE HUMAN RED CELL GHOST MEMBRANE MONITORED BY THE FLUORESCENT PROBE, 6-METHOXY-N-(3-SULFOPROPYL)QUINOLINIUM (SPQ)

Teresa M. Calafut¹ and James A. Dix
Department of Chemistry, Box 6016, State University of New York, Binghamton, NY 13902-6016
¹ Present address: College Misericordia, 301 Lake Street, Dallas, PA 18612

Address correspondence to: James A. Dix, Department of Chemistry, Box 6016, State University of New York, Binghamton, NY 13902-6016; Phone: (607) 777-2480, Fax: (607) 777-4478, e-mail dix@binghamton.edu

• Abstract
• Introduction
• Materials and Methods
• Results and Discussion
• Quenching of SQP fluorescence
• Time course of fluorescence quenching
• Hydration and dehydration of CO₂
• Effect of anion transport inhibitors
• Temperature dependence of anion exchange
• Dependence of time course on anion concentration and gradient
• Acknowledgements
• References
• Figures

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ABSTRACT

The exchange of chloride and bicarbonate across the human red cell membrane has been characterized by quenching of an intracellular fluorophore, 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). In 20 mM HEPES, pH 7.4, and varying concentrations of chloride and bicarbonate with total anion concentration of 150 mM, SPQ is quenched dynamically by chloride with an apparent Stern-Volmer quenching constant 0.071 0.016 mM^-1 at 25 °C. HEPES alone quenched SPQ fluorescence with a quenching constant of 0.030 0.003 mM^-1. Stopped-flow kinetic experiments give fluorescence time courses that, when corrected for Stern-Volmer quenching, are first-order. Disulfonic stilbenes (inhibitors of anion exchange) decrease the rate of fluorescence equilibration. Transport of bicarbonate via hydration-dehydration of CO2 does not contribute to the observed kinetics. The chloride-bicarbonate exchange rate is independent of the anion concentration gradient, but increases at 25 °C from 1 s^-1 to 4 s^-1 as equilibrium chloride concentration increases from 20 to 130 mM (with concomitant decrease in bicarbonate concentration from 130 mM to 20 mM). The data indicate that the translocation rate of the chloride-loaded transport protein is greater than that of bicarbonate-loaded transport protein, and that bicarbonate has a higher affinity for the transport protein than chloride. Our results validate the use of SPQ to measure quantitatively chloride-bicarbonate exchange in red cell ghosts. The methods we describe should be applicable to other systems exhibiting chloride-bicarbonate exchange.

[Introduction] [Title Page]