Abstract
Chloride ions subserve many physiological functions, including regulation of cell volume, intracellular pH, fluid secretion, and stabilization of the resting membrane potential. Cl− is absorbed from the gastrointestinal tract is mediated by Cl−/HCO3 − exchanger. Recent studies have suggested that a major Cl−/HCO3 − exchanger is SLC26A3. Since multiple isoforms of the Cl−/HCO3− exchanger are co-expressed in an intact colonic cell, complicating the functional analysis of an individual isoform, we generated an N-terminal hemagglutinin epitope-tagged human SLC26A3 construct and expressed transiently in CHO cells by using inducible gene expression systems. Using this system, we have previously characterized SLC26A3 by measuring of its activity with fluorescent pH-sensitive indicators, BCECF. To assess the validity of pH measurements, we measured the Cl−/HCO3− exchange activity by using chloride-sensitive dye, MQAE. We first measured Cl−/HCO3− exchange activity by MQAE and then measured its activity by using pH sensitive dye in the same cells. Cl−/HCO3− exchange rate measured by using MQAE was 10-20-fold greater than the rate measured by using BCECF. In addition, a carbonic anhydrase inhibitor acetazolamide partially inhibited Cl−/HCO3− exchange activity. These results suggest that even in the presence of a carbonic anhydrase, its reaction rate is not enough for intracellular pH measurements to assess the Cl−/HCO3− exchange activity in CHO cells. [J Physiol Sci. 2006;56 Suppl:S114]
