Parsing apical oxalate exchange in Caco-2BBe1 monolayers: siRNA knockdown of SLC26A6 reveals the role and properties of PAT-1

Freel RW, Morozumi M, Hatch M. Parsing apical oxalate exchange in Caco-2BBe1 monolayers: siRNA knockdown of SLC26A6 reveals the role and properties of PAT-1. Am J Physiol Gastrointest Liver Physiol 297: G918-G929, 2009. First published August 27, 2009; doi:10.1152/ajpgi.00251.2009.-The purpose of this investigation was to quantitate the contribution of the anion exchanger PAT-1 (putative anion transporter-1), encoded by SLC26A6, to oxalate transport in a model intestinal epithelium and to discern some characteristics of this exchanger expressed in its native environment. Control (Con) Caco-2 BBe1 monolayers, 6-8 days postseeding, were compared with those transfected with a small interfering RNA targeted to SLC26A6 (A6KD). Radiotracer and Ussing chamber techniques were used to determine the transepithelial unidirectional fluxes of Ox(2-), Cl-, and SO42- whereas fluorometric/BCECF measurements of intracellular pH were used to assess HCO3- exchange. PAT-1 was functionally targeted to the apical membrane, and SLC26A6 knockdown reduced PAT-1 protein (> 60%) and mRNA (> 75%) expression in A6KD. No net flux of Ox(2-), Cl-, or SO42- was detected in Con or A6KD monolayers, yet the unidirectional fluxes in A6KD were reduced 50, 35, and 15%, respectively. Cl--dependent HCO3- efflux from A6KD was reduced 50% compared with Con. The difference between Con and A6KD properties represents that mediated solely by PAT-1, and by this approach we found that PAT-1-mediated oxalate influx and efflux are inhibited equally by mucosal DIDS (EC50 similar to 5 mu M) and that mucosal Cl- inhibits oxalate uptake with an EC50 < 20 mM. Transepithelial Cl- gradients supported large, DIDS-sensitive net absorptive or secretory fluxes of oxalate in a direction opposite that of the imposed Cl- gradient. The overall symmetry of PAT-1-mediated oxalate exchange suggests that vectorial oxalate transport observed in vivo is principally dependent on the magnitude and direction of counterion gradients.