Molecular characterization of Slc26a3 and Slc26a6 anion transporters in guinea pig pancreatic duct

Abstract

HCO3 in pancreatic juice arises from the pancreatic duct cells. Secretin stimulates HCO3 secretion via a mechanism that involves activation of the adenylate cyclase pathway, activation of both basolateral K channels and the apical CFTR Cl channel, and stimulation of an apical Slc26mediated Cl/HCO3 exchanger (1). The Slc26 anion exchangers, Slc26a3 and Slc26a6, have both been localized to the apical membrane of human pancreatic duct and mouse pancreatic duct although their functional roles in pancreatic HCO3 secretion remain uncertain. The high rates of pancreatic bicarbonate secretion shared by guinea pig (gp) and human, in contrast to the low rates in mouse, prompted us to study the functional characteristics of gp Slc26 anion transporters. Furthermore, recent data from isolated guinea pig pancreatic duct experiments demonstrated that during stimulated HCO3 secretion an apical Slc26a6-like Cl/HCO3 exchanger is functionally coupled with CFTR (2). This study prompted us to clone cDNAs encoding Slc26a3 and Slc26a6 polypeptides from the gp pancreas and to compare their functional characteristics in Xenopus oocytes with those of their mouse and human orthologs (3-5), and with those of luminal Cl/HCO3 exchange measured in isolated gp pancreatic interlobular ducts (2). Whereas gp Slc26a6 mediated Cl/Cl exchange and bidirectional exchange of Cl for oxalate and HCO3, gp Slc26a6 mediated only sulfate influx but not sulfate efflux in preliminary experiments. Gp Slc26a3 mediated robust Cl/Cl exchange and minimal oxalate efflux in exchange for Cl, but did not mediate detectable influx of oxalate or sulfate. Measurement of intracellular pH by BCECF ratio fluorimetry in HEK cells transfected with Slc26a6 or in Slc26a3-expressing Xenopus oocytes during removal and restoration of Clo in the presence of HCO3 indicated that both gp Slc26a6 and Slc26a3 function as Cl/HCO3 exchangers. These substrate specificity experiments reveal that whereas Slc26a3 functions predominantly as a Cl/HCO3 exchanger, Slc26a6 can exchange a wider range of substrates, functioning as a Cl/HCO3 exchanger or a Cl/anion exchanger. This finding allows for the possibility that Slc26a6 may have alternative roles in transepithelial solute transport in the pancreatic duct. The apparent affinity of gp Slc26a6 for extracellular Cl (K1/2) was 2 mM during Cli/Clo exchange and 30 mM during C-oxalatei/Clo exchange. These experiments reveal that binding and/ or translocation rates of extracellular Cl with respect to gp slc26a6 is/are dependent on the internal transanion substrate and show species-specificity (5). The high affinity for extracellular Clo suggests that even when duct luminal Cl is low, such as might occur with passage of pancreatic juice along the duct lumen, Slc26a6-mediated Cl/HCO3 exchange would still be active. Whereas gp Slc26a6-mediated isotopic flux was moderately sensitive to DNDS (500 μM), niflumic acid (100 μM) and DIDS (500 μM), gp Slc26a3 was minimally sensitive to inhibition by DIDS and DNDS but strongly sensitive to niflumic acid. These pharmacological data, combined with EXPANDED ABSTRACT