W1654 Increased Transepithelial Anion Current in the SLC26A3 (Down-Regulated in Adenoma, DRA) Knockout Jejunum Is Not Associated with Loss of Electrogenic C −/HCO 3− Exchange

Abstract

Dra is the major apical membrane Cl/HCO3 exchanger in the murine small intestine. Studies of recombinant DRA have proposed an electrogenic 2Cl:1HCO3 stoichiometry (J. Gen. Physiol. 127: 511, 2006). In Dra knockout jejunum (DraKO), loss of Clabsorption is associated with an increase in the baseline short-circuit current (Isc) that may result from loss of an inward anion current due to electrogenic Cl/HCO3 exchange. Therefore, we investigated the ionic basis of the increased Isc in isolated DraKO jejunal preparations using intracellular pH (pHi) microfluorimetry and Ussing chamber studies. As described previously (Gastroenterology 136: 1645, 2008), pHi of the DraKO villous epithelium was alkaline relative to wild type (WT), ostensibly due to loss of Cl/HCO3 exchange. Studies also revealed little Cl-/OHexchange in villous epithelium by measurements performed in the nominal absence of extracellular HCO3. In Ussing chamber studies, removal of extracellular Cleliminated the elevated baseline Isc whereas removal of extracellular HCO3 had no effect in DraKO jejunum, indicating that Cl/HCO3 exchange was not responsible for the elevated Isc. The contribution of the cystic fibrosis transmembrane conductance regulator (Cftr) anion channel was investigated usingDra/Cftr double knockout micewhich demonstrated a baseline Isc that was reduced by 40% relative to the DraKO jejunum. Epithelial alkalinity in the DraKO jejunal epithelium may increase the electrical gradient for Clsecretion by hyperpolarization secondary to activation of basolateral K+ channels (Am. J. Physiol. 258:C336, 1990). Therefore, epithelial acidification was induced by exposing the luminal surface to pH 6.4 by replacing HCO3 with TES buffer in the presence of 5% CO2: 95% O2. Under this condition, the elevated baseline Isc in the DraKO was abolished. On-going studies are investigating the effect of basolateral K+ channel blockers in the DraKO jejunum. We conclude that the elevated baseline Isc in the DraKO intestine does not result from loss of electrogenic Cl-/ HCO3 exchange but from increased anion secretion associated with epithelial alkalinity that may activate basolateral membrane K+ channels. Supported by NIDDK and the Cystic Fibrosis Foundation.