Reduced surface pH and upregulated AE2 anion exchange in SLC26A3-deleted polarized intestinal epithelial cells

Abstract

Loss of function mutations in the SLC26A3 gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question, SLC26A3 was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pH_i and apical and basolateral Cl^-/HCO₃^- exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl^-/HCO₃^- exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pH_i was not significantly different from that of WT cells, but basolateral Cl^-/HCO₃^- exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl^-/HCO₃^- exchange rate and apical surface pH, but sustained a normal steady-state pH_i due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide.NEW & NOTEWORTHY SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl^-/HCO₃^- exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.

Keywords: Caco2BBe; alkalosis; bicarbonate secretion; chloride-losing diarrhea; intestine; pHi-regulation.