cAMP stimulates SLC26A3 activity in human colon by a CFTR-dependent mechanism that does not require CFTR activity

Background & Aims: DRA (SLC26A3) is an electroneutral Cl-/HCO3- exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute cAMP-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO3- secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates or does not affect its activity. Methods: This study reevaluated cAMP regulation of DRA using new "tools" including a successful knockout cell model, a specific DRA inhibitor (DRAinh-A250), specific antibodies, and a transport assay that did not rely on non-specific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2. Results: DRA is an apical protein in human proximal colon, differentiated colonoid monolayers and Caco-2 cells. It is glycosylated and appears as two bands. cAMP (forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl-/HCO3- exchanger and is inhibited by DRAinh-A250 with IC50 of 0.5 μmol/L and 0.2 μmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked CFTR. When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTRinh-172. Conclusions: DRA is acutely stimulated by cAMP by a process that is CFTR-dependent but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity.