IRBIT and LIMA1 associate with and are necessary for epithelial cell SLC26A3 (DRA) stimulation by cAMP/ATP

The epithelial brush border (BB) Cl^-/[Formula: see text] exchanger SLC26A3 [down-regulated in adenoma (DRA)] is part of two separate intestinal transport processes, neutral NaCl absorption (linked to NHE3) and anion secretion (interacting with CFTR). There is a gap in understanding the regulation of DRA in digestive physiology and in the secretory diarrheal diseases, in which there is elevation of cAMP and/or Ca²⁺. The acute stimulatory regulation of DRA in Caco-2 cells by elevated cAMP (forskolin) and Ca²⁺ (ATP) was studied. As previously reported, DRA was maximally stimulated similarly by forskolin, ATP, and their combination at concentrations that alone maximally stimulated DRA (not additive) and also by their combination at concentrations that alone had no effect (called synergistic stimulation). DRA was phosphorylated at baseline on a single amino acid (S⁵⁶³) and this markedly decreased with acute cAMP/ATP stimulation. Immunoprecipitation (IP) of DRA identified two associating proteins, inositol 1,4,5-trisphosphate receptor-binding protein released with inositol 1,4,5-trisphosphate (IRBIT) and LiM domain and actin-binding protein 1 (LIMA1), which were shown involved in the cAMP/Ca²⁺ stimulation. KD of IRBIT1 or LIMA1 reduced the cAMP plus ATP stimulation of DRA but did not alter basal DRA activity. Maximum ATP, but not maximum forskolin stimulation of DRA, was IRBIT dependent. Also, the elevation in intracellular Ca²⁺ by cAMP/ATP was IRBIT dependent. IRBIT and LIMA1 bound DRA under basal conditions, and the amount bound increased with cAMP/ATP stimulation. cAMP/ATP stimulation increased the coprecipitation of LIMA1 with both IRBIT and DRA. With acute stimulation, there was also more BB DRA, IRBIT, but not LIMA1. These results identify a DRA-IRBIT-LIMA1 complex that is involved in acute stimulation of DRA activity.NEW & NOTEWORTHY Insights into acute DRA regulation relevant to secretory diarrhea studied synergistic cAMP and Ca²⁺-induced DRA stimulation. DRA was phosphorylated under baseline conditions, which decreased with acute stimulation. Acute stimulation, but not basal activity, required the presence of both IRBIT and LIMA1 (an actin-binding scaffold), involved more plasma membrane DRA, and also increased DRA association with IRBIT and LIMA1, indicating a role for a DRA-IRBIT-LIMA1 plasma membrane complex in acute DRA stimulation.