IRBIT functionally enhances the electroneutral Na + ‐coupled bicarbonate transporter NCBE by sequestering an N‐terminal autoinhibitory domain

Abstract

IRBIT binds to and functionally enhances the electrogenic Na-HCO3 cotransporter NBCe1-B (Shirakabe et al., PNAS, 103, 2006). In an accompanying abstract, we further demonstrate that IRBIT functionally enhances the electroneutral Na-coupled HCO3 transporters NBCn1, NDCBE and NCBE. Taking the NCBE/IRBIT pair as our model, we co-expressed NCBE with a C-terminal EGFP tag (NCBE-EGFP) and IRBIT with an N-terminal HA-tag (HA-IRBIT) in Xenopus oocytes. The interaction was confirmed in oocytes by co-immunoprecipitation of HA-IRBIT (but not the S71A mutant known not to bind to NBCe1-B) with an anti-GFP antibody. We assayed the function of NCBE-EGFP by exposing oocytes to a CO2/HCO3 solution and monitoring the rate of the subsequent pHi recovery from the CO2-induced acid-load using pH-sensitive microelectrodes. NCBE-EGFP activity was enhanced by co-expression of HA-IRBIT but not the S71A mutant. Truncating the N terminus of NCBE by removing protein encoded by exons 1 (residues 1-16) or exons 1 and 2 (residues 1-43), affected neither NCBE-EGFP activity nor its enhancement by co-expressed HA-IRBIT. Removing the protein encoded by exons 1, 2 and 3 (residues 1-92) doubled the rate of pHi recovery—compared to full-length NCBE in the absence of co-expressed HA-IRBIT—and negated the enhancement in the presence of HA-IRBIT. Our data suggest that exon 3 of NCBE encodes an autoinhibitory domain (AID), and that IRBIT enhances the activity of NCBE by sequestering the AID.