IRBIT binds to and functionally enhances the electroneutral Na + ‐coupled bicarbonate transporters NBCn1, NDCBE and NCBE

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IRBIT—a soluble protein released from the IP3 receptor by IP3—binds to and functionally enhances the electrogenic Na/HCO3 cotransporter NBCe1-B (Shirakabe et al., PNAS, 103, 2006), a member of the SLC4 family of HCO3 transporters. Sequence comparisons led us to suspect that IRBIT might also interact with a region in the cytoplasmic N terminus of the electroneutral Na-coupled HCO3 transporters NBCn1, NDCBE, and NCBE, all of which play an important role in regulating neuronal pHi. Using an anti-IRBIT antibody we were able to co-immunoprecipitate all four transporters-as well as the brain specific variant NBCe1-C-from mouse brain lysate, suggesting an in vivo association. We individually expressed NBCn1, NDCBE, or NCBE with a C-terminal EGFP tag (collectively described as NBCnX-EGFP) ± IRBIT with an N-terminal HA-tag (HA-IRBIT) in Xenopus oocytes. While exposing cells to a CO2/HCO3 solution, we monitored the rate of pHi recovery from the CO2-induced acid-load using pH-sensitive microelectrodes. For each NBCnX-EGFP, co-expressing HA-IRBIT doubled the pHi-recovery rate. In addition, HA-IRBIT also doubled the magnitude of the Na conductance associated with NBCn1—evidence that the conductance is intrinsic to the transporter. Our data show that IRBIT is capable of enhancing the activities of all three known electroneutral Na+-coupled HCO3 transporters.