Cotransporter NBCe1-B Research Articles

IRBIT activates NBCe1-B by releasing the auto-inhibition module from the transmembrane domain.

Key points The electrogenic Na+/HCO3 −cotransporter NBCe1‐B is widely expressed in many tissues, including pancreas, submandibular gland, brain, heart, etc. NBCe1‐B has very low activity under basal condition due to auto‐inhibition, but can be fully activated by protein interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT).The structural components of the auto‐inhibition domain and the IRBIT‐binding domain of NBCe1‐B are finely characterized based on systematic mutations in the present study and data from previous studies.Reducing negative charges on the cytosol side of the transmembrane domain greatly decreases the magnitude of the auto‐inhibition of NBCe1‐B.We propose that the auto‐inhibition domain functions as a brake module that inactivates NBCe1‐B by binding to, via electrostatic attraction, the transmembrane domain; IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain via competitive binding to the auto‐inhibition domain. The electrogenic Na+/HCO3 − cotransporter NBCe1‐B is widely expressed in many tissues in the body. NBCe1‐B exhibits only basal activity due to the action of the auto‐inhibition domain (AID) in its unique amino‐terminus. However, NBCe1‐B can be activated by interaction with the IP3R‐binding protein released with inositol 1,4,5‐trisphosphate (IRBIT). Here, we investigate the molecular mechanism underlying the auto‐inhibition of NBCe1‐B and its activation by IRBIT. The IRBIT‐binding domain (IBD) of NBCe1‐B spans residues 1−52, essentially consisting of two arms, one negatively charged (residues 1−24) and the other positively charged (residues 40−52). The AID mainly spans residues 40−85, overlapping with the IBD in the positively charged arm. The magnitude of auto‐inhibition of NBCe1‐B is greatly decreased by manipulating the positively charged residues in the AID or by replacing a set of negatively charged residues with neutral ones in the transmembrane domain. The interaction between IRBIT and NBCe1‐B is abolished by mutating a set of negatively charged Asp/Glu residues (to Asn/Gln) plus a set of Ser/Thr residues (to Ala) in the PEST domain of IRBIT. However, this interaction is not affected by replacing the same set of Ser/Thr residues in the PEST domain with Asp. We propose that: (1) the AID, acting as a brake, binds to the transmembrane domain via electrostatic interaction to slow down NBCe1‐B; (2) IRBIT activates NBCe1‐B by releasing the brake from the transmembrane domain.

Activation of mouse NBCe1-B by Xenopus laevis and mouse IRBITs: Role of the variable Nt appendage of IRBITs

The IP3 receptor binding protein released with inositol 1,4,5-trisphosphate (IRBIT) plays important roles in the regulation of intracellular Ca2+ signaling and intracellular pH. The mammals express two IRBIT paralogs, i.e., IRBIT1 (encoded by AHCYL1) and IRBIT2 (encoded by AHCYL2). The clawed frog Xenopus laevis oocyte is widely used for biophysical studies on ion channels and transporters. It remains unknown whether endogenous IRBIT is expressed in Xenopus oocytes. Here, we cloned from frog oocyte irbit2.L and irbit2.S, orthologs of mammalian IRBIT2. When over-expressed, the frog IRBITs powerfully stimulate the electrogenic Na+/HCO3– cotransporter NBCe1-B as mouse IRBIT2-V2 does. Expression of an isolated Nt fragment of NBCe1-B containing the IRBIT-binding domain greatly decreases NBCe1-B activity in oocytes, suggesting that the basal activity of NBCe1-B contains a large component derived from the stimulation by endogenous frog IRBIT. The frog IRBITs are highly homologous to the mammalian ones in the carboxyl-terminal region, but varies greatly in the amino-terminal (Nt) appendage. Interestingly, truncation study showed that the Nt appendage of IRBIT1 and the long Nt appendage of IRBIT2-V2 modestly enhance, whereas the short Nt appendage of IRBIT2-V4 greatly inhibits the functional interaction between IRBIT and NBCe1-B. Finally, Ala-substitution of Ser68, a key phosphorylation site in the PEST domain of IRBIT, causes distinct functional consequences depending on the structural context of the Nt appendage in different IRBIT isoforms. We conclude that the Nt appendage of IRBITs is not necessary for, but plays an important regulatory role in the functional interaction between IRBIT and NBCe1-B.

Expression, Localization, and Regulation of the Sodium Bicarbonate Cotransporter NBCe1 in the Thyroid.

The intrafollicular space of thyroid follicles is the storage compartment for thyroid hormones. Its pH has been established at around 7.6 at least after thyrotropin (TSH) stimulation. This alkaline intrafollicular pH is thought to be critical for iodide coupling to thyroglobulin and internalization of iodinated thyroglobulin. At least in mice, this alkalinization requires the expression of pendrin (Slc26a4) within the apical membrane, and a lack of pendrin results in acidic follicular lumen pH. Yet, the mechanism importing HCO3- into the cytoplasm is unknown. This study investigated whether the rather ubiquitous sodium bicarbonate cotransporter NBCe1 (SLC4A4) might play this role. It also examined which variant was expressed and where it was localized in both rat and human thyroid tissue. Lastly, the dependence of its expression on TSH was studied. Reverse transcription polymerase chain reaction, immunofluorescence, and Western blotting were used to test whether TSH stimulated NBCe1 protein expression in vivo. Subcellular localization of NBCe1 was performed using immunofluorescence in both rat and human thyroid. Cultured thyroid cells were also used to attempt to define how TSH affects NBCe1 expression. Only transcripts of the NBCe1-B variant were detected in both rat and human thyroid. Of interest, NBCe1-C was not detected in human tissues, not even in the brain. On immunofluorescence microscopy, the immunostaining of NBCe1 mainly appeared in the basolateral membrane upon stimulation with TSH. This TSH induction of basolateral membrane expression of NBCe1 protein was confirmed in vivo in rat thyroid and in vitro on human thyroid slices. This study demonstrates the expression of the sodium bicarbonate cotransporter NBCe1-B in rat and human thyroid. Additionally, the data suggest that TSH blocks the degradation of NBCe1 protein by trafficking it to the basolateral membrane. Hence, TSH increases NBCe1 half-life without increasing its synthesis.

IRBIT Interacts with the Catalytic Core of Phosphatidylinositol Phosphate Kinase Type Iα and IIα through Conserved Catalytic Aspartate Residues.

Phosphatidylinositol phosphate kinases (PIPKs) are lipid kinases that generate phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a critical lipid signaling molecule that regulates diverse cellular functions, including the activities of membrane channels and transporters. IRBIT (IP3 R-binding protein released with inositol 1,4,5-trisphosphate) is a multifunctional protein that regulates diverse target proteins. Here, we report that IRBIT forms signaling complexes with members of the PIPK family. IRBIT bound to all PIPK isoforms in heterologous expression systems and specifically interacted with PIPK type Iα (PIPKIα) and type IIα (PIPKIIα) in mouse cerebellum. Site-directed mutagenesis revealed that two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are involved in the interaction with IRBIT. Furthermore, phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfered with the interaction, suggesting that IRBIT interacts with catalytic cores of PIPKs. Mutations of phosphorylation sites in the serine-rich region of IRBIT affected the selectivity of its interaction with PIPKIα and PIPKIIα. The structural flexibility of the serine-rich region, located in the intrinsically disordered protein region, is assumed to underlie the mechanism of this interaction. Furthermore, in vitro binding experiments and immunocytochemistry suggest that IRBIT and PIPKIα interact with the Na+/HCO3 − cotransporter NBCe1-B. These results suggest that IRBIT forms signaling complexes with PIPKIα and NBCe1-B, whose activity is regulated by PI(4,5)P2.

AHCYL2 (long-IRBIT) as a potential regulator of the electrogenic Na+-HCO3− cotransporter NBCe1-B

Although AHCYL2 (long-IRBIT) is highly homologous to IRBIT, which regulates ion-transporting proteins including the electrogenic Na+-HCO3− cotransporter NBCe1-B, its functions are poorly understood. Here, we found that AHCYL2 interacts with NBCe1-B in bovine parotid acinar cells using yeast two-hybrid, immunofluorescence confocal microscopy and co-immunoprecipitation analyses. Whole-cell patch-clamp experiments revealed that co-expression of AHCYL2 reduces the apparent affinity for intracellular Mg2+ in inhibition of NBCe1-B currents specifically in a HCO3−-deficient cellular condition. Our data unveil AHCYL2 as a potential regulator of NBCe1-B in mammalian cells. We propose that cytosolic ionic condition appropriate for AHCYL2 to function might be different from IRBIT.

Mechanism and synergism in epithelial fluid and electrolyte secretion

A central function of epithelia is the control of the volume and electrolyte composition of bodily fluids through vectorial transport of electrolytes and the obligatory H2O. In exocrine glands, fluid and electrolyte secretion is carried out by both acinar and duct cells, with the portion of fluid secreted by each cell type varying among glands. All acinar cells secrete isotonic, plasma-like fluid, while the duct determines the final electrolyte composition of the fluid by absorbing most of the Cl(-) and secreting HCO3 (-). The key transporters mediating acinar fluid and electrolyte secretion are the basolateral Na(+)/K(+) /2Cl(-) cotransporter, the luminal Ca(2+)-activated Cl(-) channel ANO1 and basolateral and luminal Ca(2+)-activated K(+) channels. Ductal fluid and HCO3 (-) secretion are mediated by the basolateral membrane Na(+)-HCO3 (-) cotransporter NBCe1-B and the luminal membrane Cl(-)/HCO3 (-) exchanger slc26a6 and the Cl(-) channel CFTR. The function of the transporters is regulated by multiple inputs, which in the duct include major regulation by the WNK/SPAK pathway that inhibit secretion and the IRBIT/PP1 pathway that antagonize the effects of the WNK/SPAK pathway to both stimulate and coordinate the secretion. The function of these regulatory pathways in secretory glands acinar cells is yet to be examined. An important concept in biology is synergism among signaling pathways to generate the final physiological response that ensures regulation with high fidelity and guards against cell toxicity. While synergism is observed in all epithelial functions, the molecular mechanism mediating the synergism is not known. Recent work reveals a central role for IRBIT as a third messenger that integrates and synergizes the function of the Ca(2+) and cAMP signaling pathways in activation of epithelial fluid and electrolyte secretion. These concepts are discussed in this review using secretion by the pancreatic and salivary gland ducts as model systems.

Convergence of IRBIT, phosphatidylinositol (4,5) bisphosphate, and WNK/SPAK kinases in regulation of the Na + -HCO 3 − cotransporters family

Fluid and HCO3(-) secretion is a vital function of secretory epithelia, involving basolateral HCO3(-) entry through the Na(+)-HCO3(-) cotransporter (NBC) NBCe1-B, and luminal HCO3(-) exit mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and solute carrier family 26 (SLC26) Cl(-)/HCO3(-) exchangers. HCO3(-) secretion is highly regulated, with the WNK/SPAK kinase pathway setting the resting state and the IRBIT/PP1 pathway setting the stimulated state. However, we know little about the relationships between the WNK/SPAK and IRBIT/PP1 sites in the regulation of the transporters. The first 85 N-terminal amino acids of NBCe1-B function as an autoinhibitory domain. Here we have identified a positively charged module within NBCe1-B(37-65) that is conserved in NBCn1-A and all 20 members of the NBC superfamily except NBCe1-A. This module is required for the interaction and activation of NBCe1-B and NBCn1-A by IRBIT and their regulation by phosphatidylinositol 4,5-bisphosphate (PIP2). Activation of the transporters by IRBIT and PIP2 is nonadditive but complementary. Phosphorylation of Ser65 mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 is required for regulation by IRBIT and SPAK. Sequence searches using the NBCe1-B regulatory module as a template identified a homologous sequence in the CFTR R domain and Slc26a6 sulfat transporter and antisigma factor antagonist (STAS) domain. Accordingly, the R and STAS domains bind IRBIT, and the R domain is required for activation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in a conserved domain of the NBC that may be present in other HCO3(-) transporters and thus in the regulation of epithelial fluid and HCO3(-) secretion.

Regulation of NBCe1-B by IRBIT, PIP2 and the WNK/SPAK Kinases

The electrogenic Na+-HCO3− co-transporter NBCe1-B plays a crucial role in pHin regulation and epithelial HCO3− secretion by mediating basolateral HCO3− entry, when the luminal HCO3− exit mediated by CFTR and members of the SLC26 transporters.The regulation of NBCe1-B is poorly understood. IRBIT, (inositol 1,4,5-trisphosphate receptor binding protein released with IP3), binds to N terminus of NBCe1-B to markedly increase its activity, while the WNK/SPAK pathway setting the resting state by reducing surface expression of NBCe1-B. NBCe1-B may also be regulated by PIP2 interacting with highly charge module in the N terminus. The site and mechanisms for the regulation by IRBIT, PIP2, and SPAK and the relationship between them are not known.We identified a positive clustered module in NBCe1-B and three arginines within required for regulation by IRBIT and PIP2. The effect of IRBIT and PIP2 are not additive but complementary. The module contains two critical phosphorylation sites. The constitutively cAMP phosphorylated Thr49 is required for all form of NBCe1-B regulations, while Ser65 mediates the function of SPAK. The module is conserved in most NBC superfamily and have the same roles in NBCn1-A. These findings suggest the sites of regulation on NBCe1-B by IRBIT/PIP2 and SPAK and provide a molecular mechanism by which these regulatory factors converge to coordinate epithelia HCO3− secretion.

IRBIT reduces the apparent affinity for intracellular Mg2+ in inhibition of the electrogenic Na+-HCO3− cotransporter NBCe1-B

The electrogenic Na+-HCO3− cotransporter NBCe1-B can be regulated by intracellular Mg2+ (Mg2+i). We previously reported that under whole-cell voltage-clamp conditions, bovine NBCe1-B (bNBCe1-B) currents heterologously expressed in mammalian cells are strongly inhibited by Mg2+i, and the inhibition is likely mediated by electrostatic interaction and relieved by truncation of the cytosolic NBCe1-B specific N-terminal region. Intriguingly, NBCe1-B-like currents natively expressed in bovine parotid acinar (BPA) cells are much less sensitive to Mg2+i inhibition than bNBCe1-B currents. Here, we hypothesized that this apparent discrepancy may involve IRBIT, a previously identified NBCe1-B-interacting protein. RT-PCR, Western blot and immunofluorescence confocal microscopy revealed that IRBIT was not only expressed in the cytosol, but also colocalized with NBCe1-B in the region of plasma membranes of BPA cells. IRBIT was coimmunoprecipitated with NBCe1-B by an anti-NBCe1 antibody in bovine parotid cell lysate. Whole-cell patch-clamp experiments showed that coexpression of IRBIT lowered the Mg2+i sensitivity of bNBCe1-B currents stably expressed in HEK293 cells. Collectively, these results suggest that IRBIT may reduce the apparent affinity for Mg2+i in inhibition of NBCe1-B activity in mammalian cells.

Corrigendum

CorrigendaCorrigendumPublished Online:15 Mar 2012https://doi.org/10.1152/ajpcell.zh0-6921-corr.2012Original articleMoreSectionsPDF (30 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations Volume 302, February 2012Volume 71, February 2012Lee SK, Boron WF, Parker MD. Am J Physiol Cell Physiol 302: , 2012. First published October 19, 2011; doi:10.1152/ajpcell.00352.2011 (http://ajpcell.physiology.org/content/302/3/C518).—The title of the article was printed incorrectly and should read as follows: “Relief of autoinhibition of the electrogenic Na-HCO3 cotransporter NBCe1-B: role of IRBIT vs. amino-terminal truncation.” The title has been corrected online.This article has no references to display. Download PDF Previous Back to Top FiguresReferencesRelatedInformation Related ArticlesRelief of autoinhibition of the electrogenic Na-HCO3 cotransporter NBCe1-B: role of IRBIT vs. amino-terminal truncation 01 Feb 2012American Journal of Physiology-Cell Physiology More from this issue > Volume 302Issue 6March 2012Pages C941-C941 Copyright & PermissionsCopyright © 2012 the American Physiological Societyhttps://doi.org/10.1152/ajpcell.zh0-6921-corr.2012History Published online 15 March 2012 Published in print 15 March 2012 Metrics

The electrogenic [formula omitted] cotransporter NBCe1-B is regulated by intracellular Mg2+

NBCe1-B, a major splice variant of the electrogenic Na+–HCO3- cotransporter (NBCe1) fulfills basic cellular functions including regulation of intracellular pH and epithelial HCO3- secretion. However, its cellular regulatory mechanism still remains elusive. Here, we provide evidence for the first time that NBCe1-B activity can be controlled by intracellular Mg2+ (Mg2+i), the physiologically most abundant intracellular divalent cation. Using the whole-cell patch–clamp technique, we found that recombinant NBCe1-B currents expressed in HEK293 and NIH3T3 cells were inhibited voltage-independently by Mg2+i in a concentration-dependent manner (Ki≈0.01mM). The Mg2+i inhibition was partially relieved by truncation of the NBCe1-B specific N-terminal region (Ki≈0.3mM), and was also observed for native electrogenic Na+–HCO3- cotransporter current in bovine parotid acinar cells that endogenously express NBCe1-B (Ki≈1mM). These results suggest that Mg2+ may be a cytosolic factor that limits intrinsic cotransport activity of NBCe1-B in mammalian cells.

Adaptive redistribution of NBCe1-A and NBCe1-B in rat kidney proximal tubule and striated ducts of salivary glands during acid-base disturbances

The cellular distribution of the NH2-terminal electrogenic Na+-HCO3(-) cotransporter (NBCe1) variants NBCe1-A and NBCe1-B has been investigated in rat kidney and submandibular gland (SMG) under physiological conditions and after systemic acid-base perturbations. Moreover, the in vivo data were complemented in vitro by using an immortalized cell line derived from the S1 segment of the proximal tubule (PT) of normotensive Wistar-Kyoto rats (WKPT-0293 Cl.2). NBCe1-A was basolaterally localized in PT cells, whereas NBCe1-B exhibited intracellular and basolateral distribution. SMG showed transcript and protein expression for NBCe1-A and NBCe1-B. NBCe1-B was basolaterally localized in duct cells; NBCe1-A was found intracellularly in salivary striated ducts and apically in main duct cells. Acute metabolic acidosis significantly increased cells that showed basolateral NBCe1-A in the PT, indicating increased HCO3(-) reabsorption, and significantly decreased cells that exhibited basolateral NBCe1-B in the salivary ducts, suggesting decreased HCO3(-) secretion. Chronic acidosis had no effect on NBCe1 distribution in PT but significantly increased the percentage of cells with basolateral NBCe1-A in salivary striated duct cells, suggesting increased HCO3(-) reabsorption. In contrast, chronic alkalosis caused adaptive redistribution of NBCe1-A and NBCe1-B in renal PT, favoring decreased HCO3(-) reabsorption. In vitro, WKPT-0293 Cl.2 cells expressed key acid-base transporters. Extracellular alkalosis downregulated NBCe1-A protein. WKPT-0293 Cl.2 cells are therefore a useful model to study renal acid-base regulation in vitro. The results propose redistribution of the transporters as a potential posttranslational regulation modus during acid-base disturbances. Moreover, the data demonstrate that renal PT and salivary duct epithelia respond to acid-base disturbances by an opposite redistribution pattern for NBCe1-A and NBCe1-B, reflecting specialized functions as the HCO3(-)-reabsorbing and HCO3(-)-secreting epithelium, respectively.

Distinct expression and subcellular localization patterns of Na +/HCO 3− cotransporter (SLC 4A4) variants NBCe1-A and NBCe1-B in mouse brain

The electrogenic Na +/HCO 3 − cotransporter (NBCe1) has been identified as a key player for regulation of intracellular pH in several cell types. The present study was undertaken to determine expression and subcellular localization of the NH 2-terminal solute carrier (SLC) 4A4 variants NBCe1-A and NBCe1-B in mouse brain using variant-specific antibodies by immunohistochemistry and immunoelectron microscopy. In addition, distribution of NBCe1 variants and activity-dependent regulation was investigated in mouse embryonic day 17.5 (E17.5) hippocampal primary cultures in vitro. The results showed NBCe1-A and NBCe1-B transcript expression in the mouse olfactory bulb, cerebral cortex, hippocampus and cerebellum. NBCe1-A was predominantly expressed in Purkinje cells, granule cells of the dentate gyrus, non-pyramidal cell bodies in cerebral cortex, and in periglomerular and mitral cells in the olfactory bulb. Pyramidal neurons in cerebral cortex and apical cell dendrites in the hippocampus were stained for both NBCe1-A and NBCe1-B. Moreover, NBCe1-B was present in Bergmann glia. At the ultrastructural level, NBCe1-B was preferentially expressed in perivascular astroglial lamellae, whereas both NBCe1 NH 2-terminal variants were localized in pre- and postsynaptic compartments. Except for the olfactory bulb, NBCe1-A was always colocalized with calbindin. Treatment of E17.5 primary hippocampal cultures with KCl, showed dramatic downregulation of NBCe1-B mRNA and protein after 60 min, whereas NBCe1-A expression remained unchanged. These data demonstrate for the first time distinct cellular distribution of NBCe1 NH 2-terminal variants in mouse brain. NBCe1 may be involved in neuronal modulation, and pH regulation during neuronal activity.

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

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.

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

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.