Hormones and neurotransmitters regulate cell functions by binding to their receptors, which activate intracellular signaling and produce the physiological response.¹ There are several intracellular pathways, including but not limited to, leading to the activation in protein kinases or phosphatases and increase of intracellular calcium (Ca²⁺).¹ Calcium signaling controls many cellular responses (e.g. exocytosiss, cell spreading, muscle contraction). Therefore it is important to understand the mechanisms regulating intracellular Ca²⁺. Two major sources exist to raise intracellular ca²⁺: extracellular medium and internal stores (e.g., endoplasmic reticulum, mitochondria). While Ca²⁺ channels at the plasma membrane regulate Ca²⁺ entry in the cell, the inositol-1,4,5-trisphosphate receptor (IP₃R) controls the release of Ca²⁺ from the endoplasmic reticulum.^2,3 Many proteins are known to bind to the IP₃R and regulate Ca²⁺ release.^3,4 These include IRBIT (inositol-1,4,5-trisphosphate (IP₃) receptors binding protein released with IP₃), a protein discovered by Mikoshiba’s group.^4,5 Over the past decade IRBIT has emerged not only as an IP₃R regulator but also as an important regulator of other cellular functions.

The IP₃R interacts with specific proteins (e.g. Huntingtin, RACK1) and forms a macrocomplex that is believed to provide the diversity and specificity of the Ca²⁺signaling.⁴ IRBIT was discovered while searching for novel binding partners of the IP₃R.⁵ IRBIT negatively regulates the IP₃R by competing with IP₃.⁶ IRBIT interaction with the IP₃R (thus regulation of IP₃ binding to its receptor) is decreased when IRBIT is phosphorylated as protein phosphatase (e.g. protein phosphatase 1) lowers IRBIT affinity to the IP₃R. Furthermore, phosphorylation of IRBIT’s serine 68 plays a critical role in IRBIT inhibiting IP₃ from binding to its receptor.^6,7 In addition to the phosphorylation state of IRBIT, two motifs (PEST and PDZ) were identified by in-silico analysis.⁸ Pull-down assays with IRBIT missing the PEST or PDZ motif demonstrated that these two motifs are important for the binding of IRBIT to the IP₃R.⁸

As pointed above, IRBIT is released from the IP₃R after IP₃ binds to its receptor. Thus Mikoshiba’s group hypothesized that IRBIT could be a new signaling molecule that interacts with downstream targets. By screening proteins that can bind to IRBIT they found that IRBIT interacts with the electrogenic sodium bicarbonate, NBCe1-B variant (aka pancreatic isoform). Using a heterologous system to co-express IRBIT and NBCe1-B, they showed by electrophysiological measurements that the transport activity of NBCe1-B is substantially increased in the presence of IRBIT.⁹ Another study demonstrated that IRBIT increases NBCe1-B activity by masking in part the auto inhibitory domain of NBCe1-B.¹⁰ These experiments suggest that IRBIT by increasing the activity of NBCe1-B, plays an important role in acid-base homeostasis.

The Na/H exchanger 3 (NHE3) participates in sodium (Na⁺) reabsorption in the kidney. Angiotensin II (a hormone elevating Ca²⁺ via IP₃) increases Na⁺ reabsorption by stimulating NHE3 in the kidney,¹¹ but the mechanisms by which angiotensin II activates NHE3 are not clear. Using a yeast two-hybrid system He et al. found that IBIRT interacts with NHE3.¹² In addition, knocking down IRBIT abrogates the stimulatory effect of Ca²⁺ and angiotensin II on the activity of NHE3.^12,13 These studies provide new ways of better understanding how angiotensin II,via the Ca²⁺ signaling stimulates Na⁺ reabsorption in the kidney.

In the pancreatic duct, fluid secretion is driven by an active transcellular secretion of bicarbonate (HCO–3). The basolateral NBCe1-B pumps HCO^–₃ inside the cell while the apical cystic fibrosis transmembrane regulator (CFTR) and Cl/HCO₃exchanger SLC26a6 secrete HCO^–₃ in the pancreatic duct lumen. In addition to NBCe1-B, it was found that IRBIT binds to CFTR and SLC26a6 and increases transport activity of both transporters. The authors proposed that IRBIT coordinates the activity of basolateral and apical transporters involved in HCO–3-mediated fluid secretion.^14,15 The results from these studies describe a new process allowing the crosstalk between the apical and basolateral membrane.

Finally, IRBIT has also been reported to bind with the Na-K-2Cl cotransporter 1 (NKCC1) in human T84 cells . Silencing IRBIT in this model decreased the cAMP-stimulated Cl⁻ secretion presumably by decreasing the activity of NKCC1.¹⁶

In secretory epithelia, Ca²⁺ and cAMP signaling pathways control secretion. Both pathways can have an additive or synergistic action.^17–19 For a more detailed discussion on how intracellular Ca²⁺ potentiates the cAMP pathway and vice versa and synergistic effect of Ca²⁺ and cAMP we refer the reader to the review by Ahuja et al.¹⁹ To date, the mechanistic details behind the synergistic action of Ca²⁺ and cAMP remain elusive, but work in the pancreatic duct and the salivary glands has shown that IRBIT can mediate the synergistic activation of the CFTR and SLC26a6 by cAMP and Ca²⁺.¹⁵ Further work is now needed to test whether IRBIT mediates the synergistic effect of cAMP and Ca²⁺ in other tissues and whether additional proteins participate in the synergistic mechanism.

Because of the numerous downstream targets of Ca²⁺ it is not surprising that IRBIT has additional roles other than the ones discussed above. For instance, IRBIT interacts with the cleavage and polyadenylation specificity factor, which plays a role in mRNA processing. This study suggests that IRBIT could regulate gene expression.²⁰ IRBIT also binds to the phosphatidyl inositol phosphate kinase members, who are potent regulators of ion transporters and would provide another level of regulation for electrolyte and solute transport in cells.²¹ IRBIT has been reported to inhibit the calcium calmodulin-dependent kinase II alpha in the brain and thus, would contribute to the regulation of the amount of catecholamine in the brain.²² IRBIT null mice show abnormal social behavior and hyperactivity that can be attributed to the level of catecholamine.²² Most recently IRBIT has been demonstrated to be involved with cell death by blocking the Bcl2l10 protein and facilitating large movements of Ca²⁺ to the mitochondria and thus promoting apoptosis.²³

Although some of IRBIT’s cellular functions have started to be uncovered, many unknowns about IRBIT still exist. For instance, there are two isoforms of IRBIT a short and long N-terminus IRBIT.²⁴ Most of the studies have focused on the short N-terminus, but the long N-terminus is also capable of binding to NBCe1-B and decreases the Mg²⁺-sensitivity of NBCe1-B inhibition during low HCO^–₃.²⁵ The expression and distribution of the two isoforms within tissues is not known. It remains to be determined whether the long and short IRBIT share common target proteins, and if so what is the impact of each isoform on the function of the target protein. Experiments have shown that a phosphorylation state of IRBIT is critical for carrying out its functional role; however, beside this rapid regulation we know very little about the rapid regulation of IRBIT by other proteins (e.g. kinases) or IRBIT gene expression. One study shows that in avian’s oviduct, estrogen agonist induced IRBIT expression in luminal and glandular epithelial cells of the oviduct.²⁶ Because of IRBIT involvement in Na⁺ and HCO^–₃ transport we may expect that acid-base status (e.g. metabolic acidosis, and alkalosis), blood pressure or hormones involved in Na⁺ homeostasis (e.g. aldosterone, angiotensin II)influence IRBIT expression. Because of its role as a third messenger released from the IP₃R it is expected that IRBIT will play essential roles in health and diseases.

The authors would like to thank Lynn Wellsand for her administrative assistance. PBG is supported by the Start up funds from Valparaiso University Art & Science College and the Wheat Ridge Ministries O.P. Kretzmann Grant for Research in the Healing Arts and Sciences.

The author declares no conflict of interest.

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