Cloning Of Xenopus Laevis IRBIT And Functional Regulation Of NBCe1 By IRBIT

The IRBIT(IP3 receptor-binding protein released with inositol 1,4,5-trisphosphate)family includes short IRBIT1 and long IRBIT2,encoded by AHCYL1 and AHCYL2,respectively.The IRBITs are widely expressed in diverse tissues in the body and play important physiological roles in intracellular Ca²⁺sginalling,regulation of intracellular p H,etc.The electrogenic Na⁺/HCO₃^-cotransporter NBCe1-B(encoded by SLC4A4)plays an important role in transepithelial movement of electrolytes and p H regulation.It is well known that IRBIT is able to activate NBCe1-B.However,little is known about the molecular mechanism underlying the regulation of NBCe1-B by IRBIT.In the present study,we examined the expression of IRBIT in Xenopus oocytes—a model system widely used for biophysical studies on ion channels and transporters,and the structural basis and Ca²⁺-dependence of IRBIT for its interaction with NBCe1-B.First,two IRBIT homologs,irbit2-L and irbit2-S,were cloned from Xenopus laevis oocytes.Sequence alignment shows that the Xenopus IRBIT are highly homologous to the mouse IRBIT,except for the(Nt)appendage domain.Two-electrode voltage clamp studies show that,when over-expressed,irbit2-L and irbit2-S could fully activate NBCe1-B as the mouse IRBITs do.Co-immunoprecipitation shows that irbit2-L can interact with NBCe1-B when over-expressed in Xenopus oocytes.Interestingly,by interfering with the endogenous IRBIT homologue binding to NBCe1-B in oocytes,a significant decrease in the activity of NBCe1-B was detected.These phenomena can reflect that the background activity of NBCe1-B detected in oocytes may be due to the activation of endogenous Xenopus IRBIT homologue.The main difference between IRBIT homologs is the Nt appendage.The degree of activation of NBCe1-B was different among mouse IRBIT homologs due to different Nt additional domains.Deletion mutations in the Nt appendage domains of mouse IRBIT1 and IRBIT2 reduce their stimulating effect on NBCe1-B.Previous studies have shown that the activation of NBCe1-B by IRBIT1 critically depends on the phosphorylation of Ser⁶⁸ in IRBIT1.Surprinsingly,we found that,upon removing the Nt appendage,replacing the Ser⁶⁸with Ala has no effect on NBCe1-B activation,suggesting that Ser68 is important only when the Nt appendage is present.Finally,we examined the effect of Ca²⁺on the interaction between IRBIT1 and NBCe1-B.We found that,in oocytes,depleting intracellular Ca²⁺greatly decreases the activation of NBCe1-B by IRBIT1.Moreover,administration of KN93,inhibitor of calcium calmodulin-dependent kinase II alpha(Ca MKII),also decreases the activation of NBCe1-B by IRBIT1.We propose that the activation of NBCe1-B by IRBIT1 relies on the presence of Ca²⁺in a Ca MKII-dependent manner.The above studies provide important insight for understanding the molecular mechanism underlying the activation of NBCe1-B by IRBIT.