Molecular Mechanism For Auto-inhibition Of Na⁺/HCO₃^- Cotransporter NBCe1-B And Its Activation By IRBIT

Na⁺/HCO₃^-cotransporter NBCe1,encoded by Slc4a4 of the SLC4 family,plays an important role in the regulation of acid-base balance in the body.The dysfunction of NBCe1 causes metabolic acidosis,pancreatitis,hereditary epilepsy and other diseases.Recent years,great progresses have been made in understanding the structure-function relationship of the SLC4 family transporters.The structure of NBCe1 transmembrane domain（TMD）consists of a carrier domain and a scaffold domain.NBCe1 likely employs an elevator-like mechanism.The ion translocation depends on the conformational transition of the TMD between outward-open state and inward-open state via the sliding of the carrier domain relative to the scaffold domain.NBCe1 has five variants: NBCe1-A^E.NBCe1-B is expressed in the basolateral membrane of the pancreatic ductal epithelial cells,involved in HCO3-secretion that is highly regulated by hormones and neural signals.NBCe1-B contains an auto-inhibitory domain（AID）in its unique NtB.IRBIT（IP3 receptor-binding protein released with IP3）can fully stimulate NBCe1-B by protein interaction.However,it remains unknown how the AID inhibits NBCe1-B and how IRBIT activates NBCe1-B.In the present study,by using Xenopus oocytes as heterologous expression system,we investigated the molecular mechanism underlying the auto-inhibition of NBCe1-B and its activation by IRBIT by voltage clamp incombination with molecular biology and structural biology approaches.The unique amino-terminus（Nt）of NBCe1-B,which is 85 amino-acids（aa）in length,is highly hydrophilic.Region 1-24 aa is highly rich in acid residues,denoted as Arm N.Region 40-52 aa is rich in alkaline residues,denoted as Arm P.Region 53-85 aa is mixed with acid and alkaline residues,denoted as Arm M.Our study show that the auto-inhibitory domain（AID）is mainly composed of the alkaline residue cluster Arm P and the Arm M with mixing acid and alkaline residues.Arm P plays a key role in the auto-inhibition of NBCe1-B.Replacing the strongly alkaline residues Arg and Lys in Arm P with weakly alkaline residue His significantly reduces the auto-inhibition magnitude of NBCe1-B.The inhibitory effect of AID on NBCe1 activity depends on the covalent attachment of the AID to NBCe1.The IRBIT binding domain（IBD）is mainly composed of Arm N rich in acid residues and Arm P rich in alkaline residues.The two acidic motifs in Arm N and the basic residue clusters in Arm P are essential structural elements for NBCe-B to combine with IRBIT.Further studies show that the auto-inhibition of NBCe1-B depends on a set of negatively charged residues（including Glu,Asp,and Phe）in TMD.Mutation of these residues in the TMD—replacing Glu with Gln,Asp with Asn,and Phe with Ala—significantly reduces the auto-inhibition magnitude of NBCe1-B.Moreover,combined mutation to the alkaline residues in Arm P at the amino terminus of NBCe1-B and the negatively charged residues in the TMD elicits no additive effect on the auto-inhibition magnitude of NBCe1-B,suggesting that the two sets of mutations in the Arm P and the TMD may affect a same event to change the auto-inhibition of NBCe1-B.Finally,the binding sites of NBCe1-B in IRBIT were investigated in this study.The amino terminus of IRBIT has a PEST domain rich in phosphorylation sites and acid residues.In the present study,mutation to Ser/Thr residues and/or the acid residues in the PEST domain greatly reduces the protein interaction between IRBIT and NBCe1-B.We propose that the PEST domain rich in Ser/Thr residues and acid residues is the binding sites of Arm P,the alkaline residue cluster in NBCe1-B.Based on the above results,we propose the following model for the auto-inhibition of NBCe1-B and its stimulation by IRBIT: the AID binds to TMD via electrostatic interaction,blocks the conformational transition of the TMD of the transporter,thus inhibiting the activity of NBCe1.IRBIT removes AID from TMD by competitive binding,therefore eliminating the auto-inhibition and activating NBCe1-B.In other words,AID regulates NBCe1-B activity as a “brake” element,and IRBIT releases the “brake” by protein interaction.Our study provides key insight into understanding the molecular mechanism for functional regulation of NBCe1 and also provides an important basis for understanding the role of IRBIT in pancreatic secretion.Auto-inhibition is common in ion channels and transporters,our study is a implication for studying the auto-inhibition mechanism of other transporters（NBCe1-C,NBCn1,NBCn2,NDCBE,etc.）.