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

Abstract

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. Abstract 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.

Molecular insight into coordination sites for substrates and their coupling kinetics in Na+/HCO3− cotransporter NBCe1

“…of NBCe1-B under a given [Na + ]. Note that, the single molecular activity of NBCe1-A is several-fold greater than that of NBCe1-B (McAlear et al 2006;Su et al 2021). Again, the data are consistent with our conclusion that the substrate affinity is an intrinsic property of NBCe1.…”

“…In a previous study, if an oocyte expressing NBCe1-A was continuously perfused in CO 2 /HCO 3 − solution and two electrophysiology recordings were sequentially acquired at the beginning of and 4 min after the perfusion in CO 2 /HCO 3 − , a large shift towards positive in the V rev was observed for the second I NBC , eliciting a great inward current at −160 mV (Fig. 1D; Su et al 2021). This last observation provides a clear evidence for the effect of intracellular [Na + ] and [HCO 3 − ] on V rev .…”

“…2006; Su et al . 2021). Both NBCe1‐A and NBCe1‐B were tagged with enhanced green fluorescent protein (EGFP) at the N‐terminus.…”

“…The Xenopus expression vectors containing cDNA encoding NBCe1-A (NM_003759) and NBCe1-B (NM_018760.2) have been described previously (Lu et al 2006;Su et al 2021). Both NBCe1-A and NBCe1-B were tagged with enhanced green fluorescent protein (EGFP) at the N-terminus.…”

Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+/H+ antiporter NHA2

“…It was found that in the hydrophilic N-terminus of the Na + /HCO 3 − co-transporter, the presence of charged (positively as well as negatively) residues is essential to fulfilling the autoinhibitory function of the transporter (Su et al, 2021). To corroborate the role of the N-terminus in the regulation of Hs NHA2 activity, we compared the sequences of unique homologs of NHA2 from various metazoan species (Fig.…”