Elucidating the Autoinhibitory Domain and IRBIT‐Binding Motifs of the Electrogenic Na/HCO 3 Transporter NBCe1‐B

Abstract

NBCe1 is widely expressed throughout the body, playing crucial roles in transepithelial HCO3− transport (e.g., pancreatic ducts) and regulation of intracellular pH (e.g., central nervous system [CNS]). Of the five known variants, NBCe1-A is expressed mainly in proximal tubules, B is ubiquitous, C is in the CNS, and D and E are in reproductive organs. The extreme NH2-terminus (Nt) is a major determinant of transport activity, with the unique initial 41 amino acids (aa) of A (and perhaps D) containing an autostimulatory domain, and the unique initial 85 aa of B and C (and perhaps E) containing an autoinhibitory domain (AID). The cytosolic protein IRBIT [inositol trisphosphate (IP3)-receptor binding protein released with IP3] binds somewhere within the initial 85-aa of NBCe1-B (and perhaps C & E), largely nullifying the AID. In an earlier study of residues 28–62, inclusive, of NBCe1-B (e1B), we found that a 9-residue cationic cluster (40–48) is an essential component of the AID. In the present study, we: (a) determine the extent to which the remainder of the initial 85-aa of e1B (i.e., residues 2–27 & 63–85) contribute to the AID, (b) extend our analysis of the cationic cluster, and (c) elucidate the structural components of the IRBIT-binding site within the initial 85-aa of e1B. Our main experimental tool is the Xenopus oocyte, in which we express one of dozens of e1B constructs ± a highly active IRBIT construct (super-IRBIT [sIRBIT]) that lacks the PP1 binding site. We assess e1B functional activity by two-electrode voltage-clamping, and determine plasma-membrane expression of e1B and high-affinity sIRBIT binding to e1B by extracellular surface biotinylation, followed by NeutrAvidin pull-down of plasma-membrane proteins, and western blotting. We find that sIRBIT copurifies with biotinylated e1B, but only when the e1B AID is intact (i.e., e1B activity is low in the absence of sIRBIT). For example, sIRBIT does not copurify with the e1B-D940-48, which lacks the cationic cluster. We find that residues 63–85 are not necessary for either the AID or sIRBIT binding. We next used alanine scanning to explore residues 2–27. We find that a His-His motif (residues 26–27) is not essential for the AID, but is required both for stimulation of e1B activity by sIRBIT and for sIRBIT copurification with e1B. Thus, replacing HH with AA or DD produces an e1B construct with high e1B surface abundance, but low baseline activity, no stimulation by sIRBIT, and little sIRBIT copurification. Replacing HH with RR restores robust stimulation of e1B activity by sIRBIT, even though e1B surface abundance is markedly reduced. Regarding the cationic cluster, we replace the WT cation cluster—mostly made of arginine (R) residues—with increasing numbers (N) of lysine residues (KN) or histidine residues (HN). We observe that (1) the KN and HN constructs also produce autoinhibition, (2) sIRBIT relieves the autoinhibition, (3) sIRBIT co-purifies with the KN and HN constructs, and (4) the amount of co-purified sIRBIT gradually increases with increasing N for both KN and HN constructs, as is the case with RN constructs. Thus, a cluster of positive charge—contributed by RN, KN, or HN—is essential both for autoinhibition and sIRBIT binding.