The electrogenic Na/HCO3 cotransporter NBCe1 is expressed widely throughout the body, and plays crucial roles in trans‐epithelial HCO3− transport (e.g., renal proximal tubule, PT) and regulation of intracellular pH (e.g., cells of central nervous system, CNS). The five known NBCe1 variants are: NBCe1‐A variant, mainly in PT; B, ubiquitous; C, CNS; and D and E, murine reproductive organs. For each variant, the extreme NH2‐terminus (Nt) markedly affects transport activity. For the A and D variants, the initial 41 amino acids (aa) are unique and, at least in A, confer high transport activity. For the B, C, and E variants, the initial 85 aa are likewise unique and, at least in B and C, confer low transport activity. Among these variants, at least B interacts with IRBIT [inositol trisphosphate (IP3)‐receptor binding protein released with IP3], which binds to the unique Nt and markedly enhances transport activity. In a previous study, we examined NBCe1‐B (e1B) residues 28–62, inclusive, and found that the cationic cluster (residues 40–48, inclusive) is an essential element of the AID that confers low activity to e1B. The purpose of the present study is to: (a) determine whether other portions of the unique Nt of e1B (i.e., residues 2–27 and 63–85, inclusive) contribute to the AID, (b) better characterize the cationic cluster, and (c) refine the structural requirements of the IRBIT‐e1B interaction under physiological conditions. Our approach is to co‐express in Xenopus oocytes one of several e1B constructs ± super‐IRBIT (sIRBIT, which lacks the PP1 binding site), followed by two‐electrode voltage‐clamping, extracellular surface biotinylation, and NeutrAvidin pull‐down of plasma‐membrane proteins. As assayed by western blot, we find that sIRBIT co‐purifies with biotinylated e1B protein, but only for e1B constructs with an intact AID (i.e., low‐activity constructs that sIRBIT strongly stimulates). For example, sIRBIT does not copurify with the Δ940‐48 e1B construct, which lacks the cationic cluster (i.e., AID disrupted). We find that residues 63–85, inclusive, are unnecessary for both the AID and for sIRBIT binding. Currently, we are using alanine scanning to explore residues 2–27, inclusive. When we replace the normal cation cluster—mostly made of arginine (R) residues—with increasing numbers (N) of lysine residues (KN) or histidine residues (HN), we observe that (a) the KN and HN constructs also produce autoinhibition, (b) IRBIT relieves the autoinhibition, and (c) sIRBIT co‐purifies with the KN and HN constructs, and (d) 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, the cationic cluster—whether made up of RN, KN, or HN—is essential both for autoinhibition and sIRBIT binding.Support or Funding InformationThe Office of Naval Research, N00014‐15‐1‐2060,N00014‐16‐1‐2535