Evaluating Physiological Interactions between the Electrogenic Na/HCO3 Transporter NBCe1‐B and its Cytosolic Binding Partner IRBIT
Abstract
The electrogenic Na/HCO3 transporter NBCe1 is expressed in numerous organs, where it plays many important roles, including trans‐epithelial HCO3− transport (e.g., pancreatic ducts) and maintenance of intracellular pH (e.g., cells in CNS). Presently, five NBCe1 variants are known: the A variant, mainly in kidney; B, ubiquitous; C, CNS; and D and E, murine reproductive organs. The extreme NH2‐terminus (Nt) of each variant is a strong determinant of transport activity. A and D have a unique Nt of 41 amino acids (aa) that, at least in A, confers high transport activity. B, C, and E have a unique Nt of 85 aa that, at least in B and C, confers low transport activity. However, B interacts with IRBIT [inositol trisphosphate (IP3)‐receptor binding protein released with IP3], which binds to the unique Nt of NBCe1‐B, and markedly enhances the transport activity of NBCe1‐B. Previously, others examined the interaction by fusing a fragment of the unique Nt of NBCe1‐B/C/E to maltose‐binding protein (MBP) for use in a MBP pull‐down assay, and found that NBCe1‐B (e1B) residues 1–62 are necessary for the interaction. In a recent study, we showed that the cationic cluster (residues 40–48 inclusive) is an essential element of the autoinhibitory domain (AID) that confers low activity on e1B. The purpose of the present study is to refine the structural requirements of the IRBIT‐e1B interaction under physiological conditions. Here, we co‐express 7 constructs of e1B (one at a time) together with super‐IRBIT (sIRBIT, which lacks the PP1 binding site) in Xenopus oocytes, followed by two‐electrode voltage‐clamp, extracellular‐surface‐biotinylation, and NeutrAvidin pull‐down of plasma‐membrane proteins. We find that sIRBIT copurifies, as assayed by western blot, with biotinylated e1B protein, but only for low‐activity e1B constructs (i.e., AID intact) for which sIRBIT enhances transport activity. sIRBIT does not copurify with the Δ940–48 e1B construct, which lacks the cationic cluster (i.e., AID disrupted). Thus, the cationic cluster is not only essential for autoinhibition, but also for sIRBIT binding. Our approach will be useful for elucidating other motifs that may be necessary for the interaction of IRBIT with NBCe1‐B, ‐C, or ‐E.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Apr 01, 2019
- Source ID
- 10.1096/fasebj.2019.33.1_supplement.544.6
Entities
People
- Seong‐ki Lee
- Walter Boron
Organizations
- Case Western Reserve University
- Office of Naval Research