Abstract
The Na/HCO3 cotransporter NBCe1 is a member of SLC4A transporters that move HCO3− across cell membranes and regulate intracellular pH or transepithelial HCO3 transport. NBCe1 is highly selective to HCO3− and does not transport other anions; the molecular mechanism of anion selectivity is presently unclear. We previously reported that replacing Asp555 with a Glu (D555E) in NBCe1 induces increased selectivity to other anions, including Cl−. This finding is unexpected because all SLC4A transporters contain either Asp or Glu at the corresponding position and maintain a high selectivity to HCO3−. In this study, we tested whether the Cl− transport in D555E is mediated by an interaction between residues in the ion binding site. Human NBCe1 and mutant transporters were expressed in Xenopus oocytes, and their ability to transport Cl− was assessed by two-electrode voltage clamp. The results show that the Cl− transport is induced by a charge interaction between Glu555 and Lys558. The bond length between the two residues is within the distance for a salt bridge, and the ionic strength experiments confirm an interaction. This finding indicates that the HCO3− selectivity in NBCe1 is established by avoiding a specific charge interaction in the ion binding site, rather than maintaining such an interaction.
Highlights
Selectivity in NBCe1 is established by avoiding a specific charge interaction in the ion binding site, rather than maintaining such an interaction
We have previously reported that substituting Asp555 with a Glu (D555E) causes the transporter to be permissive to other anions, including Cl−, NO3 −, SCN−, I−, and Br− [17]
We investigated the effect of charge interactions between residues in site 2 on anion selectivity
Summary
NBCe1 is a membrane protein that mediates electrogenic Na+ -HCO3 − and/or CO3 2−. transport across cell membrane and regulates intracellular and extracellular pH, as well as transepithelial HCO3 − transport in many cells [1,2,3,4]. Zhekova et al [20] performed site identification by ligand competitive saturation (SILCS) mapping of the binding pockets in human AE1 and NBCe1, followed by molecular dynamics (MD) simulations, and proposed two putative anion binding sites in the ion accessibility pathway of the proteins: central site S1 and entrance site S2. All members of Na+ coupled bicarbonate transporters contain either Asp or Glu at the corresponding site, implicating that the geometrical difference in the carboxyl side chain is not the only cause for altered anion selectivity and an additional mechanism should be involved. The two residues involved in the interaction are not simultaneously present in any member of the SLC4A bicarbonate transporters; the HCO3 − selectivity is maintained by avoiding a charge interaction in the ion binding site, located at the entrance of the ion accessibility pathway. We find that Na+ is required for HCO3 − access to the transporter, consistent with a conventional concept of a Na+ prerequisite for substrate movement in secondary active transporters
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