Mutation of Aspartate 555 of the Sodium/Bicarbonate Transporter SLC4A4/NBCe1 Induces Chloride Transport

• Type of Material: Article
• Authors: Han Soo Yang, Emory University; Eunjin Kim, Emory University; Soojung Lee, Emory University; Hae Jeong Park, Emory University; Deborah S. Cooper, Emory University; Ira Rajbhandari, Emory University; Inyeong Choi, Emory University
• Language: English
• Date: 2009-06-05
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://www.jbc.org/content/284/23/15970
• Title of Journal or Parent Work: Journal of Biological Chemistry
• Volume: 284
• Issue: 23
• Start Page: 15970
• End Page: 15979
• Grant/Funding Information: This work was also supported by a National Kidney Foundation Young Investigator Grant (to I. C.).; This work was supported, in whole or in part, by National Institutes of Health Grant GM078502.
• Research Categories: Chemistry, Biochemistry; Biology, Physiology

Abstract

To understand the mechanism for ion transport through the sodium/bicarbonate transporter SLC4A4 (NBCe1), we examined amino acid residues, within transmembrane domains, that are conserved among electrogenic Na/HCO3 transporters but are substituted with residues at the corresponding site of all electroneutral Na/HCO3 transporters. Point mutants were constructed and expressed in Xenopus oocytes to assess function using two-electrode voltage clamp. Among the mutants, D555E (charge-conserved substitution of the aspartate at position 555 with a glutamate) produced decreasing HCO3− currents at more positive membrane voltages. Immunohistochemistry showed D555E protein expression in oocyte membranes. D555E induced Na/HCO3-dependent pH recovery from a CO2-induced acidification. Current-voltage relationships revealed that D555E produced an outwardly rectifying current in the nominally CO2/HCO3−-free solution that was abolished by Cl− removal from the bath. In the presence of CO2/HCO3−, however, the outward current produced by D555E decreased only slightly after Cl− removal. Starting from a Cl−-free condition, D555E produced dose-dependent outward currents in response to a series of chloride additions. The D555E-mediated chloride current decreased by 70% in the presence of CO2/HCO3−. The substitution of Asp555 with an asparagine also produced a Cl− current. Anion selectivity experiments revealed that D555E was broadly permissive to other anions including NO3−. Fluorescence measurements of chloride transport were done with human embryonic kidney HEK 293 cells expressing NBCe1 and D555E. A marked increase in chloride transport was detected in cells expressing D555E. We conclude that Asp555 plays a role in HCO3− selectivity.