A novel I551F variant of the Naþ/HCO3- cotransporter NBCe1-A shows reduced cell surface expression, resulting in diminished transport activity

Osamu Yamazaki, Maho Yamashita, Jinping Li, Fumika Ochiai-Homma, Tadashi Yoshida, Junichi Hirahashi, Taiji Furukawa, Ken Kozuma, Yoshihide Fujigaki, George Seki, Matsuhiko Hayashi, Shigeru Shibata

Research output: Contribution to journalArticlepeer-review

Abstract

Homozygous mutations in SLC4A4, which encodes the electrogenic Naþ/HCO3- cotransporter (NBCe1), cause proximal renal tubular acidosis associated with extrarenal symptoms. Although 17` mutated sites in SLC4A4 have thus far been identified among patients with proximal renal tubular acidosis, the physiological significance of other nonsynonymous single-nucleotide variants (SNVs) remains largely undetermined. Here, we investigated the functional properties of SNVs in NBCe1. From the National Center for Biotechnology Information dbSNP database, we identified 13 SNVs that have not previously been characterized in the highly conserved, transmembrane domains of NBCe1-A. Immunocytochemical analysis revealed that the I551F variant was present predominantly in the cytoplasm in human embryonic kidney (HEK)-293 cells, whereas all other SNVs did not show as dramatic a change in subcellular distribution. Western blot analysis in HEK-293 cells demonstrated that the I551F variant showed impaired glycosylation and a 69% reduction in cell surface levels. To determine the role of I551 in more detail, we examined the significance of various artificial mutants in both nonpolarized HEK-293 cells and polarized Madin-Darby canine kidney cells, which indicated that only I551F substitution resulted in cytoplasmic retention. Moreover, functional analysis using Xenopus oocytes demonstrated that the I551F variant had a significantly reduced activity corresponding to 39% of that of the wild-type, whereas any other SNVs and artificial I551 mutants did not show significant changes in activity. Finally, immunofluorescence experiments in HEK-293 cells indicated that the I551F variant retained wild-type NBCe1-A in the cytoplasm. These data demonstrate that the I551F variant of NBCe1-A shows impaired transport activity predominantly through cytoplasmic retention and suggest that the variant can have a dominant negative effect by forming complexes with wild-type NBCe1-A.

Original languageEnglish
Pages (from-to)F771-F784
JournalAmerican Journal of Physiology - Renal Physiology
Volume321
Issue number6
DOIs
Publication statusPublished - 2021 Dec
Externally publishedYes

Keywords

  • Acid/base
  • Blood pressure
  • Electrogenic Na-HCO cotransporter
  • Glycosylation
  • Proximal tubule

ASJC Scopus subject areas

  • Physiology
  • Urology

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