Abstract: PO1077
A Novel I551F Variant of Na+/HCO3- Cotransporter NBCe1 Shows Reduced Cell Surface Expression and May Exert a Dominant Negative Activity
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolyte, and Acid-Base Disorders
- 901 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Yamazaki, Osamu, Teikyo Daigaku, Itabashi-ku, Tokyo, Japan
- Yoshida, Tadashi, Keio Gijuku Daigaku Byoin, Shinjuku-ku, Tokyo, Japan
- Hirahashi, Junichi, Keio Gijuku Daigaku Byoin, Shinjuku-ku, Tokyo, Japan
- Hayashi, Matsuhiko, Kawakita Sogo Byoin, Suginami-ku, Tokyo, Japan
- Shibata, Shigeru, Teikyo Daigaku, Itabashi-ku, Tokyo, Japan
Background
Homozygous mutations in SLC4A4, encoding the electrogenic Na+/HCO3- cotransporter NBCe1, cause proximal renal tubular acidosis (pRTA) associated with extrarenal symptoms. Although 17 mutated sites in SLC4A4 have thus far been identified among pRTA patients, physiological significance of other nonsynonymous single nucleotide variants (SNVs) in this gene remains largely undetermined.
Methods
We investigated the functional properties of SNVs in NBCe1 using immunocytochemical, western blotting, and electrophysiological assays. From NCBI data base, we identified 13 SNVs that have not previously been characterized in highly conserved, transmembrane domains of NBCe1-A.
Results
Immunocytochemical analysis revealed that I551F variant was present predominantly in the cytoplasm in HEK293 cells, whereas all other SNVs did not show obvious changes in subcellular distribution. Western blot analysis in HEK293 cells demonstrated that the I551F variant showed impaired glycosylation and a 69% reduction in cell surface levels. To determine the role of Ile551 in more detail, we examined the significance of various artificial mutants both in non-polarized HEK293 cells and polarized MDCK 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 wild-type, whereas any other SNVs and artificial I551 mutants did not show significant changes in activity. Finally, immunofluorescence study in HEK293 cells indicated that the I551F variant retains wild-type NBCe1-A in the cytoplasm.
Conclusion
These data demonstrate that I551F-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.
Funding
- Government Support – Non-U.S.