A Novel R881S Mutant of Transporter NBCe1 Has Cytosolic Retention and Lacks Dominant Negative Effect
- Fluid, Electrolyte, Acid-Base Disorders: Basic
November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolytes, and Acid-Base Disorders
- 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic
- Yamazaki, Osamu, Division of Nephrology, Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan
- Fujii, Wataru, Division of Nephrology, Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan
- Fujigaki, Yoshihide, Division of Nephrology, Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan
- Shibata, Shigeru, Division of Nephrology, Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan
The electrogenic Na+/HCO3- cotransporter NBCe1 is expressed in many organs including the brain, eye, and the kidney, where it controls sodium and bicarbonate reabsorption. NBCe1 homozygous mutation causes not only severe hypotension but also proximal renal tubular acidosis (RTA) in human and rodents. Here we characterized the functional significance of SNVs reported from NCBI database.
We identified the missense mutation R881S in NBCe1 variant A (kidney type) from the database. Given the report of R881C mutation in patients with RTA (JASN 2005), we conducted a comparative analysis of cellular localization and cell protein expression between R881S and R881C using confocal microscopy and Western blotting.
Immunofluorescence analysis with confocal microscopy revealed that the R881S variant was present exclusively in the cytoplasm in both HEK293 cells and MDCK cells. Biotinylated western blotting in HEK293 cells confirmed that the cell-surface expression was completely abolished in R881S mutant. In total cell lysates, R881S-NBCe1 showed a lower molecular weight compared with wild-type, and deglycosylation study confirmed that R881S substitution impaired N-glycosylation. Moreover, co-immunoprecipitation study revealed that the interaction with wild-type NBCe1 was severely impaired in R881S compared with R881C.
R881S mutation inactivates the NBCe1 function without lack of dominant-negative effect. These data illustrate the diverse physiological consequences of distinct SNVs and underscore the importance of functional characterization in membrane transport proteins.
- Government Support – Non-U.S.