Kidney Week

Abstract: SA-PO1005

A Novel I551F Mutant of Na+/HCO3- Cotransporter NBCe1 Has Cytosolic Retention and Diminishes Transport Activity

Session Information

• Fluid and Electrolytes: Basic - II
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Yamazaki, Osamu, Keio University of Medicine, Tokyo, Japan

Osamu Yamazaki,

• Yamashita, Maho, Keio University of Medicine, Tokyo, Japan

Maho Yamashita,

• Li, Jinping, Teikyo University School of Medicine, Tokyo, Japan

Jinping Li,

• Yoshida, Tadashi, Keio University of Medicine, Tokyo, Japan

Tadashi Yoshida,

• Hirahashi, Junichi, Keio University of Medicine, Tokyo, Japan

Junichi Hirahashi,

• Shibata, Shigeru, Teikyo University School of Medicine, Tokyo, Japan

Shigeru Shibata,

• Hayashi, Matsuhiko, Kawakita General Hospital, Suginami-ku, Japan

Matsuhiko Hayashi,

Background

Homozygous mutations in SLC4A4 encoding the electrogenic Na⁺/HCO₃^- cotransporter cause proximal renal tubular acidosis associated with ocular abnormalities. Although up to 14 SLC4A4 mutations have been identified, the mechanism of NBCe1 inactivation due to the individual mutations has not been completely clarified.

Methods

In the present study we investigated the impact of SNP (Single Nucleotide Polymorphism) mutations on NBCe1 function. We identified 14 mutations in NBCe1A, resulting in the single amino acid substitutions, A425T, F461L, A465S, L494F, N503H, A518T, A518G, I523V, V533D, Y535H, I551F, Y554H, M753V, and L785I.

Results

Immunohistological analysis with confocal microscopy revealed that almost all of SNP mutants except I551F were predominantly expressed in the plasma membrane of HEK293 cells and MDCK cells. On the other hand, I551F mutant was expressed only in the cytoplasm of HEK 293 cells and MDCK cells.
Functional analysis using Xenopus oocytes revealed that I551F mutant had a significantly reduced activity corresponding to 52% of that of wild-type (p<0.01). Western blotting in HEK293 cells confirmed that the surface expression of I551F mutant was significantly reduced and showed immaure band. To examine the role of I551 in more details, we also examined the properties of artificial mutants I551A, I551P, I551K, I551R, I551D and I551E in HEK293 cells. Artificial mutants were properly expressed in the plasma membrane. By contrast, only I551F mutant was predominantly expressed in cytoplasmic regions.

Conclusion

These results indicate that I551F mutation inactivates the NBCe1 function with cytoplasmic retention. Because NBCe1 plays a major role in renal proximal sodium and bicarbonate reabsorption, I551F SNP may be associated with the disturbance in systemic acid-base balance or the changes in blood pressure.