Abstract P2038: A Nobel I551F Mutant of Sodium Handling Transporter NBCe1 Has Cytosolic Retention and Diminishes Transport Activity

Abstract

The electrogenic Na + /HCO 3 - cotransporter NBCe1 is expressed in many organs and controls sodium handling. NBCe1 homozygous mutation causes not only severe hypotension but also proximal renal tubular acidosis in human and rodents. Here we shows the functional significance of the mutations reported from NCBI database on NBCe1 function. We identified 14 mutations in NBCe1 variant A(kidney type), resulting in the single amino acid substitutions, A425T, F461L, A465S, L494F, N503H, A518T, A518G, I523V, V533D, Y535H, I551F, Y554H, M753V, and L785I. Immunocytological analysis with confocal microscopy revealed only I551F mutant was expressed predominantly 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). Whole and biotinylated western blotting in HEK293 cells confirmed that the surface expression of I551F mutant was significantly reduced. Deglycosylation study revealed that I551F has impaired N-glycosylation. To examine the role of I551 in more detail, we also examined the properties of artificial mutants I551A, I551P, I551K, I551R, I551D and I551E in HEK293 and MDCK cells. All of artificial mutants were properly expressed in the plasma membrane. By contrast, only I551F mutant was predominantly expressed in cytoplasmic regions. 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 mutation may be associated with the disturbance in systemic acid-base balance or the changes in blood pressure.