Abstract P427: Novel SNVs of NBCe1, D405E and I803M mutants, reduced cell-surface expression and transport activity.

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Introduction: The electrogenic Na + /HCO 3 - cotransporter NBCe1 is mainly expressed in proximal renal tubule, and regulate pH homeostasis and plasma volume. NBCe1 homozygous mutation causes many symptoms, including severe hypotension, proximal renal tubular acidosis (RTA), and extrarenal phenotype (migraine, mental retardation, mineral bone disorder and ocular abnormalities). Hypothesis: Undetermined single nucleotide variants (SNVs) in NBCe1 will be crucial role of sodium and bicarbonate transport. Methods: We identified the missense mutations (D405E and I803M) in NBCe1 variant A (kidney type) from the NCBI database. We conducted a comparative analysis of cellular localization and cell protein expression using confocal microscopy and western blotting. Furthermore, we performed functional analysis in D405E and I803M SNVs using two-electrode voltage clamp method. Results: Immunofluorescence analysis with confocal microscopy revealed that the D405E and I803M variants were present predominantly in the cell-surface in HEK293 cells. Western blotting in HEK293 cells confirmed that the D405E and I803M variants were significantly reduced protein expression level (25% in D405E and 57% in I803M) compared with WT, respectively. Moreover, electrophysiological analysis revealed that D405E and I803M variants were significantly reduced transport activity. These consisting data suggest that cell-expression level and transport activity are comparable. Conclusion: These data illustrate the diverse physiological consequences of distinct SNVs and underscore the importance of functional characterization in membrane transport proteins.