A novel I551F variant of Na+/HCO3- cotransporter NBCe1-A shows reduced cell surface expression, resulting in diminished transport activity
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) remains largely undetermined. Here, we investigated the functional properties of SNVs in NBCe1. From NCBI dbSNP database, we identified 13 SNVs that have not previously been characterized in highly conserved, transmembrane domains of NBCe1-A. Immunocytochemical analysis revealed that I551F variant was present predominantly in the cytoplasm in HEK293 cells, whereas all other SNVs did not show as dramatic a change 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 I551 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. 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.