A previously uncharacterized R881S variant of transporter NBCe1 exhibits intracellular retention and virtually no plasma membrane expression

Mutations in the Na + /HCO 3 - cotransporter NBCe1 (SLC4A4) cause proximal renal tubular acidosis (pRTA) and extrarenal symptoms including glaucoma, band kelatopathy, migraine, growth disorder, and abnormal tooth enamel. From NCBI dbSNP database, we recently identified a previously uncharacterized single nucleotide variant (SNV) R881S in NBCe1, located in hydrophilic helix 4. R881S NBCe1-A showed intracellular retention in human embryonic kidney 293 cells and its plasma membrane expression was profoundly reduced in polarized Madin-Darby canine kidney cells. Unlike WT and R881C NBCe1-A (which causes pRTA), Western blot analysis demonstrated that the R881S NBCe1-A showed a single, low-molecular-weight signal at above 100kDa. Deglycosylation study showed that R881S NBCe1-A was scarcely deglycosylated by PNGase F. Coimmunoprecipitation study demonstrated that wild-type and R881S NBCe1-A did not form heterodimer. Moreover, functional analysis using Xenopus oocytes revealed that the R881S variant had markedly reduced transport activity compared with wild-type NBCe1-A. Thus, R881S NBCe1-A shows no detectable transport activity, likely due to defective trafficking and reduced glycosylation. In contrast to the R881C mutant, however, R881S NBCe1-A fails to form dimers with wild-type NBCe1-A, suggesting the absence of a dominant-negative effect and underscoring the need for functional characterization of reported genetic variants.