The electrogenic Na+-HCO3- cotransporter NBCe1 is important for the regulation of intracellular pH (pHi) and for epithelial HCO3- transport in many tissues, including kidney, pancreas, and brain. In the present study, we investigate glycosylation sites in NBCe1. Treatment of rat kidney membrane extracts with peptide N-glycosidase F (PNGase F) shifted the apparent molecular weight (MW) of NBCe1 from 130 to 116, the MW predicted from the deduced amino acid sequence. Treatment with endoglycosidase F2 or H or O-glycosidase did not affect the MW of NBCe1. Lectin-binding studies, together with the enzyme data, suggest that the N-linked carbohydrates are of tri- or tetra-antennary type. To localize glycosylation sites, we individually mutated the seven consensus N-glycosylation sites by replacing asparagine (N) with glutamine (Q) and assessing mutant transporters in Xenopus laevis oocytes. Immunoblotting of oocyte membrane extracts treated with PNGase F indicates that NBCe1 is normally glycosylated at N597 and N617 (both on the third extracellular loop). However, N592 (on the same loop) is glycosylated when the other two sites are mutated. The triple mutant (N592Q/N597Q/N617Q) is completely unglycosylated but, based on microelectrode measurements of membrane potential and pHi in oocytes, preserves the Na+ and HCO3- dependence and electrogenicity of wild-type NBCe1.
|Original language||English (US)|
|Journal||American Journal of Physiology - Renal Physiology|
|Issue number||6 53-6|
|State||Published - Jun 1 2003|
- Acid-base mechanism
- PH measurement
ASJC Scopus subject areas