title: IRBIT reduces the apparent affinity for intracellular Mg²⁺ in inhibition of the electrogenic Na⁺-HCO₃⁻ cotransporter NBCe1-B.
abstract: The electrogenic Na(+)-HCO(3)(-) cotransporter NBCe1-B can be regulated by intracellular Mg(2+) (Mg(2+)(i)). We previously reported that under whole-cell voltage-clamp conditions, bovine NBCe1-B (bNBCe1-B) currents heterologously expressed in mammalian cells are strongly inhibited by Mg(2+)(i), and the inhibition is likely mediated by electrostatic interaction and relieved by truncation of the cytosolic NBCe1-B specific N-terminal region. Intriguingly, NBCe1-B-like currents natively expressed in bovine parotid acinar (BPA) cells are much less sensitive to Mg(2+)(i) inhibition than bNBCe1-B currents. Here, we hypothesized that this apparent discrepancy may involve IRBIT, a previously identified NBCe1-B-interacting protein. RT-PCR, Western blot and immunofluorescence confocal microscopy revealed that IRBIT was not only expressed in the cytosol, but also colocalized with NBCe1-B in the region of plasma membranes of BPA cells. IRBIT was coimmunoprecipitated with NBCe1-B by an anti-NBCe1 antibody in bovine parotid cell lysate. Whole-cell patch-clamp experiments showed that coexpression of IRBIT lowered the Mg(2+)(i) sensitivity of bNBCe1-B currents stably expressed in HEK293 cells. Collectively, these results suggest that IRBIT may reduce the apparent affinity for Mg(2+)(i) in inhibition of NBCe1-B activity in mammalian cells.
authors: Laboratory of Physiology, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
authors: Soichiro
authors: S
authors: Yamaguchi
authors: Toru
authors: T
authors: Ishikawa
mesh_terms: D000818:Animals; D002417:Cattle; D057809:HEK293 Cells; D006801:Humans; D037181:Lectins, C-Type / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D008565:Membrane Proteins; D010306:Parotid Gland / Q000166:cytology / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: C470184:CLECL1 protein, human; D037181:Lectins, C-Type; D008565:Membrane Proteins; D027982:Sodium-Bicarbonate Symporters; D008274:Magnesium