IRBIT reduces the apparent affinity for intracellular Mg²⁺ in inhibition of the electrogenic Na⁺-HCO₃⁻ cotransporter NBCe1-B

Biochem Biophys Res Commun. 2012 Aug 3;424(3):433-8. doi: 10.1016/j.bbrc.2012.06.127. Epub 2012 Jul 3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • HEK293 Cells
  • Humans
  • Lectins, C-Type / metabolism*
  • Magnesium / metabolism*
  • Membrane Proteins
  • Parotid Gland / cytology
  • Parotid Gland / metabolism
  • Sodium-Bicarbonate Symporters / antagonists & inhibitors
  • Sodium-Bicarbonate Symporters / metabolism*

Substances

  • CLECL1 protein, human
  • Lectins, C-Type
  • Membrane Proteins
  • Sodium-Bicarbonate Symporters
  • Magnesium