Format

Send to

Choose Destination
J Physiol. 2011 Apr 1;589(Pt 7):1551-69. doi: 10.1113/jphysiol.2010.201483. Epub 2011 Feb 7.

Cytosolic H+ microdomain developed around AE1 during AE1-mediated Cl-/HCO3- exchange.

Author information

1
Membrane Protein Research Group, Department of Physiology, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

Abstract

Microdomains, regions of discontinuous cytosolic solute concentration enhanced by rapid solute transport and slow diffusion rates, have many cellular roles. pH-regulatory membrane transporters, like the Cl−/HCO3− exchanger AE1, could develop H+ microdomains since AE1 has a rapid transport rate and cytosolic H+ diffusion is slow. We examined whether the pH environment surrounding AE1 differs from other cellular locations. As AE1 drives Cl−/HCO3− exchange, differences in pH, near and remote from AE1, were monitored by confocal microscopy using two pH-sensitive fluorescent proteins: deGFP4 (GFP) and mNectarine (mNect). Plasma membrane (PM) pH (defined as ∼1 μm region around the cell periphery) was monitored by GFP fused to AE1 (GFP.AE1), and mNect fused to an inactive mutant of the Na+-coupled nucleoside co-transporter, hCNT3 (mNect.hCNT3). GFP.AE1 to mNect.hCNT3 distance was varied by co-expression of different amounts of the two proteins in HEK293 cells. As the GFP.AE1–mNect.hCNT3 distance increased, mNect.hCNT3 detected the Cl−/HCO3− exchange-associated cytosolic pH change with a time delay and reduced rate of pH change compared to GFP.AE1. We found that a H+ microdomain 0.3 μm in diameter forms around GFP.AE1 during physiological HCO3− transport. Carbonic anhydrase isoform II inhibition prevented H+ microdomain formation. We also measured the rate of H+ movement from PM GFP.AE1 to endoplasmic reticulum (ER), using mNect fused to the cytosolic face of ER-resident calnexin (CNX.mNect). The rate of H+ diffusion through cytosol was 60-fold faster than along the cytosolic surface of the plasma membrane. The pH environment surrounding pH regulatory transport proteins may differ as a result of H+ microdomain formation, which will affect nearby pH-sensitive processes.

PMID:
21300752
PMCID:
PMC3099015
DOI:
10.1113/jphysiol.2010.201483
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Wiley Icon for PubMed Central
Loading ...
Support Center