title: Cytosolic H+ microdomain developed around AE1 during AE1-mediated Cl-/HCO3- exchange.
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.
authors.affiliation: Membrane Protein Research Group, Department of Physiology, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Danielle E
authors.initials: DE
authors.lastname: Johnson
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Casey
mesh_terms: D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001639:Bicarbonates / Q000378:metabolism; D024402:Carbonic Anhydrase II / Q000037:antagonists & inhibitors; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D038761:Photobleaching; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D002712:Chlorides; D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D024402:Carbonic Anhydrase II