title: An intramolecular transport metabolon: fusion of carbonic anhydrase II to the COOH terminus of the Cl(-)/HCO(3)(-)exchanger, AE1.
abstract: Anion exchanger 1 (AE1) is the plasma membrane Cl(-)/HCO(3)(-) exchanger of erythrocytes. Carbonic anhydrases (CA) provide substrate for AE1 by catalyzing the reaction, H(2)O + CO(2) ↔ HCO(3)(-) + H(+). The physical complex of CAII with AE1 has been proposed to maximize anion exchange activity. To examine the effect of CAII catalysis on AE1 transport rate, we fused either CAII-wild type or catalytically inactive CAII-V143Y to the cytoplasmic COOH terminus of AE1 to form AE1.CAII and AE1.CAII-V143Y, respectively. When expressed in transfected human embryonic kidney 293 cells, AE1.CAII had a similar Cl(-)/HCO(3)(-) exchange activity to AE1 alone, as assessed by the flux of H(+) equivalents (87 ± 4% vs. AE1) or rate of change of intracellular Cl(-) concentration (93 ± 4% vs. AE1), suggesting that CAII does not activate AE1. In contrast, AE1.CAII-V143Y displayed transport rates for H(+) equivalents and Cl(-) of 55 ± 2% and of 40 ± 2%, versus AE1. Fusion of CAII to AE1 therefore reduces anion transport activity, but this reduction is compensated for during Cl(-)/HCO(3)(-) exchange by the presence of catalytically active CAII. Overexpression of free CAII-V143Y acts in a dominant negative manner to reduce AE1-mediated HCO(3)(-) transport by displacement of endogenous CAII-wild type from its binding site on AE1. To examine whether AE1.CAII bound endogenous CAII, we coexpressed CAII-V143Y along with AE1 or AE1.CAII. The bicarbonate transport activity of AE1 was inhibited by CAII-V143Y, whereas the activity of AE1.CAII was unaffected by CAII-V143Y, suggesting impaired transport activity upon displacement of functional CAII from AE1 but not AE1.CAII. Taken together, these data suggest that association of functional CAII with AE1 increases Cl(-)/HCO(3)(-) exchange activity, consistent with the HCO(3)(-) transport metabolon model.
authors.affiliation: Membrane Protein Disease Research Group, Department of Physiology, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Daniel
authors.initials: D
authors.lastname: Sowah
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Casey
mesh_terms: D000704:Analysis of Variance; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism*; D001692:Biological Transport; D024402:Carbonic Anhydrase II / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D002712:Chlorides / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014162:Transfection
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D001639:Bicarbonates; D002712:Chlorides; D011993:Recombinant Fusion Proteins; C454201:SLC4A1 protein, human; D024402:Carbonic Anhydrase II