Transport properties of the human intestinal anion exchanger DRA (down-regulated in adenoma) in transfected HEK293 cells.
Electroneutral NaCl absorption in the intestine is mediated by parallel Na+/H+ and Cl-/HCO3- exchange. Mutations in the down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea but the transport characteristics of human DRA have not been studied in a heterologous human expression system. A N-terminal enhanced green fluorescence protein (EGFP)-tagged human DRA construct was therefore expressed stably in HEK293 cells. Cl-/HCO3- exchange was assessed by measuring intracellular pH and intracellular Cl- using fluorescent dyes. Expression of DRA resulted in the appearance of EGFP fluorescence and DRA immunoreactivity consistent with a location in the plasma membrane and possibly structures below the plasma membrane. DRA mediated electroneutral Cl-/HCO3- exchange but OH- was not transported and SO4(2-)/HCO3- exchange was minimal. In the presence of 5% CO2/HCO3- the apparent affinity of DRA for Cl- in transfected HEK cells was 23-36 mM, which is lower than that reported for rabbit ileal brush border membrane vesicles and for oocytes injected with human DRA. DRA was inhibited by 4 mM DIDS (45+/-11%), by 50 microM tenidap (71+/-8%) and by 100 microM glibenclamide (59+/-22% inhibition of HCO3- transport and 79+/-3% inhibition of Cl- transport). The effects of DIDS and tenidap were not additive to those of glibenclamide.
1st Department of Medicine, University of Tübingen, 72076, Tübingen, Germany. hans-georg.lamprecht@uni-tuebingen.de
Georg
G
Lamprecht
Susannah
S
Baisch
Elena
E
Schoenleber
Michael
M
Gregor
D017920:Antiporters / Q000235:genetics* / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology; D000076988:Sulfate Transporters; D013431:Sulfates / Q000378:metabolism; D014162:Transfection
D017920:Antiporters; D001639:Bicarbonates; C505032:CFTR protein, human; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; C081011:SLC26A3 protein, human; D000076988:Sulfate Transporters; D013431:Sulfates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins