Expression of cystic fibrosis transmembrane conductance regulator in a model epithelium.
Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation and by intracellular nucleotides. The function of CFTR, like other recombinant ion channels, has generally been studied in single cells using voltage-clamp techniques. However, because CFTR is normally located in the apical membrane of epithelia we wanted to develop a system to study the function of recombinant CFTR expressed in an epithelium. We chose Fischer rat thyroid (FRT) epithelia for two reasons. First, when grown on permeable filter supports, FRT cells form polarized epithelia with a high transepithelial resistance. Second, they have no endogenous cAMP-regulated Cl- channels in their apical membrane. We expressed CFTR in FRT epithelia either transiently, using recombinant vaccinia virus, or stably, using a retrovirus. To measure apical membrane Cl- currents, we permeabilized the basolateral membrane to monovalent ions with nystatin and imposed a large transepithelial Cl- concentration gradient. cAMP agonists stimulated apical membrane Cl- currents in FRT epithelia infected with wild-type CFTR (vTF-CFTR) but not in FRT epithelia infected with either control virus (vTF7-3) or CFTR containing the delta F508 mutation (vTF-delta F508). These Cl- currents had properties similar to those of cAMP-activated Cl- currents in cells expressing endogenous or recombinant CFTR.(ABSTRACT TRUNCATED AT 250 WORDS)
Sheppard
D N
DN
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
Carson
M R
MR
Ostedgaard
L S
LS
Denning
G M
GM
Welsh
M J
MJ
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D018118:Chloride Channels / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004553:Electric Conductivity; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism / Q000502:physiology; D005821:Genetic Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism* / Q000502:physiology
D018118:Chloride Channels; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator