Transfection of wild-type CFTR into cystic fibrosis lymphocytes restores chloride conductance at G1 of the cell cycle.
We complemented the Cl- conductance defect in cystic fibrosis lymphocytes by transfection with wild-type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non-CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector-specific sequences. Both fluorescence and patch-clamp assays showed that transfectants expressing wild-type CFTR acquired properties previously associated with Cl- conductance (GCl) regulation in non-CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist-induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild-type CFTR corrects the defects in Cl- conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR-mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR.
Gregory Fleming James Cystic Fibrosis Research Center, Department of Microbiology, University of Alabama, Birmingham 35294.
R D
RD
Krauss
J K
JK
Bubien
M L
ML
Drumm
T
T
Zheng
S C
SC
Peiper
F S
FS
Collins
K L
KL
Kirk
R A
RA
Frizzell
T A
TA
Rado
D001402:B-Lymphocytes / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D002463:Cell Membrane Permeability; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA / Q000235:genetics; D016193:G1 Phase; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012194:RNA-Directed DNA Polymerase; D016196:S Phase; D014162:Transfection
C505032:CFTR protein, human; D002712:Chlorides; D008565:Membrane Proteins; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA; D000242:Cyclic AMP; D012194:RNA-Directed DNA Polymerase