Cyclic AMP-dependent protein kinase activation of cystic fibrosis transmembrane conductance regulator chloride channels in planar lipid bilayers.
Membrane vesicles, prepared from mouse NIH-3T3 fibroblasts and Chinese hamster ovary cells expressing high levels of cystic fibrosis transmembrane conductance regulator (CFTR), were fused with Mueller-Rudin planar lipid bilayers. Upon addition of the catalytic subunit of cAMP-dependent protein kinase and ATP, low conductance Cl(-)-selective ion channels were observed in 10 of 16 experiments. The channels had a linear current-voltage relationship and a unitary conductance of approximately 6.5 pS. The channels were more permeable to Cl- than to I- and showed no appreciable time-dependent voltage activation. In contrast, addition of cAMP-dependent protein kinase and ATP to lipid bilayers fused with vesicles prepared from mock transfected (n = 14) cells failed to activate Cl- channels. These data support the conclusion that CFTR is a Cl- channel. They indicate that it can be reconstituted in a planar lipid bilayer and that the biophysical and regulatory properties are very similar to those observed in the native cell membrane. These data also argue against the requirement for loosely associated factors for regulation or function of the channel.
Tilly
B C
BC
Howard Hughes Medical Institute, Department of Internal Medicine and Physiology, University of Iowa College of Medicine, Iowa City 52242.
Winter
M C
MC
Ostedgaard
L S
LS
O'Riordan
C
C
Smith
A E
AE
Welsh
M J
MJ
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels; D006224:Cricetinae; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006801:Humans; D007473:Ion Channels / Q000502:physiology; D008051:Lipid Bilayers; D008561:Membrane Fusion; D008563:Membrane Lipids / Q000378:metabolism; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology*; D051379:Mice; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D014162:Transfection
C505032:CFTR protein, human; D018118:Chloride Channels; D007473:Ion Channels; D008051:Lipid Bilayers; D008563:Membrane Lipids; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011189:Potassium Chloride; D011494:Protein Kinases