Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR).
Circumstantial evidence has accumulated suggesting that CFTR is a regulated low-conductance Cl- channel. To test this postulate directly, we have purified to homogeneity a recombinant CFTR protein from a high-level baculovirus-infected insect cell line. Evidence of purity included one- and two-dimensional gel electrophoresis, N-terminal peptide sequence, and quantitative amino acid analysis. Reconstitution into proteoliposomes at less than one molecule per vesicle was accomplished by established procedures. Nystatin and ergosterol were included in these vesicles, so that nystatin conductance could serve as a quantitative marker of vesicle fusion with a planar lipid bilayer. Upon incorporation, purified CFTR exhibited regulated chloride channel activity, providing evidence that the protein itself is the channel. This activity exhibited the basic biophysical and regulatory properties of the type of Cl- channel found exclusively in CFTR-expressing cell types and believed to underlie cAMP-evoked secretion in epithelial cells.
Bear
C E
CE
Department of Physiology, University of Toronto, Ontario, Canada.
Li
C H
CH
Kartner
N
N
Bridges
R J
RJ
Jensen
T J
TJ
Ramjeesingh
M
M
Riordan
J R
JR
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D018118:Chloride Channels; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006801:Humans; D007313:Insecta / Q000235:genetics; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*
C505032:CFTR protein, human; D018118:Chloride Channels; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator