Effects of pyrophosphate and nucleotide analogs suggest a role for ATP hydrolysis in cystic fibrosis transmembrane regulator channel gating.
Single channel analysis of artificial lipid planar bilayers reconstituted with wild-type human cystic fibrosis transmembrane regulator (CFTR) revealed a 10.3 pS Cl- selective channel that was activated upon phosphorylation with protein kinase A. Gating of this channel was described by a simple kinetic model consisting of a single open burst state and two closed states. The open probability of CFTR channels in bilayers increased as a function of increasing Mg-ATP concentration and exhibited negative cooperativity, suggesting the interaction of two or more ATP binding sites in channel gating. Mg-ATP increased channel open probability by decreasing the duration of the long-lived closed state but had no effect on either the mean open time or the fast closed state. ADP inhibited channel opening by precisely antagonizing the effect of ATP, suggesting that ADP inhibits the CFTR channel by competing with ATP for binding. Poorly hydrolyzable ATP analogs such as AMP-PNP and ATP gamma S, polyphosphates such as pyrophosphate (PPi) and tripolyphosphate (PPPi), and orthovanadate failed to support channel activity alone. When applied in the presence of ATP, these compounds all caused the CFTR channel to "lock" into a prolonged open burst state. These data support a model in which hydrolysis of ATP leads to closure of channels that have been opened by ATP.
Gunderson
K L
KL
Department of Biological Sciences, Stanford University, California 94305-5020.
Kopito
R R
RR
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011756:Diphosphates / Q000494:pharmacology; D004553:Electric Conductivity; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D011756:Diphosphates; D008051:Lipid Bilayers; D008565:Membrane Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D014638:Vanadates; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate