CFTR, the protein defective in cystic fibrosis, functions as a Cl− channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [α32P]8-N3ATP or [γ32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0°C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30°C, with extensive washing, also at 30°C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30°C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore.
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1 September 2003
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August 25 2003
Prolonged Nonhydrolytic Interaction of Nucleotide with CFTR's NH2-terminal Nucleotide Binding Domain and its Role in Channel Gating
Claudia Basso,
Claudia Basso
1Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021
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Paola Vergani,
Paola Vergani
1Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021
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Angus C. Nairn,
Angus C. Nairn
2Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021
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David C. Gadsby
David C. Gadsby
1Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021
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Claudia Basso
1Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021
Paola Vergani
1Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021
Angus C. Nairn
2Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021
David C. Gadsby
1Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021
Address correspondence to David C. Gadsby, Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, 1230 York Ave., New York, NY 10021. Fax: (212) 327-7589; email: [email protected]
Claudia Basso's present address is Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Independencia 1027, Santiago, Chile.
Abbreviation used in this paper: NBD, nucleotide-binding domain.
Received:
January 15 2003
Accepted:
July 21 2003
Online ISSN: 1540-7748
Print ISSN: 0022-1295
The Rockefeller University Press
2003
J Gen Physiol (2003) 122 (3): 333–348.
Article history
Received:
January 15 2003
Accepted:
July 21 2003
Citation
Claudia Basso, Paola Vergani, Angus C. Nairn, David C. Gadsby; Prolonged Nonhydrolytic Interaction of Nucleotide with CFTR's NH2-terminal Nucleotide Binding Domain and its Role in Channel Gating . J Gen Physiol 1 September 2003; 122 (3): 333–348. doi: https://doi.org/10.1085/jgp.200308798
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