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Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating

Basso, C; Vergani, P; Nairn, AC; Gadsby, DC; (2003) Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating. The Journal of General Physiology , 122 (3) 333 - 348. 10.1085/jgp.200308798. Green open access

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Abstract

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 [alpha(32)P]8-N(3)ATP or [gamma(32)P]8-N(3)ATP 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 0degreesC with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30degreesC, with extensive washing, also at 30degreesC. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30degreesC 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-N(3)ATP 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.

Type: Article
Title: Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating
Open access status: An open access version is available from UCL Discovery
DOI: 10.1085/jgp.200308798
Publisher version: http://dx.doi.org/10.1085/jgp.200308798
Language: English
Additional information: Publication available under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported license described at http://creativecommons.org/licenses/by-nc-sa/3.0/ and http://creativecommons.org/licenses/by-nc-sa/3.0/legalcode. This provides the non-exclusive right to copy, distribute, or display the Work is granted after six months of publication.
Keywords: ABC transporters, photolabeling, 8-azidonucleotides, atp binding and hydrolysis, orthovanadate, transmembrane conductance regulator, multidrug-resistance protein, human p-glycoprotein, atp-binding, abc transporter, sulfonylurea receptor, chloride channel, kinetic-analysis, catalytic cycle, cl-channels
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Neuro, Physiology and Pharmacology
URI: https://discovery.ucl.ac.uk/id/eprint/125767
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