scholarly journals Mutations That Change the Position of the Putative γ-Phosphate Linker in the Nucleotide Binding Domains of CFTR Alter Channel Gating

2001 ◽  
Vol 277 (3) ◽  
pp. 2125-2131 ◽  
Author(s):  
Allan L. Berger ◽  
Mutsuhiro Ikuma ◽  
John F. Hunt ◽  
Philip J. Thomas ◽  
Michael J. Welsh
Keyword(s):  
Channel Gating ◽  
Nucleotide Binding ◽  
Binding Domains

Cooperation of nucleotide binding domains of sur is required for cardaic KATP channel gating

2001 ◽  
Vol 33 (6) ◽  
pp. A138
Author(s):  
L.V. Zingman ◽  
A.E. Alekseev ◽  
M. Bienengraeber ◽  
A. Kim ◽  
D. Hodgson ◽  
...  
Keyword(s):  
Katp Channel ◽  
Channel Gating ◽  
Nucleotide Binding ◽  
Binding Domains

scholarly journals Functional Roles of Nonconserved Structural Segments in CFTR's NH2-terminal Nucleotide Binding Domain

2004 ◽  
Vol 125 (1) ◽  
pp. 43-55 ◽  
Author(s):  
László Csanády ◽  
Kim W. Chan ◽  
Angus C. Nairn ◽  
David C. Gadsby
Keyword(s):  
Cystic Fibrosis ◽  
Conformational Changes ◽  
Single Channel ◽  
Channel Gating ◽  
Essential Element ◽  
Nucleotide Binding ◽  
Transmembrane Conductance Regulator ◽  
Functional Roles ◽  
Binding Domains ◽  
Excised Patches

The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282–293) identified two regions absent from structures of all other NBDs determined so far, a “regulatory insertion” (residues 404–435) and a “regulatory extension” (residues 639–670), both positioned to impede formation of the putative NBD1–NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1–414 with residues 433–1480, or residues 1–633 with 668–1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.

scholarly journals Prolonged Nonhydrolytic Interaction of Nucleotide with CFTR's NH2-terminal Nucleotide Binding Domain and its Role in Channel Gating

2003 ◽  
Vol 122 (3) ◽  
pp. 333-348 ◽  
Author(s):  
Claudia Basso ◽  
Paola Vergani ◽  
Angus C. Nairn ◽  
David C. Gadsby
Keyword(s):  
Channel Gating ◽  
Nucleotide Binding ◽  
Nucleoside Triphosphate ◽  
Binding Domains ◽  
Rate Limiting Step ◽  
Channel Opening ◽  
Nucleotide Interactions ◽  
Dependent Protein ◽  
Opening And Closing ◽  
Ion Pore

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.

scholarly journals Intracellular ATP Increases Capsaicin-Activated Channel Activity by Interacting with Nucleotide-Binding Domains

2000 ◽  
Vol 20 (22) ◽  
pp. 8298-8304 ◽  
Author(s):  
Jiyeon Kwak ◽  
Myeong Hyeon Wang ◽  
Sun Wook Hwang ◽  
Tae-Yoon Kim ◽  
Soon-Youl Lee ◽  
...  
Keyword(s):  
Nucleotide Binding ◽  
Channel Activity ◽  
Intracellular Atp ◽  
Binding Domains

Identification of a Functionally Important Negatively Charged Residue Within the Second Catalytic Site of the SUR1 Nucleotide-Binding Domains

Diabetes ◽  
2004 ◽  
Vol 53 (Supplement 3) ◽  
pp. S123-S127 ◽  
Author(s):  
J. D. Campbell ◽  
P. Proks ◽  
J. D. Lippiat ◽  
M. S.P. Sansom ◽  
F. M. Ashcroft
Keyword(s):  
Nucleotide Binding ◽  
Catalytic Site ◽  
Charged Residue ◽  
Binding Domains
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