|
|
|
|
|
|
|
||
Original Article |
gadsby{at}rockvax.rockefeller.edu
Opening and closing of a CFTR Cl– channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634–836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1–633 and 837–1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.
Key Words: ATP-binding cassette transporter • chloride ion channel • phosphorylation • gating regulation • kinetic model
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Csanady, P. Vergani, and D. C. Gadsby Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations PNAS, January 19, 2010; 107(3): 1241 - 1246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Muallem and P. Vergani ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator Phil Trans R Soc B, January 27, 2009; 364(1514): 247 - 255. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Seavilleklein, N. Amer, A. Evagelidis, F. Chappe, T. Irvine, J. W. Hanrahan, and V. Chappe PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR Am J Physiol Cell Physiol, November 1, 2008; 295(5): C1366 - C1375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Lewarchik, K. W. Peters, J. Qi, and R. A. Frizzell Regulation of CFTR Trafficking by Its R Domain J. Biol. Chem., October 17, 2008; 283(42): 28401 - 28412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.-Y. Chen and T.-C. Hwang CLC-0 and CFTR: Chloride Channels Evolved From Transporters Physiol Rev, April 1, 2008; 88(2): 351 - 387. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Fuller, C. H. Thompson, Z.-R. Zhang, C. S. Freeman, E. Schay, G. Szakacs, E. Bakos, B. Sarkadi, D. McMaster, R. J. French, et al. State-dependent Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels by a Novel Peptide Toxin J. Biol. Chem., December 28, 2007; 282(52): 37545 - 37555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ramu, Y. Xu, and Z. Lu Inhibition of CFTR Cl- channel function caused by enzymatic hydrolysis of sphingomyelin PNAS, April 10, 2007; 104(15): 6448 - 6453. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Csanady, A. C. Nairn, and D. C. Gadsby Thermodynamics of CFTR Channel Gating: A Spreading Conformational Change Initiates an Irreversible Gating Cycle J. Gen. Physiol., November 1, 2006; 128(5): 523 - 533. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhou, X. Wang, H.-Y. Liu, X. Zou, M. Li, and T.-C. Hwang The Two ATP Binding Sites of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Play Distinct Roles in Gating Kinetics and Energetics J. Gen. Physiol., October 1, 2006; 128(4): 413 - 422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Zhou, X. Wang, M. Li, Y. Sohma, X. Zou, and T.-C. Hwang High affinity ATP/ADP analogues as new tools for studying CFTR gating J. Physiol., December 1, 2005; 569(2): 447 - 457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. O. Randak and M. J. Welsh Adenylate Kinase Activity in ABC Transporters J. Biol. Chem., October 14, 2005; 280(41): 34385 - 34388. [Full Text] [PDF]
|
|
|
|
|
|
S. G. Bompadre, T. Ai, J. H. Cho, X. Wang, Y. Sohma, M. Li, and T.-C. Hwang CFTR Gating I: Characterization of the ATP-dependent Gating of a Phosphorylation-independent CFTR Channel ({Delta}R-CFTR) J. Gen. Physiol., March 28, 2005; 125(4): 361 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Csanady, D. Seto-Young, K. W. Chan, C. Cenciarelli, B. B. Angel, J. Qin, D. T. McLachlin, A. N. Krutchinsky, B. T. Chait, A. C. Nairn, et al. Preferential Phosphorylation of R-domain Serine 768 Dampens Activation of CFTR Channels by PKA J. Gen. Physiol., January 31, 2005; 125(2): 171 - 186. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Csanady, K. W. Chan, A. C. Nairn, and D. C. Gadsby Functional Roles of Nonconserved Structural Segments in CFTR's NH2-terminal Nucleotide Binding Domain J. Gen. Physiol., December 28, 2004; 125(1): 43 - 55. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Csanady and V. Adam-Vizi Antagonistic Regulation of Native Ca2+- and ATP-sensitive Cation Channels in Brain Capillaries by Nucleotides and Decavanadate J. Gen. Physiol., June 1, 2004; 123(6): 743 - 757. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ai, S. G. Bompadre, X. Wang, S. Hu, M. Li, and T.-C. Hwang Capsaicin Potentiates Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride-Channel Currents Mol. Pharmacol., June 1, 2004; 65(6): 1415 - 1426. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Cormet-Boyaka, M. Jablonsky, A. P. Naren, P. L. Jackson, D. D. Muccio, and K. L. Kirk Rescuing cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by transcomplementation PNAS, May 25, 2004; 101(21): 8221 - 8226. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. W. Hanrahan and M.-A. Wioland Revisiting Cystic Fibrosis Transmembrane Conductance Regulator Structure and Function Proceedings of the ATS, January 1, 2004; 1(1): 17 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Basso, P. Vergani, A. C. Nairn, and D. C. Gadsby Prolonged Nonhydrolytic Interaction of Nucleotide with CFTR's NH2-terminal Nucleotide Binding Domain and its Role in Channel Gating J. Gen. Physiol., August 25, 2003; 122(3): 333 - 348. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Vergani, A. C. Nairn, and D. C. Gadsby On the Mechanism of MgATP-dependent Gating of CFTR Cl- Channels J. Gen. Physiol., December 30, 2002; 121(1): 17 - 36. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G. Dousmanis, A. C. Nairn, and D. C. Gadsby Distinct Mg2+-dependent Steps Rate Limit Opening and Closing of a Single CFTR Cl- Channel J. Gen. Physiol., June 1, 2002; 119(6): 545 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. S. Ostedgaard, J. Zabner, D. W. Vermeer, T. Rokhlina, P. H. Karp, A. A. Stecenko, C. Randak, and M. J. Welsh CFTR with a partially deleted R domain corrects the cystic fibrosis chloride transport defect in human airway epithelia in vitro and in mouse nasal mucosa in vivo PNAS, February 14, 2002; (2002) 261714599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Fu, H.-L. Ji, A. P Naren, and K. L Kirk A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating J. Physiol., October 15, 2001; 536(2): 459 - 470. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-M. Chen, C. Cutler, C. Jacques, E. Denamur, G. Lecointre, B. Mercier, G. Cramb, and C. Ferec A Combined Analysis of the Cystic Fibrosis Transmembrane Conductance Regulator: Implications for Structure and Disease Models Mol. Biol. Evol., September 1, 2001; 18(9): 1771 - 1788. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. S. Ostedgaard, O. Baldursson, and M. J. Welsh Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Cl- Channel by Its R Domain J. Biol. Chem., March 9, 2001; 276(11): 7689 - 7692. [Full Text] [PDF]
|
|
|
|
|
|
L. S. Ostedgaard, J. Zabner, D. W. Vermeer, T. Rokhlina, P. H. Karp, A. A. Stecenko, C. Randak, and M. J. Welsh CFTR with a partially deleted R domain corrects the cystic fibrosis chloride transport defect in human airway epithelia in vitro and in mouse nasal mucosa in vivo PNAS, March 5, 2002; 99(5): 3093 - 3098. [Abstract] [Full Text] [PDF]
|
|
|
|
