|
|
|
|
|
|
|
||
Original Article |
gadsby{at}rockvax.rockefeller.edu
The cystic fibrosis transmembrane conductance regulator is a Cl– channel that belongs to the family of ATP-binding cassette proteins. The CFTR polypeptide comprises two transmembrane domains, two nucleotide binding domains (NBD1 and NBD2), and a regulatory (R) domain. Gating of the channel is controlled by kinase-mediated phosphorylation of the R domain and by ATP binding, and, likely, hydrolysis at the NBDs. Exon 13 of the CFTR gene encodes amino acids (aa's) 590–830, which were originally ascribed to the R domain. In this study, CFTR channels were severed near likely NH2- or COOH-terminal boundaries of NBD1. CFTR channel activity, assayed using two-microelectrode voltage clamp and excised patch recordings, provided a sensitive measure of successful assembly of each pair of channel segments as the sever point was systematically shifted along the primary sequence. Substantial channel activity was taken as an indication that NBD1 was functionally intact. This approach revealed that the COOH terminus of NBD1 extends beyond aa 590 and lies between aa's 622 and 634, while the NH2 terminus of NBD1 lies between aa's 432 and 449. To facilitate biochemical studies of the expressed proteins, a Flag epitope was added to the NH2 termini of full length CFTR, and of CFTR segments truncated before the normal COOH terminus (aa 1480). The functionally identified NBD1 boundaries are supported by Western blotting, coimmunoprecipitation, and deglycosylation studies, which showed that an NH2-terminal segment representing aa's 3–622 (Flag3-622) or 3–633 (Flag3-633) could physically associate with a COOH-terminal fragment representing aa's 634–1480 (634-1480); however, the latter fragment was glycosylated to the mature form only in the presence of Flag3-633. Similarly, 433-1480 could physically associate with Flag3-432 and was glycosylated to the mature form; however, 449-1480 protein seemed unstable and could hardly be detected even when expressed with Flag3-432. In excised-patch recordings, all functional severed CFTR channels displayed the hallmark characteristics of CFTR, including the requirement of phosphorylation and exposure to MgATP for gating, ability to be locked open by pyrophosphate or AMP-PNP, small single channel conductances, and high apparent affinity of channel opening by MgATP. Our definitions of the boundaries of the NBD1 domain in CFTR are supported by comparison with the solved NBD structures of HisP and RbsA.
Key Words: adenosine triphosphate-binding cassette transporter • domain structure • chloride channel • gating kinetics • coimmunoprecipitation
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  M.-H. Chang, C. Plata, A. Sindic, W. K. Ranatunga, A.-P. Chen, K. Zandi-Nejad, K. W. Chan, J. Thompson, D. B. Mount, and M. F. Romero Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain J. Biol. Chem., October 9, 2009; 284(41): 28306 - 28318. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Du and G. L. Lukacs Cooperative Assembly and Misfolding of CFTR Domains In Vivo Mol. Biol. Cell, April 1, 2009; 20(7): 1903 - 1915. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Csanady and B. Torocsik Four Ca2+ Ions Activate TRPM2 Channels by Binding in Deep Crevices near the Pore but Intracellularly of the Gate J. Gen. Physiol., February 1, 2009; 133(2): 189 - 203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. F. N. Rosser, D. E. Grove, L. Chen, and D. M. Cyr Assembly and Misassembly of Cystic Fibrosis Transmembrane Conductance Regulator: Folding Defects Caused by Deletion of F508 Occur Before and After the Calnexin-dependent Association of Membrane Spanning Domain (MSD) 1 and MSD2 Mol. Biol. Cell, November 1, 2008; 19(11): 4570 - 4579. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Artigas, S. J. Al'Aref, E. A. Hobart, L. F. Diaz, M. Sakaguchi, S. Straw, and O. S. Andersen 2,3-Butanedione Monoxime Affects Cystic Fibrosis Transmembrane Conductance Regulator Channel Function through Phosphorylation-Dependent and Phosphorylation-Independent Mechanisms: The Role of Bilayer Material Properties Mol. Pharmacol., December 1, 2006; 70(6): 2015 - 2026. [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]
|
|
|
|
 |
|
 K. Fang, L. Csanady, and K. W. Chan The N-terminal transmembrane domain (TMD0) and a cytosolic linker (L0) of sulphonylurea receptor define the unique intrinsic gating of KATP channels J. Physiol., October 15, 2006; 576(2): 379 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Liu, C. Alexander, J. Serrano, E. Borg, and D. C. Dawson Variable Reactivity of an Engineered Cysteine at Position 338 in Cystic Fibrosis Transmembrane Conductance Regulator Reflects Different Chemical States of the Thiol J. Biol. Chem., March 24, 2006; 281(12): 8275 - 8285. [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]
|
|
|
|
|
|
Z.-R. Zhang, G. Cui, X. Liu, B. Song, D. C. Dawson, and N. A. McCarty Determination of the Functional Unit of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel: ONE POLYPEPTIDE FORMS ONE PORE J. Biol. Chem., January 7, 2005; 280(1): 458 - 468. [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]
|
|
|
|
 |
|
 Y. Chen, G. A. Altenberg, and L. Reuss Mechanism of activation of Xenopus CFTR by stimulation of PKC Am J Physiol Cell Physiol, November 1, 2004; 287(5): C1256 - C1263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chen, B. Button, G. A. Altenberg, and L. Reuss Potentiation of effect of PKA stimulation of Xenopus CFTR by activation of PKC: role of NBD2 Am J Physiol Cell Physiol, November 1, 2004; 287(5): C1436 - C1444. [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]
|
|
|
|
|
|
V Chappe, D A Hinkson, T Zhu, X-B Chang, J R Riordan, and J W Hanrahan Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA J. Physiol., April 1, 2003; 548(1): 39 - 52. [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. C Powe Jr, L. Al-Nakkash, M. Li, and T.-C. Hwang Mutation of Walker-A lysine 464 in cystic fibrosis transmembrane conductance regulator reveals functional interaction between its nucleotide-binding domains J. Physiol., March 1, 2002; 539(2): 333 - 346. [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]
|
|
|
|
|
|
B. Button, L. Reuss, and G. A. Altenberg Pkc-Mediated Stimulation of Amphibian Cftr Depends on a Single Phosphorylation Consensus Site. Insertion of This Site Confers Pkc Sensitivity to Human Cftr J. Gen. Physiol., May 1, 2001; 117(5): 457 - 468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Csanady, K. W. Chan, D. Seto-Young, D. C. Kopsco, A. C. Nairn, and D. C. Gadsby Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains J. Gen. Physiol., September 1, 2000; 116(3): 477 - 500. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ottolia, S. John, Z. Qiu, and K. D. Philipson Split Na+-Ca2+ Exchangers. IMPLICATIONS FOR FUNCTION AND EXPRESSION J. Biol. Chem., May 25, 2001; 276(22): 19603 - 19609. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Fu and K. L. Kirk Cysteine Substitutions Reveal Dual Functions of the Amino-terminal Tail in Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating J. Biol. Chem., September 14, 2001; 276(38): 35660 - 35668. [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]
|
|
|
|
|
|
W. Wang, Z. He, T. J. O'Shaughnessy, J. Rux, and W. W. Reenstra Domain-domain associations in cystic fibrosis transmembrane conductance regulator Am J Physiol Cell Physiol, May 1, 2002; 282(5): C1170 - C1180. [Abstract] [Full Text] [PDF]
|
|
|
|
