|
|
|
|
|
|
|
||
The Journal of General Physiology, Vol 107, 103-119, Copyright © 1996 by The Rockefeller University Press
ARTICLES |
DJ Wilkinson, MK Mansoura, PY Watson, LS Smit, FS Collins and DC Dawson
Department of Physiology, University of Michigan, Ann Arbor, 48109, USA.
The functional roles of the two nucleotide binding folds, NBF1 and NBF2, in the activation of the cystic fibrosis transmembrane conductance regulator (CFTR) were investigated by measuring the rates of activation and deactivation of CFTR Cl- conductance in Xenopus oocytes. Activation of wild-type CFTR in response to application of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was described by a single exponential. Deactivation after washout of the cocktail consisted of two phases: an initial slow phase, described by a latency, and an exponential decline. Rate analysis of CFTR variants bearing analogous mutations in NBF1 and NBF2 permitted us to characterize amino acid substitutions according to their effects on the accessibility and stability of the active state. Access to the active state was very sensitive to substitutions for the invariant glycine (G551) in NBF1, where mutations to alanine (A), serine (S), or aspartic acid (D) reduced the apparent on rate by more than tenfold. The analogous substitutions in NBF2 (G1349) also reduced the on rate, by twofold to 10-fold, but substantially destabilized the active state as well, as judged by increased deactivation rates. In the putative ATP-binding pocket of either NBF, substitution of alanine, glutamine (Q), or arginine (R) for the invariant lysine (K464 or K1250) reduced the on rate similarly, by two- to fourfold. In contrast, these analogous substitutions produced opposite effects on the deactivation rate. NBF1 mutations destabilized the active state, whereas the analogous substitutions in NBF2 stabilized the active state such that activation was prolonged compared with that seen with wild-type CFTR. Substitution of asparagine (N) for a highly conserved aspartic acid (D572) in the ATP-binding pocket of NBF1 dramatically slowed the on rate and destabilized the active state. In contrast, the analogous substitution in NBF2 (D1370N) did not appreciably affect the on rate and markedly stabilized the active state. These results are consistent with a hypothesis for CFTR activation that invokes the binding and hydrolysis of ATP at NBF1 as a crucial step in activation, while at NBF2, ATP binding enhances access to the active state, but the rate of ATP hydrolysis controls the duration of the active state. The relatively slow time courses for activation and deactivation suggest that slow processes modulate ATP-dependent gating.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  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]
|
|
|
|
 |
|
 E. J. Beck, Y. Yang, S. Yaemsiri, and V. Raghuram Conformational Changes in a Pore-lining Helix Coupled to Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating J. Biol. Chem., February 22, 2008; 283(8): 4957 - 4966. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. G. Bompadre, Y. Sohma, M. Li, and T.-C. Hwang G551D and G1349D, Two CF-associated Mutations in the Signature Sequences of CFTR, Exhibit Distinct Gating Defects J. Gen. Physiol., March 26, 2007; 129(4): 285 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Zhao and X.-b. Chang Mutation of the Aromatic Amino Acid Interacting with Adenine Moiety of ATP to a Polar Residue Alters the Properties of Multidrug Resistance Protein 1 J. Biol. Chem., November 19, 2004; 279(47): 48505 - 48512. [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]
|
|
|
|
|
|
X. Liu, S. S. Smith, F. Sun, and D. C. Dawson Cftr: Covalent Modification of Cysteine-Substituted Channels Expressed in Xenopus Oocytes Shows That Activation Is Due to the Opening of Channels Resident in the Plasma Membrane J. Gen. Physiol., October 1, 2001; 118(4): 433 - 446. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Smith, X. Liu, Z.-R. Zhang, F. Sun, T. E. Kriewall, N. A. McCarty, and D. C. Dawson Cftr: Covalent and Noncovalent Modification Suggests a Role for Fixed Charges in Anion Conduction J. Gen. Physiol., October 1, 2001; 118(4): 407 - 432. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. W. Chan, L. Csanady, D. Seto-Young, A. C. Nairn, and D. C. Gadsby Severed Molecules Functionally Define the Boundaries of the Cystic Fibrosis Transmembrane Conductance Regulator's Nh2-Terminal Nucleotide Binding Domain J. Gen. Physiol., August 1, 2000; 116(2): 163 - 180. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Akabas Cystic Fibrosis Transmembrane Conductance Regulator. STRUCTURE AND FUNCTION OF AN EPITHELIAL CHLORIDE CHANNEL J. Biol. Chem., February 11, 2000; 275(6): 3729 - 3732. [Full Text] [PDF]
|
|
|
|
|
|
J. R. Hume, D. Duan, M. L. Collier, J. Yamazaki, and B. Horowitz Anion Transport in Heart Physiol Rev, January 1, 2000; 80(1): 31 - 81. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Naren, E. Cormet-Boyaka, J. Fu, M. Villain, J. E. Blalock, M. W. Quick, and K. L. Kirk CFTR Chloride Channel Regulation by an Interdomain Interaction Science, October 15, 1999; 286(5439): 544 - 548. [Abstract] [Full Text]
|
|
|
|
|
|
S. Zeltwanger, F. Wang, G.-T. Wang, K. D. Gillis, and T.-C. Hwang Gating of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels by Adenosine Triphosphate Hydrolysis: Quantitative Analysis of a Cyclic Gating Scheme J. Gen. Physiol., April 1, 1999; 113(4): 541 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. N. SHEPPARD and M. J. WELSH Structure and Function of the CFTR Chloride Channel Physiol Rev, January 1, 1999; 79(1): 23 - 45. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. DAWSON, S. S. SMITH, and M. K. MANSOURA CFTR: Mechanism of Anion Conduction Physiol Rev, January 1, 1999; 79(1): 47 - 75. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. GADSBY and A. C. NAIRN Control of CFTR Channel Gating by Phosphorylation and Nucleotide Hydrolysis Physiol Rev, January 1, 1999; 79(1): 77 - 107. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. D. SCHULTZ, A. K. SINGH, D. C. DEVOR, and R. J. BRIDGES Pharmacology of CFTR Chloride Channel Activity Physiol Rev, January 1, 1999; 79(1): 109 - 144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. S. Seibert, P. Linsdell, T. W. Loo, J. W. Hanrahan, J. R. Riordan, and D. M. Clarke Cytoplasmic Loop Three of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Regulation of Chloride Channel Activity J. Biol. Chem., November 1, 1996; 271(44): 27493 - 27499. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ikuma and M. J. Welsh Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis PNAS, July 18, 2000; 97(15): 8675 - 8680. [Abstract] [Full Text] [PDF]
|
|
|
|
