The roles played by ATP binding and hydrolysis in the complex mechanisms that open and close cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels remain controversial. In this work, the contributions made by ATP and Mg2+ ions to the gating of phosphorylated cardiac CFTR channels were evaluated separately by measuring the rates of opening and closing of single channels in excised patches exposed to solutions in which [ATP] and [Mg2+] were varied independently. Channel opening was found to be rate-limited not by the binding of ATP alone, but by a Mg2+-dependent step that followed binding of both ATP and Mg2+. Once a channel had opened, sudden withdrawal of all Mg2+ and ATP could prevent it from closing for tens of seconds. But subsequent exposure of such an open channel to Mg2+ ions alone could close it, and the closing rate increased with [Mg2+] over the micromolar range (half maximal at ∼50 μM [Mg2+]). A simple interpretation is that channel closing is stoichiometrically coupled to hydrolysis of an ATP molecule that remains tightly associated with the open CFTR channel despite continuous washing. If correct, that ATP molecule appears able to reside for over a minute in the catalytic site that controls channel closing, implying that the site must entrap, or have an intrinsically high apparent affinity for, ATP, even without a Mg2+ ion. Such stabilization of the open-channel conformation of CFTR by tight binding, or occlusion, of an ATP molecule echoes the stabilization of the active conformation of a G protein by GTP.
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1 June 2002
Article|
May 28 2002
Distinct Mg2+-dependent Steps Rate Limit Opening and Closing of a Single CFTR Cl− Channel
Athanasios G. Dousmanis,
Athanasios G. Dousmanis
1Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021
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Angus C. Nairn,
Angus C. Nairn
2Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021
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David C. Gadsby
David C. Gadsby
1Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021
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Athanasios G. Dousmanis
1Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021
Angus C. Nairn
2Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021
David C. Gadsby
1Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021
Address correspondence to David C. Gadsby Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, 1230 York Avenue, New York, NY 10021. Fax: (212) 327-7589; E-mail: [email protected]
Dr. Dousmanis' present address is Neurological Institute, 710 West 168th Street, New York, NY 10032.
*
Abbreviations used in this paper: ABC, ATP binding cassette; CDTA, 2 trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid; CFTR, cystic fibrosis transmembrane conductance regulator; MRP, multidrug resistance–related protein.
Received:
March 13 2002
Revision Received:
April 05 2002
Accepted:
April 11 2002
Online ISSN: 1540-7748
Print ISSN: 0022-1295
The Rockefeller University Press
2002
J Gen Physiol (2002) 119 (6): 545–559.
Article history
Received:
March 13 2002
Revision Received:
April 05 2002
Accepted:
April 11 2002
Citation
Athanasios G. Dousmanis, Angus C. Nairn, David C. Gadsby; Distinct Mg2+-dependent Steps Rate Limit Opening and Closing of a Single CFTR Cl− Channel . J Gen Physiol 1 June 2002; 119 (6): 545–559. doi: https://doi.org/10.1085/jgp.20028594
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