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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: gadsby{at}mail.rockefeller.edu
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.
Key Words: single channels • gating kinetics • nucleotide binding domains • ATP binding • free [Mg2+]
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