Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl(-) channel.
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 Mg(2+) 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 [Mg(2+)] were varied independently. Channel opening was found to be rate-limited not by the binding of ATP alone, but by a Mg(2+)-dependent step that followed binding of both ATP and Mg(2+). Once a channel had opened, sudden withdrawal of all Mg(2+) and ATP could prevent it from closing for tens of seconds. But subsequent exposure of such an open channel to Mg(2+) ions alone could close it, and the closing rate increased with [Mg(2+)] over the micromolar range (half maximal at approximately 50 microM [Mg(2+)]). 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 Mg(2+) 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.
Dousmanis
Athanasios G
AG
Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021, USA.
Nairn
Angus C
AC
Gadsby
David C
DC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000502:physiology*; D004533:Egtazic Acid / Q000494:pharmacology; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000166:cytology; D015640:Ion Channel Gating / Q000187:drug effects*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D009206:Myocardium / Q000166:cytology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D017434:Protein Structure, Tertiary
D002614:Chelating Agents; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000266:Adenylyl Imidodiphosphate; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D008274:Magnesium