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Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl(-) channel.

Dousmanis AG, Nairn AC, Gadsby DC - J. Gen. Physiol. (2002)

Bottom Line: 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+).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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
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.

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At constant high (1.2 mM) free [Mg2+], [ATP] (≈ [MgATP]) determines opening rate of cardiac CFTR channels. (A and B) Records of unitary currents in two representative inside-out patches (after PKA washout) containing ≥5 (A) phosphorylated CFTR channels (Vh = 20 mV), or only one (B; Vh = 0 mV), respectively. Standard ATP-free bath (cytoplasmic) solution was replaced by one containing a test [MgATP] (1, 5, 50 μM), between exposures to the reference [MgATP] (2 mM), as indicated; solution was exchanged within ∼1 s. Relative Po was determined in each patch as the ratio of that at the test [MgATP] to the mean of the two bracketing Po values at 2 mM MgATP. (C) Summary of relative Po (rel Po) at test [MgATP] of 0, 1, 5, 50, and 200 μM, averaged from 5–20 measurements at each [MgATP] of phosphorylated channels but after withdrawal of PKA. The curve shows the least-squares fit to the Hill equation: rel Po = Po([MgATP])/Po(2 mM) = rel Pomax/{1+(K0.5/[ATP])n}, where n = 0.9 ± 0.2, K0.5 = 35 ± 11 μM, and rel Pomax = 1.04 ± 0.06.
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fig1: At constant high (1.2 mM) free [Mg2+], [ATP] (≈ [MgATP]) determines opening rate of cardiac CFTR channels. (A and B) Records of unitary currents in two representative inside-out patches (after PKA washout) containing ≥5 (A) phosphorylated CFTR channels (Vh = 20 mV), or only one (B; Vh = 0 mV), respectively. Standard ATP-free bath (cytoplasmic) solution was replaced by one containing a test [MgATP] (1, 5, 50 μM), between exposures to the reference [MgATP] (2 mM), as indicated; solution was exchanged within ∼1 s. Relative Po was determined in each patch as the ratio of that at the test [MgATP] to the mean of the two bracketing Po values at 2 mM MgATP. (C) Summary of relative Po (rel Po) at test [MgATP] of 0, 1, 5, 50, and 200 μM, averaged from 5–20 measurements at each [MgATP] of phosphorylated channels but after withdrawal of PKA. The curve shows the least-squares fit to the Hill equation: rel Po = Po([MgATP])/Po(2 mM) = rel Pomax/{1+(K0.5/[ATP])n}, where n = 0.9 ± 0.2, K0.5 = 35 ± 11 μM, and rel Pomax = 1.04 ± 0.06.

Mentions: Average Po was determined by dividing the area under open channel currents by that expected if the largest number of simultaneously observed channels were all open. Closed (interburst) times were measured only in patches that showed no simultaneous openings at 2 mM [MgATP] and 1.2 mM free [Mg2+] (average Po = 0.41 ± 0.04 for the 15 single-channel patches of Fig. 1 C); open burst times were predominantly taken from single-channel patches, but were occasionally (at low [MgATP]) measured in patches containing two or three channels, though only during periods devoid of simultaneous openings. Burst and interburst durations were measured directly from amplified high-speed chart recordings using the half-amplitude criterion to identify openings and closings; control recordings of rectangular test pulses showed that openings or closings ≥35 ms long could be detected, i.e., for burst durations, the method is equivalent to suppressing brief “flickery” closures <35 ms (compare Winter et al., 1994; Zeltwanger et al., 1999; Csanády et al., 2000).


Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl(-) channel.

Dousmanis AG, Nairn AC, Gadsby DC - J. Gen. Physiol. (2002)

At constant high (1.2 mM) free [Mg2+], [ATP] (≈ [MgATP]) determines opening rate of cardiac CFTR channels. (A and B) Records of unitary currents in two representative inside-out patches (after PKA washout) containing ≥5 (A) phosphorylated CFTR channels (Vh = 20 mV), or only one (B; Vh = 0 mV), respectively. Standard ATP-free bath (cytoplasmic) solution was replaced by one containing a test [MgATP] (1, 5, 50 μM), between exposures to the reference [MgATP] (2 mM), as indicated; solution was exchanged within ∼1 s. Relative Po was determined in each patch as the ratio of that at the test [MgATP] to the mean of the two bracketing Po values at 2 mM MgATP. (C) Summary of relative Po (rel Po) at test [MgATP] of 0, 1, 5, 50, and 200 μM, averaged from 5–20 measurements at each [MgATP] of phosphorylated channels but after withdrawal of PKA. The curve shows the least-squares fit to the Hill equation: rel Po = Po([MgATP])/Po(2 mM) = rel Pomax/{1+(K0.5/[ATP])n}, where n = 0.9 ± 0.2, K0.5 = 35 ± 11 μM, and rel Pomax = 1.04 ± 0.06.
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Related In: Results  -  Collection

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fig1: At constant high (1.2 mM) free [Mg2+], [ATP] (≈ [MgATP]) determines opening rate of cardiac CFTR channels. (A and B) Records of unitary currents in two representative inside-out patches (after PKA washout) containing ≥5 (A) phosphorylated CFTR channels (Vh = 20 mV), or only one (B; Vh = 0 mV), respectively. Standard ATP-free bath (cytoplasmic) solution was replaced by one containing a test [MgATP] (1, 5, 50 μM), between exposures to the reference [MgATP] (2 mM), as indicated; solution was exchanged within ∼1 s. Relative Po was determined in each patch as the ratio of that at the test [MgATP] to the mean of the two bracketing Po values at 2 mM MgATP. (C) Summary of relative Po (rel Po) at test [MgATP] of 0, 1, 5, 50, and 200 μM, averaged from 5–20 measurements at each [MgATP] of phosphorylated channels but after withdrawal of PKA. The curve shows the least-squares fit to the Hill equation: rel Po = Po([MgATP])/Po(2 mM) = rel Pomax/{1+(K0.5/[ATP])n}, where n = 0.9 ± 0.2, K0.5 = 35 ± 11 μM, and rel Pomax = 1.04 ± 0.06.
Mentions: Average Po was determined by dividing the area under open channel currents by that expected if the largest number of simultaneously observed channels were all open. Closed (interburst) times were measured only in patches that showed no simultaneous openings at 2 mM [MgATP] and 1.2 mM free [Mg2+] (average Po = 0.41 ± 0.04 for the 15 single-channel patches of Fig. 1 C); open burst times were predominantly taken from single-channel patches, but were occasionally (at low [MgATP]) measured in patches containing two or three channels, though only during periods devoid of simultaneous openings. Burst and interburst durations were measured directly from amplified high-speed chart recordings using the half-amplitude criterion to identify openings and closings; control recordings of rectangular test pulses showed that openings or closings ≥35 ms long could be detected, i.e., for burst durations, the method is equivalent to suppressing brief “flickery” closures <35 ms (compare Winter et al., 1994; Zeltwanger et al., 1999; Csanády et al., 2000).

Bottom Line: 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+).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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cardiac/Membrane Physiology, The Rockefeller University, New York, NY 10021, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus