Limits...
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.

Show MeSH

Related in: MedlinePlus

Influence of free [Mg2+] on rate of channel closing from prolonged open bursts. (A) Semilog survivor plots of probability channel stayed open, summarizing measurements from records like those in Fig. 5. Dashed lines show least-squares fits, yielding mean channel closing rates of 0.29 ± 0.01 s−1 at 1.2 mM Mg2+ (▴), 0.21 ± 0.01s−1 in 200 μM Mg2+ (▿), 0.16 ± 0.01 s−1 in 40 μM Mg2+ (♦), 0.05 ± 0.001 s−1 in 5 μM Mg2+ (○), and 0.03 ± 0.002 s−1 in 0 μM Mg2+ (•). (B) Closing rates (kobs) from data in A plotted against free [Mg2+], showing fit to kobs = k0 + {kMg[Mg2+]/([Mg2+] + K0.5)}, with k0 (closing rate in 0 Mg2+) set at 0.03, yielding kMg = 0.26 ± 0.02 s−1, and K0.5 = 51 ± 17 μM. (Inset) Semilog survivor plot of 60 open-state dwell times (○) before channel closure after sudden ATP withdrawal at constant 1.2 mM free [Mg2+] from 33 patches; single exponential fit (dashed line) gives closing rate of 0.26 ± 0.01 s−1 (○ in main graph).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2233863&req=5

fig6: Influence of free [Mg2+] on rate of channel closing from prolonged open bursts. (A) Semilog survivor plots of probability channel stayed open, summarizing measurements from records like those in Fig. 5. Dashed lines show least-squares fits, yielding mean channel closing rates of 0.29 ± 0.01 s−1 at 1.2 mM Mg2+ (▴), 0.21 ± 0.01s−1 in 200 μM Mg2+ (▿), 0.16 ± 0.01 s−1 in 40 μM Mg2+ (♦), 0.05 ± 0.001 s−1 in 5 μM Mg2+ (○), and 0.03 ± 0.002 s−1 in 0 μM Mg2+ (•). (B) Closing rates (kobs) from data in A plotted against free [Mg2+], showing fit to kobs = k0 + {kMg[Mg2+]/([Mg2+] + K0.5)}, with k0 (closing rate in 0 Mg2+) set at 0.03, yielding kMg = 0.26 ± 0.02 s−1, and K0.5 = 51 ± 17 μM. (Inset) Semilog survivor plot of 60 open-state dwell times (○) before channel closure after sudden ATP withdrawal at constant 1.2 mM free [Mg2+] from 33 patches; single exponential fit (dashed line) gives closing rate of 0.26 ± 0.01 s−1 (○ in main graph).

Mentions: Could this ∼7-fold slower opening of CFTR channels at 5 μM free Mg2+ be accounted for by the expected reduced concentration of MgATP complex available for binding at the NBDs? Under the conditions of these experiments, the concentration of the MgATP complex is calculated to be ∼100 μM at 5 μM free [Mg2+] and 2 mM total [ATP]. For comparison, Fig. 1 C shows that, at 1.2 mM free [Mg2+], channel Po (and hence opening rate) was already ∼75% of its maximal value when [MgATP] was 100 μM (compare Venglarik et al., 1994; Zeltwanger et al., 1999; Csanády et al., 2000). In other words, at high free [Mg2+] a reduction of [MgATP] from 2 mM to 100 μM would not be expected to appreciably lower the rate of channel opening. The greatly slowed opening of the CFTR channels observed at 5 μM free Mg2+ cannot, therefore, be attributed to diminished availability of MgATP complex per se. Nor can it be attributed to reduced availability of uncomplexed ATP because, on the contrary, when [MgATP] was ∼100 μM in the experiments of Fig. 3 (5 μM free Mg2+) the free [ATP] was far higher (∼1.9 mM) than it was in the 100 μM [MgATP] solution used in the experiments of Fig. 1 (free [Mg2+] 1.2 mM, free [ATP] <10 μM). The simplest interpretation, then, is that at 5 μM free [Mg2+] and 2 mM total [ATP] competition between the ∼100 μM MgATP and the ∼1.9 mM free ATP results in the NBDs being mostly occupied by ATP without a Mg2+ ion, a condition under which channel opening is demonstrably impaired (Fig. 2 A). We therefore conclude that the speed of CFTR channel opening is controlled not by the probability of ATP binding at the responsible site, but by the probability of that site (or sites) being occupied simultaneously by ATP and a Mg2+ ion. The severalfold slowing of channel opening (Figs. 3 and 4) attributed to this competition between MgATP and the almost 20-fold more plentiful free ATP further implies that free ATP may not only bind at the active site that controls opening of CFTR channels, but may do so nearly as well as MgATP. Thus, the K0.5(Po) of ∼40 μM (Fig. 1 C) implies (Csanády et al., 2000) an apparent affinity, K0.5(rCO), of ∼60 μM for MgATP binding at the site that controls opening rate (rCO), from the relation K0.5(rCO) = K0.5(Po) × (1+[rCOmax/rOC]) using maximal opening rate, rCOmax, of ∼0.2 s−1 (Fig. 4), and closing rate, rOC, of ∼0.3 s−1 (see Fig. 6 B, below). The ∼7-fold reduction in opening rate then suggests an apparent affinity of ∼170 μM for competitive binding of free ATP at the opening site. This concurs with the moderately (∼10-fold) reduced apparent affinity for binding of 8-azidoATP without Mg2+ to CFTR at ∼0°C compared with that for binding of Mg-8-azidoATP (Travis et al., 1993; cf. Aleksandrov et al., 2002).


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)

Influence of free [Mg2+] on rate of channel closing from prolonged open bursts. (A) Semilog survivor plots of probability channel stayed open, summarizing measurements from records like those in Fig. 5. Dashed lines show least-squares fits, yielding mean channel closing rates of 0.29 ± 0.01 s−1 at 1.2 mM Mg2+ (▴), 0.21 ± 0.01s−1 in 200 μM Mg2+ (▿), 0.16 ± 0.01 s−1 in 40 μM Mg2+ (♦), 0.05 ± 0.001 s−1 in 5 μM Mg2+ (○), and 0.03 ± 0.002 s−1 in 0 μM Mg2+ (•). (B) Closing rates (kobs) from data in A plotted against free [Mg2+], showing fit to kobs = k0 + {kMg[Mg2+]/([Mg2+] + K0.5)}, with k0 (closing rate in 0 Mg2+) set at 0.03, yielding kMg = 0.26 ± 0.02 s−1, and K0.5 = 51 ± 17 μM. (Inset) Semilog survivor plot of 60 open-state dwell times (○) before channel closure after sudden ATP withdrawal at constant 1.2 mM free [Mg2+] from 33 patches; single exponential fit (dashed line) gives closing rate of 0.26 ± 0.01 s−1 (○ in main graph).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233863&req=5

fig6: Influence of free [Mg2+] on rate of channel closing from prolonged open bursts. (A) Semilog survivor plots of probability channel stayed open, summarizing measurements from records like those in Fig. 5. Dashed lines show least-squares fits, yielding mean channel closing rates of 0.29 ± 0.01 s−1 at 1.2 mM Mg2+ (▴), 0.21 ± 0.01s−1 in 200 μM Mg2+ (▿), 0.16 ± 0.01 s−1 in 40 μM Mg2+ (♦), 0.05 ± 0.001 s−1 in 5 μM Mg2+ (○), and 0.03 ± 0.002 s−1 in 0 μM Mg2+ (•). (B) Closing rates (kobs) from data in A plotted against free [Mg2+], showing fit to kobs = k0 + {kMg[Mg2+]/([Mg2+] + K0.5)}, with k0 (closing rate in 0 Mg2+) set at 0.03, yielding kMg = 0.26 ± 0.02 s−1, and K0.5 = 51 ± 17 μM. (Inset) Semilog survivor plot of 60 open-state dwell times (○) before channel closure after sudden ATP withdrawal at constant 1.2 mM free [Mg2+] from 33 patches; single exponential fit (dashed line) gives closing rate of 0.26 ± 0.01 s−1 (○ in main graph).
Mentions: Could this ∼7-fold slower opening of CFTR channels at 5 μM free Mg2+ be accounted for by the expected reduced concentration of MgATP complex available for binding at the NBDs? Under the conditions of these experiments, the concentration of the MgATP complex is calculated to be ∼100 μM at 5 μM free [Mg2+] and 2 mM total [ATP]. For comparison, Fig. 1 C shows that, at 1.2 mM free [Mg2+], channel Po (and hence opening rate) was already ∼75% of its maximal value when [MgATP] was 100 μM (compare Venglarik et al., 1994; Zeltwanger et al., 1999; Csanády et al., 2000). In other words, at high free [Mg2+] a reduction of [MgATP] from 2 mM to 100 μM would not be expected to appreciably lower the rate of channel opening. The greatly slowed opening of the CFTR channels observed at 5 μM free Mg2+ cannot, therefore, be attributed to diminished availability of MgATP complex per se. Nor can it be attributed to reduced availability of uncomplexed ATP because, on the contrary, when [MgATP] was ∼100 μM in the experiments of Fig. 3 (5 μM free Mg2+) the free [ATP] was far higher (∼1.9 mM) than it was in the 100 μM [MgATP] solution used in the experiments of Fig. 1 (free [Mg2+] 1.2 mM, free [ATP] <10 μM). The simplest interpretation, then, is that at 5 μM free [Mg2+] and 2 mM total [ATP] competition between the ∼100 μM MgATP and the ∼1.9 mM free ATP results in the NBDs being mostly occupied by ATP without a Mg2+ ion, a condition under which channel opening is demonstrably impaired (Fig. 2 A). We therefore conclude that the speed of CFTR channel opening is controlled not by the probability of ATP binding at the responsible site, but by the probability of that site (or sites) being occupied simultaneously by ATP and a Mg2+ ion. The severalfold slowing of channel opening (Figs. 3 and 4) attributed to this competition between MgATP and the almost 20-fold more plentiful free ATP further implies that free ATP may not only bind at the active site that controls opening of CFTR channels, but may do so nearly as well as MgATP. Thus, the K0.5(Po) of ∼40 μM (Fig. 1 C) implies (Csanády et al., 2000) an apparent affinity, K0.5(rCO), of ∼60 μM for MgATP binding at the site that controls opening rate (rCO), from the relation K0.5(rCO) = K0.5(Po) × (1+[rCOmax/rOC]) using maximal opening rate, rCOmax, of ∼0.2 s−1 (Fig. 4), and closing rate, rOC, of ∼0.3 s−1 (see Fig. 6 B, below). The ∼7-fold reduction in opening rate then suggests an apparent affinity of ∼170 μM for competitive binding of free ATP at the opening site. This concurs with the moderately (∼10-fold) reduced apparent affinity for binding of 8-azidoATP without Mg2+ to CFTR at ∼0°C compared with that for binding of Mg-8-azidoATP (Travis et al., 1993; cf. Aleksandrov et al., 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.

Show MeSH
Related in: MedlinePlus