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Intrinsic versus extrinsic voltage sensitivity of blocker interaction with an ion channel pore.

Martínez-François JR, Lu Z - J. Gen. Physiol. (2010)

Bottom Line: To date, no systematic investigation has been performed to distinguish between these voltage-dependent mechanisms of channel block.The most fundamental characteristic of the extrinsic mechanism, i.e., that block can be rendered voltage independent, remains to be established and formally analyzed for the case of organic blockers.Additionally, a blocker generates (at least) two blocked states, which, if related serially, may preclude meaningful application of a commonly used approach for investigating channel gating, namely, inferring the properties of the activation gate from the kinetics of channel block.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Many physiological and synthetic agents act by occluding the ion conduction pore of ion channels. A hallmark of charged blockers is that their apparent affinity for the pore usually varies with membrane voltage. Two models have been proposed to explain this voltage sensitivity. One model assumes that the charged blocker itself directly senses the transmembrane electric field, i.e., that blocker binding is intrinsically voltage dependent. In the alternative model, the blocker does not directly interact with the electric field; instead, blocker binding acquires voltage dependence solely through the concurrent movement of permeant ions across the field. This latter model may better explain voltage dependence of channel block by large organic compounds that are too bulky to fit into the narrow (usually ion-selective) part of the pore where the electric field is steep. To date, no systematic investigation has been performed to distinguish between these voltage-dependent mechanisms of channel block. The most fundamental characteristic of the extrinsic mechanism, i.e., that block can be rendered voltage independent, remains to be established and formally analyzed for the case of organic blockers. Here, we observe that the voltage dependence of block of a cyclic nucleotide-gated channel by a series of intracellular quaternary ammonium blockers, which are too bulky to traverse the narrow ion selectivity filter, gradually vanishes with extreme depolarization, a predicted feature of the extrinsic voltage dependence model. In contrast, the voltage dependence of block by an amine blocker, which has a smaller "diameter" and can therefore penetrate into the selectivity filter, follows a Boltzmann function, a predicted feature of the intrinsic voltage dependence model. Additionally, a blocker generates (at least) two blocked states, which, if related serially, may preclude meaningful application of a commonly used approach for investigating channel gating, namely, inferring the properties of the activation gate from the kinetics of channel block.

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Voltage-dependent block by intracellular bis-QAC10. (A) Chemical structure of bis-QAC10. (B) Macroscopic current traces recorded from an inside-out patch containing CNGA1 channels in the absence or presence of 5 mM of intracellular bis-QAC10. Currents were elicited by first stepping the voltage from the 0-mV holding potential to −150 mV, and then testing voltages between −150 and 150 mV in 10-mV increments before returning to the holding potential. For clarity, only traces every 20 mV are shown. Dotted line indicates 0 current level. (C) I-V curves (mean ± SEM; n = 5) determined at the end of the test pulses in the absence or presence of 5 mM bis-QAC10. (D) Fraction of current not blocked (mean ± SEM; n = 5) by 5 mM bis-QAC10 is plotted against membrane voltage. The solid curve is a fit of Eq. 10 to the data from −150 to 90 mV (arrow) with KB1 = 3.17 ± 0.05 × 10−2 M, KB2 = 2.23 ± 0.14 × 10−2, KB2-Na = 2.67 ± 0.09 M−1, and Z = 1.11 ± 0.02. Dotted curve is a fit to the data from −150 to 90 mV (arrow) of a Boltzmann function (similar to Eq. 1, except for a nonunity asymptote at hyperpolarized potentials) with parameters appKd = 1.23 ± 0.08 × 10−2 M and Z = 0.83 ± 0.03.
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fig8: Voltage-dependent block by intracellular bis-QAC10. (A) Chemical structure of bis-QAC10. (B) Macroscopic current traces recorded from an inside-out patch containing CNGA1 channels in the absence or presence of 5 mM of intracellular bis-QAC10. Currents were elicited by first stepping the voltage from the 0-mV holding potential to −150 mV, and then testing voltages between −150 and 150 mV in 10-mV increments before returning to the holding potential. For clarity, only traces every 20 mV are shown. Dotted line indicates 0 current level. (C) I-V curves (mean ± SEM; n = 5) determined at the end of the test pulses in the absence or presence of 5 mM bis-QAC10. (D) Fraction of current not blocked (mean ± SEM; n = 5) by 5 mM bis-QAC10 is plotted against membrane voltage. The solid curve is a fit of Eq. 10 to the data from −150 to 90 mV (arrow) with KB1 = 3.17 ± 0.05 × 10−2 M, KB2 = 2.23 ± 0.14 × 10−2, KB2-Na = 2.67 ± 0.09 M−1, and Z = 1.11 ± 0.02. Dotted curve is a fit to the data from −150 to 90 mV (arrow) of a Boltzmann function (similar to Eq. 1, except for a nonunity asymptote at hyperpolarized potentials) with parameters appKd = 1.23 ± 0.08 × 10−2 M and Z = 0.83 ± 0.03.

Mentions: CNGA1 channels are blocked by millimolar concentrations of QAs from the intracellular but not extracellular side (Goulding et al., 1993; Stotz and Haynes, 1996; Contreras and Holmgren, 2006). Because QAs are generally too bulky to enter the narrow selectivity filter, the voltage sensitivity of their block of CNGA1 channels must arise indirectly, as it does when they block K+ channels (Armstrong, 1971; French and Shoukimas, 1981; Spassova and Lu, 1998; Shin and Lu, 2005; Xu et al., 2009). We examined the voltage dependence of CNGA1 block by a series of QAs to learn how it differs from block by PhTx. Using decane-bis-trimethylammonium (bis-QAC10; Fig. 8 A) as an example, we will first illustrate the basic properties of QA block.


Intrinsic versus extrinsic voltage sensitivity of blocker interaction with an ion channel pore.

Martínez-François JR, Lu Z - J. Gen. Physiol. (2010)

Voltage-dependent block by intracellular bis-QAC10. (A) Chemical structure of bis-QAC10. (B) Macroscopic current traces recorded from an inside-out patch containing CNGA1 channels in the absence or presence of 5 mM of intracellular bis-QAC10. Currents were elicited by first stepping the voltage from the 0-mV holding potential to −150 mV, and then testing voltages between −150 and 150 mV in 10-mV increments before returning to the holding potential. For clarity, only traces every 20 mV are shown. Dotted line indicates 0 current level. (C) I-V curves (mean ± SEM; n = 5) determined at the end of the test pulses in the absence or presence of 5 mM bis-QAC10. (D) Fraction of current not blocked (mean ± SEM; n = 5) by 5 mM bis-QAC10 is plotted against membrane voltage. The solid curve is a fit of Eq. 10 to the data from −150 to 90 mV (arrow) with KB1 = 3.17 ± 0.05 × 10−2 M, KB2 = 2.23 ± 0.14 × 10−2, KB2-Na = 2.67 ± 0.09 M−1, and Z = 1.11 ± 0.02. Dotted curve is a fit to the data from −150 to 90 mV (arrow) of a Boltzmann function (similar to Eq. 1, except for a nonunity asymptote at hyperpolarized potentials) with parameters appKd = 1.23 ± 0.08 × 10−2 M and Z = 0.83 ± 0.03.
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fig8: Voltage-dependent block by intracellular bis-QAC10. (A) Chemical structure of bis-QAC10. (B) Macroscopic current traces recorded from an inside-out patch containing CNGA1 channels in the absence or presence of 5 mM of intracellular bis-QAC10. Currents were elicited by first stepping the voltage from the 0-mV holding potential to −150 mV, and then testing voltages between −150 and 150 mV in 10-mV increments before returning to the holding potential. For clarity, only traces every 20 mV are shown. Dotted line indicates 0 current level. (C) I-V curves (mean ± SEM; n = 5) determined at the end of the test pulses in the absence or presence of 5 mM bis-QAC10. (D) Fraction of current not blocked (mean ± SEM; n = 5) by 5 mM bis-QAC10 is plotted against membrane voltage. The solid curve is a fit of Eq. 10 to the data from −150 to 90 mV (arrow) with KB1 = 3.17 ± 0.05 × 10−2 M, KB2 = 2.23 ± 0.14 × 10−2, KB2-Na = 2.67 ± 0.09 M−1, and Z = 1.11 ± 0.02. Dotted curve is a fit to the data from −150 to 90 mV (arrow) of a Boltzmann function (similar to Eq. 1, except for a nonunity asymptote at hyperpolarized potentials) with parameters appKd = 1.23 ± 0.08 × 10−2 M and Z = 0.83 ± 0.03.
Mentions: CNGA1 channels are blocked by millimolar concentrations of QAs from the intracellular but not extracellular side (Goulding et al., 1993; Stotz and Haynes, 1996; Contreras and Holmgren, 2006). Because QAs are generally too bulky to enter the narrow selectivity filter, the voltage sensitivity of their block of CNGA1 channels must arise indirectly, as it does when they block K+ channels (Armstrong, 1971; French and Shoukimas, 1981; Spassova and Lu, 1998; Shin and Lu, 2005; Xu et al., 2009). We examined the voltage dependence of CNGA1 block by a series of QAs to learn how it differs from block by PhTx. Using decane-bis-trimethylammonium (bis-QAC10; Fig. 8 A) as an example, we will first illustrate the basic properties of QA block.

Bottom Line: To date, no systematic investigation has been performed to distinguish between these voltage-dependent mechanisms of channel block.The most fundamental characteristic of the extrinsic mechanism, i.e., that block can be rendered voltage independent, remains to be established and formally analyzed for the case of organic blockers.Additionally, a blocker generates (at least) two blocked states, which, if related serially, may preclude meaningful application of a commonly used approach for investigating channel gating, namely, inferring the properties of the activation gate from the kinetics of channel block.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

ABSTRACT
Many physiological and synthetic agents act by occluding the ion conduction pore of ion channels. A hallmark of charged blockers is that their apparent affinity for the pore usually varies with membrane voltage. Two models have been proposed to explain this voltage sensitivity. One model assumes that the charged blocker itself directly senses the transmembrane electric field, i.e., that blocker binding is intrinsically voltage dependent. In the alternative model, the blocker does not directly interact with the electric field; instead, blocker binding acquires voltage dependence solely through the concurrent movement of permeant ions across the field. This latter model may better explain voltage dependence of channel block by large organic compounds that are too bulky to fit into the narrow (usually ion-selective) part of the pore where the electric field is steep. To date, no systematic investigation has been performed to distinguish between these voltage-dependent mechanisms of channel block. The most fundamental characteristic of the extrinsic mechanism, i.e., that block can be rendered voltage independent, remains to be established and formally analyzed for the case of organic blockers. Here, we observe that the voltage dependence of block of a cyclic nucleotide-gated channel by a series of intracellular quaternary ammonium blockers, which are too bulky to traverse the narrow ion selectivity filter, gradually vanishes with extreme depolarization, a predicted feature of the extrinsic voltage dependence model. In contrast, the voltage dependence of block by an amine blocker, which has a smaller "diameter" and can therefore penetrate into the selectivity filter, follows a Boltzmann function, a predicted feature of the intrinsic voltage dependence model. Additionally, a blocker generates (at least) two blocked states, which, if related serially, may preclude meaningful application of a commonly used approach for investigating channel gating, namely, inferring the properties of the activation gate from the kinetics of channel block.

Show MeSH
Related in: MedlinePlus