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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 dependence of intracellular PhTx block under low Na+ conditions. (A) Macroscopic current traces recorded in an inside-out patch containing CNGA1 channels in the absence or presence of 5 µM of intracellular PhTx in symmetrical 30 mM Na+. Currents were activated with 2 mM of intracellular cGMP and elicited with the voltage protocol shown. Dotted line indicates 0 current level. (B) Fraction of current not blocked (mean ± SEM; n = 4–8) by the indicated concentrations of intracellular PhTx is plotted against membrane voltage. Curves are fits of Eq. 3 to the three datasets simultaneously with Z1 fixed at 0. The best-fit parameters were: K1 = 1.04 ± 0.03 × 10−5 M, K2 = 2.03 ± 0.14 × 10−3, and Z2 = 2.08 ± 0.04. (C) Fraction of current not blocked (mean ± SEM; n = 8) by 1 µM of intracellular PhTx in the presence of 0.02 mM (filled circles) or 2 mM (open circles; taken from B) cGMP is plotted against membrane voltage. Curves are fits of Eq. 3 to both datasets simultaneously, with Z1 set to 0 and K1 common to both cGMP curves. The best-fit parameters were: K1 = 7.65 ± 0.22 × 10−6 M for both cGMP concentrations; K2 = 1.29 ± 0.08 × 10−2 and Z2 = 1.92 ± 0.05 for 0.02 mM cGMP; and K2 = 1.72 ± 0.18 × 10−3 and Z2 = 2.29 ± 0.06 for 2 mM cGMP. (D) Kinetics of depolarization-induced CNGA1 block by intracellular PhTx in 30 mM Na+. Natural logarithm of kon, determined as in Fig. 3, at four concentrations (0.03, 0.1, 0.3, and 1 µM; n = 6) of intracellular PhTx is plotted against membrane voltage. The plot is fitted with the equation ln kon = ln kon (0 mV) + zonVF/RT, yielding kon (0 mV) = 8.42 ± 0.68 × 108 M−1s−1 and zon = 0.14 ± 0.01. (E) Kinetics of hyperpolarization-induced recovery from CNGA1 block by intracellular PhTx in 30 mM Na+. Natural logarithm of koff, determined as in Fig. 4, at four concentrations (0.1, 0.3, 0.6, and 1 µM; n = 6) of intracellular PhTx is plotted against membrane voltage. The plot is fitted with the equation ln koff = ln koff (0 mV) − zoffVF/RT, yielding koff (0 mV) = 27 ± 2 s−1 and zoff = 1.10 ± 0.02.
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fig6: Voltage dependence of intracellular PhTx block under low Na+ conditions. (A) Macroscopic current traces recorded in an inside-out patch containing CNGA1 channels in the absence or presence of 5 µM of intracellular PhTx in symmetrical 30 mM Na+. Currents were activated with 2 mM of intracellular cGMP and elicited with the voltage protocol shown. Dotted line indicates 0 current level. (B) Fraction of current not blocked (mean ± SEM; n = 4–8) by the indicated concentrations of intracellular PhTx is plotted against membrane voltage. Curves are fits of Eq. 3 to the three datasets simultaneously with Z1 fixed at 0. The best-fit parameters were: K1 = 1.04 ± 0.03 × 10−5 M, K2 = 2.03 ± 0.14 × 10−3, and Z2 = 2.08 ± 0.04. (C) Fraction of current not blocked (mean ± SEM; n = 8) by 1 µM of intracellular PhTx in the presence of 0.02 mM (filled circles) or 2 mM (open circles; taken from B) cGMP is plotted against membrane voltage. Curves are fits of Eq. 3 to both datasets simultaneously, with Z1 set to 0 and K1 common to both cGMP curves. The best-fit parameters were: K1 = 7.65 ± 0.22 × 10−6 M for both cGMP concentrations; K2 = 1.29 ± 0.08 × 10−2 and Z2 = 1.92 ± 0.05 for 0.02 mM cGMP; and K2 = 1.72 ± 0.18 × 10−3 and Z2 = 2.29 ± 0.06 for 2 mM cGMP. (D) Kinetics of depolarization-induced CNGA1 block by intracellular PhTx in 30 mM Na+. Natural logarithm of kon, determined as in Fig. 3, at four concentrations (0.03, 0.1, 0.3, and 1 µM; n = 6) of intracellular PhTx is plotted against membrane voltage. The plot is fitted with the equation ln kon = ln kon (0 mV) + zonVF/RT, yielding kon (0 mV) = 8.42 ± 0.68 × 108 M−1s−1 and zon = 0.14 ± 0.01. (E) Kinetics of hyperpolarization-induced recovery from CNGA1 block by intracellular PhTx in 30 mM Na+. Natural logarithm of koff, determined as in Fig. 4, at four concentrations (0.1, 0.3, 0.6, and 1 µM; n = 6) of intracellular PhTx is plotted against membrane voltage. The plot is fitted with the equation ln koff = ln koff (0 mV) − zoffVF/RT, yielding koff (0 mV) = 27 ± 2 s−1 and zoff = 1.10 ± 0.02.

Mentions: The simplest way to reveal a suspected low affinity blocking state would be to apply much higher PhTx concentrations, but this was cost prohibitive. We therefore increased the apparent affinity of the toxin by lowering the Na+ concentration on both sides of the membrane from 130 mM (used in all experiments thus far) to 30 mM. As shown in Fig. 6 (A and B), under low Na+ conditions, intracellular PhTx, as expected, produces voltage-dependent block with markedly higher affinity. Lowering Na+ now also reveals the existence of dose-dependent but voltage-independent channel block at highly hyperpolarized voltages (−100 to −150 mV). Thus, intracellular PhTx apparently produces both voltage-dependent and -independent blocked states, and the blocking curve is no longer described by a simple Boltzmann function.


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

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

Voltage dependence of intracellular PhTx block under low Na+ conditions. (A) Macroscopic current traces recorded in an inside-out patch containing CNGA1 channels in the absence or presence of 5 µM of intracellular PhTx in symmetrical 30 mM Na+. Currents were activated with 2 mM of intracellular cGMP and elicited with the voltage protocol shown. Dotted line indicates 0 current level. (B) Fraction of current not blocked (mean ± SEM; n = 4–8) by the indicated concentrations of intracellular PhTx is plotted against membrane voltage. Curves are fits of Eq. 3 to the three datasets simultaneously with Z1 fixed at 0. The best-fit parameters were: K1 = 1.04 ± 0.03 × 10−5 M, K2 = 2.03 ± 0.14 × 10−3, and Z2 = 2.08 ± 0.04. (C) Fraction of current not blocked (mean ± SEM; n = 8) by 1 µM of intracellular PhTx in the presence of 0.02 mM (filled circles) or 2 mM (open circles; taken from B) cGMP is plotted against membrane voltage. Curves are fits of Eq. 3 to both datasets simultaneously, with Z1 set to 0 and K1 common to both cGMP curves. The best-fit parameters were: K1 = 7.65 ± 0.22 × 10−6 M for both cGMP concentrations; K2 = 1.29 ± 0.08 × 10−2 and Z2 = 1.92 ± 0.05 for 0.02 mM cGMP; and K2 = 1.72 ± 0.18 × 10−3 and Z2 = 2.29 ± 0.06 for 2 mM cGMP. (D) Kinetics of depolarization-induced CNGA1 block by intracellular PhTx in 30 mM Na+. Natural logarithm of kon, determined as in Fig. 3, at four concentrations (0.03, 0.1, 0.3, and 1 µM; n = 6) of intracellular PhTx is plotted against membrane voltage. The plot is fitted with the equation ln kon = ln kon (0 mV) + zonVF/RT, yielding kon (0 mV) = 8.42 ± 0.68 × 108 M−1s−1 and zon = 0.14 ± 0.01. (E) Kinetics of hyperpolarization-induced recovery from CNGA1 block by intracellular PhTx in 30 mM Na+. Natural logarithm of koff, determined as in Fig. 4, at four concentrations (0.1, 0.3, 0.6, and 1 µM; n = 6) of intracellular PhTx is plotted against membrane voltage. The plot is fitted with the equation ln koff = ln koff (0 mV) − zoffVF/RT, yielding koff (0 mV) = 27 ± 2 s−1 and zoff = 1.10 ± 0.02.
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Related In: Results  -  Collection

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fig6: Voltage dependence of intracellular PhTx block under low Na+ conditions. (A) Macroscopic current traces recorded in an inside-out patch containing CNGA1 channels in the absence or presence of 5 µM of intracellular PhTx in symmetrical 30 mM Na+. Currents were activated with 2 mM of intracellular cGMP and elicited with the voltage protocol shown. Dotted line indicates 0 current level. (B) Fraction of current not blocked (mean ± SEM; n = 4–8) by the indicated concentrations of intracellular PhTx is plotted against membrane voltage. Curves are fits of Eq. 3 to the three datasets simultaneously with Z1 fixed at 0. The best-fit parameters were: K1 = 1.04 ± 0.03 × 10−5 M, K2 = 2.03 ± 0.14 × 10−3, and Z2 = 2.08 ± 0.04. (C) Fraction of current not blocked (mean ± SEM; n = 8) by 1 µM of intracellular PhTx in the presence of 0.02 mM (filled circles) or 2 mM (open circles; taken from B) cGMP is plotted against membrane voltage. Curves are fits of Eq. 3 to both datasets simultaneously, with Z1 set to 0 and K1 common to both cGMP curves. The best-fit parameters were: K1 = 7.65 ± 0.22 × 10−6 M for both cGMP concentrations; K2 = 1.29 ± 0.08 × 10−2 and Z2 = 1.92 ± 0.05 for 0.02 mM cGMP; and K2 = 1.72 ± 0.18 × 10−3 and Z2 = 2.29 ± 0.06 for 2 mM cGMP. (D) Kinetics of depolarization-induced CNGA1 block by intracellular PhTx in 30 mM Na+. Natural logarithm of kon, determined as in Fig. 3, at four concentrations (0.03, 0.1, 0.3, and 1 µM; n = 6) of intracellular PhTx is plotted against membrane voltage. The plot is fitted with the equation ln kon = ln kon (0 mV) + zonVF/RT, yielding kon (0 mV) = 8.42 ± 0.68 × 108 M−1s−1 and zon = 0.14 ± 0.01. (E) Kinetics of hyperpolarization-induced recovery from CNGA1 block by intracellular PhTx in 30 mM Na+. Natural logarithm of koff, determined as in Fig. 4, at four concentrations (0.1, 0.3, 0.6, and 1 µM; n = 6) of intracellular PhTx is plotted against membrane voltage. The plot is fitted with the equation ln koff = ln koff (0 mV) − zoffVF/RT, yielding koff (0 mV) = 27 ± 2 s−1 and zoff = 1.10 ± 0.02.
Mentions: The simplest way to reveal a suspected low affinity blocking state would be to apply much higher PhTx concentrations, but this was cost prohibitive. We therefore increased the apparent affinity of the toxin by lowering the Na+ concentration on both sides of the membrane from 130 mM (used in all experiments thus far) to 30 mM. As shown in Fig. 6 (A and B), under low Na+ conditions, intracellular PhTx, as expected, produces voltage-dependent block with markedly higher affinity. Lowering Na+ now also reveals the existence of dose-dependent but voltage-independent channel block at highly hyperpolarized voltages (−100 to −150 mV). Thus, intracellular PhTx apparently produces both voltage-dependent and -independent blocked states, and the blocking curve is no longer described by a simple Boltzmann function.

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