Limits...
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.

Show MeSH

Related in: MedlinePlus

cGMP activation of CNGA1 channels. (A) Macroscopic current traces recorded in symmetric 130 mM Na+ from an inside-out patch containing CNGA1 channels in the presence of the indicated concentrations of intracellular cGMP. Currents were elicited by stepping from the 0-mV holding potential to voltages between −200 and 200 mV in 50-mV increments. Currents in the absence of cGMP were used as templates for subsequent offline background current corrections. Dotted lines indicate 0 current levels. (B) Fraction of maximal current (I/Imax; mean ± SEM; n = 3–7) plotted against cGMP concentration for −100 mV (squares) and 100 mV (circles). Solid curves are Hill equation fits yielding EC50 = 79 ± 1 µM and h = 1.37 ± 0.03 at −100 mV, and EC50 = 64 ± 2 µM and h = 1.39 ± 0.05 at 100 mV.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2812505&req=5

fig1: cGMP activation of CNGA1 channels. (A) Macroscopic current traces recorded in symmetric 130 mM Na+ from an inside-out patch containing CNGA1 channels in the presence of the indicated concentrations of intracellular cGMP. Currents were elicited by stepping from the 0-mV holding potential to voltages between −200 and 200 mV in 50-mV increments. Currents in the absence of cGMP were used as templates for subsequent offline background current corrections. Dotted lines indicate 0 current levels. (B) Fraction of maximal current (I/Imax; mean ± SEM; n = 3–7) plotted against cGMP concentration for −100 mV (squares) and 100 mV (circles). Solid curves are Hill equation fits yielding EC50 = 79 ± 1 µM and h = 1.37 ± 0.03 at −100 mV, and EC50 = 64 ± 2 µM and h = 1.39 ± 0.05 at 100 mV.

Mentions: We first constructed a dose–response curve for cGMP activation of retinal CNGA1 channels in our recording system. Fig. 1 A shows currents recorded from an inside-out membrane patch in the presence of four concentrations of intracellular cGMP. Confirming previous reports (Kaupp et al., 1989; Goulding et al., 1994; Zagotta and Siegelbaum, 1996; Benndorf et al., 1999), cGMP stimulates current with an EC50 of ∼80 µM, approaching saturation at 2 mM cGMP. The dose–response curve differs little between −100 and 100 mV (Fig. 1 B).


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

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

cGMP activation of CNGA1 channels. (A) Macroscopic current traces recorded in symmetric 130 mM Na+ from an inside-out patch containing CNGA1 channels in the presence of the indicated concentrations of intracellular cGMP. Currents were elicited by stepping from the 0-mV holding potential to voltages between −200 and 200 mV in 50-mV increments. Currents in the absence of cGMP were used as templates for subsequent offline background current corrections. Dotted lines indicate 0 current levels. (B) Fraction of maximal current (I/Imax; mean ± SEM; n = 3–7) plotted against cGMP concentration for −100 mV (squares) and 100 mV (circles). Solid curves are Hill equation fits yielding EC50 = 79 ± 1 µM and h = 1.37 ± 0.03 at −100 mV, and EC50 = 64 ± 2 µM and h = 1.39 ± 0.05 at 100 mV.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812505&req=5

fig1: cGMP activation of CNGA1 channels. (A) Macroscopic current traces recorded in symmetric 130 mM Na+ from an inside-out patch containing CNGA1 channels in the presence of the indicated concentrations of intracellular cGMP. Currents were elicited by stepping from the 0-mV holding potential to voltages between −200 and 200 mV in 50-mV increments. Currents in the absence of cGMP were used as templates for subsequent offline background current corrections. Dotted lines indicate 0 current levels. (B) Fraction of maximal current (I/Imax; mean ± SEM; n = 3–7) plotted against cGMP concentration for −100 mV (squares) and 100 mV (circles). Solid curves are Hill equation fits yielding EC50 = 79 ± 1 µM and h = 1.37 ± 0.03 at −100 mV, and EC50 = 64 ± 2 µM and h = 1.39 ± 0.05 at 100 mV.
Mentions: We first constructed a dose–response curve for cGMP activation of retinal CNGA1 channels in our recording system. Fig. 1 A shows currents recorded from an inside-out membrane patch in the presence of four concentrations of intracellular cGMP. Confirming previous reports (Kaupp et al., 1989; Goulding et al., 1994; Zagotta and Siegelbaum, 1996; Benndorf et al., 1999), cGMP stimulates current with an EC50 of ∼80 µM, approaching saturation at 2 mM cGMP. The dose–response curve differs little between −100 and 100 mV (Fig. 1 B).

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