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Properties of the inner pore region of TRPV1 channels revealed by block with quaternary ammoniums.

Jara-Oseguera A, Llorente I, Rosenbaum T, Islas LD - J. Gen. Physiol. (2008)

Bottom Line: We found that all four QAs used, tetraethylammonium (TEA), tetrapropylammonium (TPrA), tetrabutylammonium, and tetrapentylammonium, block the TRPV1 channel from the intracellular face of the channel in a voltage-dependent manner, and that block by these molecules occurs with different kinetics, with the bigger molecules becoming slower blockers.We also found that TPrA and the larger QAs can only block the channel in the open state, and that they interfere with the channel's activation gate upon closing, which is observed as a slowing of tail current kinetics.The dependence of the rate constants on the size of the blocker suggests a size of around 10 A for the inner pore of TRPV1 channels.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Fisiología, Facultad de Medicina, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, D.F., 04510, México

ABSTRACT
The transient receptor potential vanilloid 1 (TRPV1) nonselective cationic channel is a polymodal receptor that activates in response to a wide variety of stimuli. To date, little structural information about this channel is available. Here, we used quaternary ammonium ions (QAs) of different sizes in an effort to gain some insight into the nature and dimensions of the pore of TRPV1. We found that all four QAs used, tetraethylammonium (TEA), tetrapropylammonium (TPrA), tetrabutylammonium, and tetrapentylammonium, block the TRPV1 channel from the intracellular face of the channel in a voltage-dependent manner, and that block by these molecules occurs with different kinetics, with the bigger molecules becoming slower blockers. We also found that TPrA and the larger QAs can only block the channel in the open state, and that they interfere with the channel's activation gate upon closing, which is observed as a slowing of tail current kinetics. TEA does not interfere with the activation gate, indicating that this molecule can reside in its blocking site even when the channel is closed. The dependence of the rate constants on the size of the blocker suggests a size of around 10 A for the inner pore of TRPV1 channels.

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Kinetics of block by intracellular TEA determined with the β distribution. (A) Single-channel openings in the absence (top trace) and in the presence of 2, 5, and 20 mM TEA obtained at 60 mV. The apparent current amplitude decreases as the TEA concentration is increased, as predicted for a fast blocker. The dashed line represents the closed-channel current level, and the dotted line is the mean open-channel level in the absence of TEA. (B) Normalized amplitude histograms obtained from traces as in A. Solid lines are fits to the β distribution with the following parameters: 2 mM, β = 26,679 s−1, α = 61,454 s−1; 5 mM, β = 43,001 s−1, α = 55,882 s−1; 20 mM, β = 156,370 s−1, α = 44,582 s−1. (C) Blocking rate constants obtained from the fits to the β distribution as in B from three separate patches at several voltages and blocker concentrations. The on-rate, kon, increases with voltage and reaches a plateau. The solid line is a fit to a double exponential function reflecting both the voltage dependence of the on-rate and of the relief of block and has the form\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}k_{on}=\frac{1}{k_{ac}(0){\mathrm{exp}}z_{ac}V/kT}+\frac{1}{k_{rel}(0){\mathrm{exp}}z_{rel}V/kT}.\end{equation*}\end{document}The values of the fit parameters are: kac(0) = 1.171 × 106 M−1s−1; zac = 1.0193; krel(0) = 4.722 × 107 M−1s−1; zrel = 0.48. The off-rate decreases with increasing voltages but starts to increase at voltages more positive than 60 mV. The values of kon and koff at 0 mV were estimated from an exponential fit to the data between 240 and 60 mV, and the parameters of the fit are: kon(0) = 1.04 × 106 M−1s−1; z = 0.94 eo; koff(0) = 28,950 s−1; z = 0.27 eo. Group data are presented as mean ± SEM.
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fig4: Kinetics of block by intracellular TEA determined with the β distribution. (A) Single-channel openings in the absence (top trace) and in the presence of 2, 5, and 20 mM TEA obtained at 60 mV. The apparent current amplitude decreases as the TEA concentration is increased, as predicted for a fast blocker. The dashed line represents the closed-channel current level, and the dotted line is the mean open-channel level in the absence of TEA. (B) Normalized amplitude histograms obtained from traces as in A. Solid lines are fits to the β distribution with the following parameters: 2 mM, β = 26,679 s−1, α = 61,454 s−1; 5 mM, β = 43,001 s−1, α = 55,882 s−1; 20 mM, β = 156,370 s−1, α = 44,582 s−1. (C) Blocking rate constants obtained from the fits to the β distribution as in B from three separate patches at several voltages and blocker concentrations. The on-rate, kon, increases with voltage and reaches a plateau. The solid line is a fit to a double exponential function reflecting both the voltage dependence of the on-rate and of the relief of block and has the form\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}k_{on}=\frac{1}{k_{ac}(0){\mathrm{exp}}z_{ac}V/kT}+\frac{1}{k_{rel}(0){\mathrm{exp}}z_{rel}V/kT}.\end{equation*}\end{document}The values of the fit parameters are: kac(0) = 1.171 × 106 M−1s−1; zac = 1.0193; krel(0) = 4.722 × 107 M−1s−1; zrel = 0.48. The off-rate decreases with increasing voltages but starts to increase at voltages more positive than 60 mV. The values of kon and koff at 0 mV were estimated from an exponential fit to the data between 240 and 60 mV, and the parameters of the fit are: kon(0) = 1.04 × 106 M−1s−1; z = 0.94 eo; koff(0) = 28,950 s−1; z = 0.27 eo. Group data are presented as mean ± SEM.

Mentions: Block of TRPV1 by TEA is a very fast process. The individual blocking events are so short-lived that they are smeared by the filter and, as a result, block appears as an apparent reduction of the single-channel conductance. For this reason and to estimate the kinetic parameters of the blocking reaction, we used an approach to analyze the all-points amplitude histogram from bursts of openings, which makes use of the β distribution. As discussed in Materials and methods, we showed that this approximation can describe a two-state process filtered by a Gaussian filter with reasonable accuracy. Fig. 4 A shows representative single-channel openings in the absence and presence of increasing concentrations of TEA obtained at 60 mV. It is apparent that the effect of the blocker is a reduction of the single channel current. Fig. 4 B shows amplitude histograms for channel openings obtained from traces as in Fig. 4 A and constructed as indicated in Materials and methods. Superimposed on the histograms are fits to Eq. 2 convolved with a Gaussian function, which describes the current amplitude distribution of the closed level in the absence of blocker.


Properties of the inner pore region of TRPV1 channels revealed by block with quaternary ammoniums.

Jara-Oseguera A, Llorente I, Rosenbaum T, Islas LD - J. Gen. Physiol. (2008)

Kinetics of block by intracellular TEA determined with the β distribution. (A) Single-channel openings in the absence (top trace) and in the presence of 2, 5, and 20 mM TEA obtained at 60 mV. The apparent current amplitude decreases as the TEA concentration is increased, as predicted for a fast blocker. The dashed line represents the closed-channel current level, and the dotted line is the mean open-channel level in the absence of TEA. (B) Normalized amplitude histograms obtained from traces as in A. Solid lines are fits to the β distribution with the following parameters: 2 mM, β = 26,679 s−1, α = 61,454 s−1; 5 mM, β = 43,001 s−1, α = 55,882 s−1; 20 mM, β = 156,370 s−1, α = 44,582 s−1. (C) Blocking rate constants obtained from the fits to the β distribution as in B from three separate patches at several voltages and blocker concentrations. The on-rate, kon, increases with voltage and reaches a plateau. The solid line is a fit to a double exponential function reflecting both the voltage dependence of the on-rate and of the relief of block and has the form\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}k_{on}=\frac{1}{k_{ac}(0){\mathrm{exp}}z_{ac}V/kT}+\frac{1}{k_{rel}(0){\mathrm{exp}}z_{rel}V/kT}.\end{equation*}\end{document}The values of the fit parameters are: kac(0) = 1.171 × 106 M−1s−1; zac = 1.0193; krel(0) = 4.722 × 107 M−1s−1; zrel = 0.48. The off-rate decreases with increasing voltages but starts to increase at voltages more positive than 60 mV. The values of kon and koff at 0 mV were estimated from an exponential fit to the data between 240 and 60 mV, and the parameters of the fit are: kon(0) = 1.04 × 106 M−1s−1; z = 0.94 eo; koff(0) = 28,950 s−1; z = 0.27 eo. Group data are presented as mean ± SEM.
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fig4: Kinetics of block by intracellular TEA determined with the β distribution. (A) Single-channel openings in the absence (top trace) and in the presence of 2, 5, and 20 mM TEA obtained at 60 mV. The apparent current amplitude decreases as the TEA concentration is increased, as predicted for a fast blocker. The dashed line represents the closed-channel current level, and the dotted line is the mean open-channel level in the absence of TEA. (B) Normalized amplitude histograms obtained from traces as in A. Solid lines are fits to the β distribution with the following parameters: 2 mM, β = 26,679 s−1, α = 61,454 s−1; 5 mM, β = 43,001 s−1, α = 55,882 s−1; 20 mM, β = 156,370 s−1, α = 44,582 s−1. (C) Blocking rate constants obtained from the fits to the β distribution as in B from three separate patches at several voltages and blocker concentrations. The on-rate, kon, increases with voltage and reaches a plateau. The solid line is a fit to a double exponential function reflecting both the voltage dependence of the on-rate and of the relief of block and has the form\documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}k_{on}=\frac{1}{k_{ac}(0){\mathrm{exp}}z_{ac}V/kT}+\frac{1}{k_{rel}(0){\mathrm{exp}}z_{rel}V/kT}.\end{equation*}\end{document}The values of the fit parameters are: kac(0) = 1.171 × 106 M−1s−1; zac = 1.0193; krel(0) = 4.722 × 107 M−1s−1; zrel = 0.48. The off-rate decreases with increasing voltages but starts to increase at voltages more positive than 60 mV. The values of kon and koff at 0 mV were estimated from an exponential fit to the data between 240 and 60 mV, and the parameters of the fit are: kon(0) = 1.04 × 106 M−1s−1; z = 0.94 eo; koff(0) = 28,950 s−1; z = 0.27 eo. Group data are presented as mean ± SEM.
Mentions: Block of TRPV1 by TEA is a very fast process. The individual blocking events are so short-lived that they are smeared by the filter and, as a result, block appears as an apparent reduction of the single-channel conductance. For this reason and to estimate the kinetic parameters of the blocking reaction, we used an approach to analyze the all-points amplitude histogram from bursts of openings, which makes use of the β distribution. As discussed in Materials and methods, we showed that this approximation can describe a two-state process filtered by a Gaussian filter with reasonable accuracy. Fig. 4 A shows representative single-channel openings in the absence and presence of increasing concentrations of TEA obtained at 60 mV. It is apparent that the effect of the blocker is a reduction of the single channel current. Fig. 4 B shows amplitude histograms for channel openings obtained from traces as in Fig. 4 A and constructed as indicated in Materials and methods. Superimposed on the histograms are fits to Eq. 2 convolved with a Gaussian function, which describes the current amplitude distribution of the closed level in the absence of blocker.

Bottom Line: We found that all four QAs used, tetraethylammonium (TEA), tetrapropylammonium (TPrA), tetrabutylammonium, and tetrapentylammonium, block the TRPV1 channel from the intracellular face of the channel in a voltage-dependent manner, and that block by these molecules occurs with different kinetics, with the bigger molecules becoming slower blockers.We also found that TPrA and the larger QAs can only block the channel in the open state, and that they interfere with the channel's activation gate upon closing, which is observed as a slowing of tail current kinetics.The dependence of the rate constants on the size of the blocker suggests a size of around 10 A for the inner pore of TRPV1 channels.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Fisiología, Facultad de Medicina, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, D.F., 04510, México

ABSTRACT
The transient receptor potential vanilloid 1 (TRPV1) nonselective cationic channel is a polymodal receptor that activates in response to a wide variety of stimuli. To date, little structural information about this channel is available. Here, we used quaternary ammonium ions (QAs) of different sizes in an effort to gain some insight into the nature and dimensions of the pore of TRPV1. We found that all four QAs used, tetraethylammonium (TEA), tetrapropylammonium (TPrA), tetrabutylammonium, and tetrapentylammonium, block the TRPV1 channel from the intracellular face of the channel in a voltage-dependent manner, and that block by these molecules occurs with different kinetics, with the bigger molecules becoming slower blockers. We also found that TPrA and the larger QAs can only block the channel in the open state, and that they interfere with the channel's activation gate upon closing, which is observed as a slowing of tail current kinetics. TEA does not interfere with the activation gate, indicating that this molecule can reside in its blocking site even when the channel is closed. The dependence of the rate constants on the size of the blocker suggests a size of around 10 A for the inner pore of TRPV1 channels.

Show MeSH
Related in: MedlinePlus