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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 TPrA block depends on the extracellular sodium concentration. (A) Single-channel openings (left) in the absence of TPrA (left trace) and the presence of 2 mM TPrA (right trace) with isometric 130-mM NaCl solutions at 40 mV. The right panel depicts the normalized amplitude histogram from multiple traces as in the left panel. The β distribution fit yielded parameters of β = 8,563.7 s−1 and α = 10,971 s−1. (B) Single-channel openings (left) in the absence of TPrA (left trace) and the presence of 2 mM TPrA (right trace) with 10 mM NaCl in the extracellular and 130 mM in the intracellular solution at 40 mV. The right panel shows the normalized amplitude histogram with the β distribution fit superimposed. The parameters of the fit are: β = 26,919 s−1, α = 11,661 s−1. The amplitude histogram under low extracellular sodium conditions is left-shifted, indicating faster blocker kinetics under these conditions. (C) On-rates obtained from fits of the β distribution to histograms as in B under low sodium conditions, obtained from two different patches (filled circles). The black line is a fit to an exponential of 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*}{\mathrm{k}}_{{\mathrm{on}}}{\mathrm{(V)}}={\mathrm{k}}_{{\mathrm{on}}}(0){\mathrm{exp}}({\mathrm{z}}_{{\mathrm{on}}}{\mathrm{V/kT}}).\end{equation*}\end{document}The values of the fit parameters are: kon(0) = 1.57 × 106 M−1 s−1; zon = 0.72 eo. The gray line corresponds to the fit to the on-rate under isometric 130 mM NaCl conditions in Fig. 5 C (open circles). Error bars are smaller than the symbols. The data indicate that the relief of block observed as a plateau for the on-rate at more positive potentials is a result of interactions between the blocker and the Na+ ions in the selectivity filter, and not due to diffusion limitation. Group data are presented as mean ± SEM.
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fig6: Kinetics of TPrA block depends on the extracellular sodium concentration. (A) Single-channel openings (left) in the absence of TPrA (left trace) and the presence of 2 mM TPrA (right trace) with isometric 130-mM NaCl solutions at 40 mV. The right panel depicts the normalized amplitude histogram from multiple traces as in the left panel. The β distribution fit yielded parameters of β = 8,563.7 s−1 and α = 10,971 s−1. (B) Single-channel openings (left) in the absence of TPrA (left trace) and the presence of 2 mM TPrA (right trace) with 10 mM NaCl in the extracellular and 130 mM in the intracellular solution at 40 mV. The right panel shows the normalized amplitude histogram with the β distribution fit superimposed. The parameters of the fit are: β = 26,919 s−1, α = 11,661 s−1. The amplitude histogram under low extracellular sodium conditions is left-shifted, indicating faster blocker kinetics under these conditions. (C) On-rates obtained from fits of the β distribution to histograms as in B under low sodium conditions, obtained from two different patches (filled circles). The black line is a fit to an exponential of 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*}{\mathrm{k}}_{{\mathrm{on}}}{\mathrm{(V)}}={\mathrm{k}}_{{\mathrm{on}}}(0){\mathrm{exp}}({\mathrm{z}}_{{\mathrm{on}}}{\mathrm{V/kT}}).\end{equation*}\end{document}The values of the fit parameters are: kon(0) = 1.57 × 106 M−1 s−1; zon = 0.72 eo. The gray line corresponds to the fit to the on-rate under isometric 130 mM NaCl conditions in Fig. 5 C (open circles). Error bars are smaller than the symbols. The data indicate that the relief of block observed as a plateau for the on-rate at more positive potentials is a result of interactions between the blocker and the Na+ ions in the selectivity filter, and not due to diffusion limitation. Group data are presented as mean ± SEM.

Mentions: The data in Fig. 6 shows that, as expected, blockade of TRPV1 by TPrA is more effective in the low extracellular Na+ (10 mM; Fig. 6 B) experiments than in the isometric (130 mM) Na+ concentration experiments (Fig. 6 A), as indicated by the larger reduction of current amplitude produced by TPrA under low extracellular sodium conditions. This reduced current amplitude translates into larger values for the on-rate constant. In addition, the on-rate constant no longer saturates at positive voltages and behaves as an exponential function of voltage for the entire range of voltages tested. The corresponding valence of this exponential is 0.7 eo (Fig. 6 C), which is the same as that measured at negative potentials under isometric 130 mM Na+ conditions.


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 TPrA block depends on the extracellular sodium concentration. (A) Single-channel openings (left) in the absence of TPrA (left trace) and the presence of 2 mM TPrA (right trace) with isometric 130-mM NaCl solutions at 40 mV. The right panel depicts the normalized amplitude histogram from multiple traces as in the left panel. The β distribution fit yielded parameters of β = 8,563.7 s−1 and α = 10,971 s−1. (B) Single-channel openings (left) in the absence of TPrA (left trace) and the presence of 2 mM TPrA (right trace) with 10 mM NaCl in the extracellular and 130 mM in the intracellular solution at 40 mV. The right panel shows the normalized amplitude histogram with the β distribution fit superimposed. The parameters of the fit are: β = 26,919 s−1, α = 11,661 s−1. The amplitude histogram under low extracellular sodium conditions is left-shifted, indicating faster blocker kinetics under these conditions. (C) On-rates obtained from fits of the β distribution to histograms as in B under low sodium conditions, obtained from two different patches (filled circles). The black line is a fit to an exponential of 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*}{\mathrm{k}}_{{\mathrm{on}}}{\mathrm{(V)}}={\mathrm{k}}_{{\mathrm{on}}}(0){\mathrm{exp}}({\mathrm{z}}_{{\mathrm{on}}}{\mathrm{V/kT}}).\end{equation*}\end{document}The values of the fit parameters are: kon(0) = 1.57 × 106 M−1 s−1; zon = 0.72 eo. The gray line corresponds to the fit to the on-rate under isometric 130 mM NaCl conditions in Fig. 5 C (open circles). Error bars are smaller than the symbols. The data indicate that the relief of block observed as a plateau for the on-rate at more positive potentials is a result of interactions between the blocker and the Na+ ions in the selectivity filter, and not due to diffusion limitation. Group data are presented as mean ± SEM.
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Related In: Results  -  Collection

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fig6: Kinetics of TPrA block depends on the extracellular sodium concentration. (A) Single-channel openings (left) in the absence of TPrA (left trace) and the presence of 2 mM TPrA (right trace) with isometric 130-mM NaCl solutions at 40 mV. The right panel depicts the normalized amplitude histogram from multiple traces as in the left panel. The β distribution fit yielded parameters of β = 8,563.7 s−1 and α = 10,971 s−1. (B) Single-channel openings (left) in the absence of TPrA (left trace) and the presence of 2 mM TPrA (right trace) with 10 mM NaCl in the extracellular and 130 mM in the intracellular solution at 40 mV. The right panel shows the normalized amplitude histogram with the β distribution fit superimposed. The parameters of the fit are: β = 26,919 s−1, α = 11,661 s−1. The amplitude histogram under low extracellular sodium conditions is left-shifted, indicating faster blocker kinetics under these conditions. (C) On-rates obtained from fits of the β distribution to histograms as in B under low sodium conditions, obtained from two different patches (filled circles). The black line is a fit to an exponential of 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*}{\mathrm{k}}_{{\mathrm{on}}}{\mathrm{(V)}}={\mathrm{k}}_{{\mathrm{on}}}(0){\mathrm{exp}}({\mathrm{z}}_{{\mathrm{on}}}{\mathrm{V/kT}}).\end{equation*}\end{document}The values of the fit parameters are: kon(0) = 1.57 × 106 M−1 s−1; zon = 0.72 eo. The gray line corresponds to the fit to the on-rate under isometric 130 mM NaCl conditions in Fig. 5 C (open circles). Error bars are smaller than the symbols. The data indicate that the relief of block observed as a plateau for the on-rate at more positive potentials is a result of interactions between the blocker and the Na+ ions in the selectivity filter, and not due to diffusion limitation. Group data are presented as mean ± SEM.
Mentions: The data in Fig. 6 shows that, as expected, blockade of TRPV1 by TPrA is more effective in the low extracellular Na+ (10 mM; Fig. 6 B) experiments than in the isometric (130 mM) Na+ concentration experiments (Fig. 6 A), as indicated by the larger reduction of current amplitude produced by TPrA under low extracellular sodium conditions. This reduced current amplitude translates into larger values for the on-rate constant. In addition, the on-rate constant no longer saturates at positive voltages and behaves as an exponential function of voltage for the entire range of voltages tested. The corresponding valence of this exponential is 0.7 eo (Fig. 6 C), which is the same as that measured at negative potentials under isometric 130 mM Na+ conditions.

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