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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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Voltage dependence and steady-state block at negative voltages. (A) Dose-response curves for the different QAs measured at a voltage of 100 mV. The solid lines represent fits to the Hill equation. The parameters are: TEA, KD = 8.7 ± 0.7 mM, s = 0.77 ± 0.08 (n = 5); TPrA, KD = 940 ± 60 μM, s = 0.79 ± 0.04 (n = 6); TBA, KD = 327 ± 25 μM, s = 0.88 ± 0.02 (n = 9); TPA, KD = 36 ± 8 μM, s = 1.15 ± 0.12 (n = 3). All recordings were performed in the presence of 4 μM capsaicin. (B) The apparent dissociation constant, KD, derived from data as in A at different voltages. The inset shows a complete dataset for TEA for voltages from −80 to 100 mV. Voltage dependence of block was determined at negative voltages by fitting Eq. 3 to the data in B up to −20 mV. (C) The parameters obtained from the fit are plotted as a function of the size of the blocker. KD at 0 mV (in units of M; top): TEA, 6.65 × 10−3 ± 3.10−3 (n = 4); TPrA, 2.31 × 10−3 ± 0.11 × 10−3 (n = 4); TBA, 21.3 × 10−5 ± 1.7 × 10−5 (n = 6); TPA, 2.2 × 10−5 ± 10−5 (n = 5). The values of Z (bottom) are (in units of eo): TEA, 1.32 ± 0.097; TPrA, 1.4 ± 0.02; TBA, 0.98 ± 0.02; TPA, 0.61 ± 0.06. The affinity of the channel for the blockers increases with blocker size. Group data are presented as mean ± SEM.
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fig3: Voltage dependence and steady-state block at negative voltages. (A) Dose-response curves for the different QAs measured at a voltage of 100 mV. The solid lines represent fits to the Hill equation. The parameters are: TEA, KD = 8.7 ± 0.7 mM, s = 0.77 ± 0.08 (n = 5); TPrA, KD = 940 ± 60 μM, s = 0.79 ± 0.04 (n = 6); TBA, KD = 327 ± 25 μM, s = 0.88 ± 0.02 (n = 9); TPA, KD = 36 ± 8 μM, s = 1.15 ± 0.12 (n = 3). All recordings were performed in the presence of 4 μM capsaicin. (B) The apparent dissociation constant, KD, derived from data as in A at different voltages. The inset shows a complete dataset for TEA for voltages from −80 to 100 mV. Voltage dependence of block was determined at negative voltages by fitting Eq. 3 to the data in B up to −20 mV. (C) The parameters obtained from the fit are plotted as a function of the size of the blocker. KD at 0 mV (in units of M; top): TEA, 6.65 × 10−3 ± 3.10−3 (n = 4); TPrA, 2.31 × 10−3 ± 0.11 × 10−3 (n = 4); TBA, 21.3 × 10−5 ± 1.7 × 10−5 (n = 6); TPA, 2.2 × 10−5 ± 10−5 (n = 5). The values of Z (bottom) are (in units of eo): TEA, 1.32 ± 0.097; TPrA, 1.4 ± 0.02; TBA, 0.98 ± 0.02; TPA, 0.61 ± 0.06. The affinity of the channel for the blockers increases with blocker size. Group data are presented as mean ± SEM.

Mentions: Fig. 3 A shows the dose-response curves for the various QAs obtained at 100 mV. Blockade was dose dependent with the apparent dissociation constant, KD, decreasing with blocker size, indicating an increase in affinity. The steepness of the Hill equation used to fit the data is close to one, suggesting that only a molecule of blocker can bind to the channel at a time (Fig. 3 A). A plot of the apparent dissociation constant, KD, versus voltage indicates that block is clearly voltage dependent; however, contrary to the expectation from a Woodhull-type model (Woodhull, 1973), the value of KD for all blockers reaches an asymptotic value at positive potentials (Fig. 3, B and inset). This apparent relief of block has been explained in other types of ion channels by several different mechanisms, including a permeant blocker mechanism (Guo and Lu, 2000), diffusion limitation of the on-rate at positive voltages (Blaustein and Finkelstein, 1990b), and permeant–ion interactions with the blocker in the conduction pathway (Heginbotham and Kutluay, 2004). For TBA, we have previously shown that relief of block can be explained by the latter mechanism (Oseguera et al., 2007), and it is very likely that the same is true for the rest of QA blockers used here. At negative voltages, the KD does behave as an exponential function of voltage, and we can estimate the valence of the blocking reaction at voltages more negative than 0 mV by fitting Eq. 3 to the data:(3)\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_{D}=K_{D}(0){\mathrm{exp}}(-Z_{app}V/kT),\end{equation*}\end{document}where KD(0) is the value of KD at zero mV and Zapp is the apparent charge associated with the blocking reaction. These parameters are shown in Fig. 3 C for each blocker. Similar to observations in voltage-dependent potassium channels (Choi et al., 1993), the apparent affinity of the blocker increases with the size and hydrophobicity of the QA derivative (Fig. 3 C). It can also be appreciated that the voltage dependence of TEA, TPrA, and TBA, represented by the value of Zapp (Fig. 3 C), is higher than or near a value of one. A similar observation of high apparent valences of blockers in potassium-permeable channels has been shown to be due to the coupling of permeant ion movement with blocker occupancy (French and Shoukimas, 1985; Spassova and Lu, 1998), and we have shown previously that in the case of TBA, a large fraction of this voltage dependence is due to movement of Na+ ions in the selectivity filter of the TRPV1 channel, which is coupled to blocker occupancy, and not due to the blocker traversing a large fraction of the transmembrane voltage (Oseguera et al., 2007).


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)

Voltage dependence and steady-state block at negative voltages. (A) Dose-response curves for the different QAs measured at a voltage of 100 mV. The solid lines represent fits to the Hill equation. The parameters are: TEA, KD = 8.7 ± 0.7 mM, s = 0.77 ± 0.08 (n = 5); TPrA, KD = 940 ± 60 μM, s = 0.79 ± 0.04 (n = 6); TBA, KD = 327 ± 25 μM, s = 0.88 ± 0.02 (n = 9); TPA, KD = 36 ± 8 μM, s = 1.15 ± 0.12 (n = 3). All recordings were performed in the presence of 4 μM capsaicin. (B) The apparent dissociation constant, KD, derived from data as in A at different voltages. The inset shows a complete dataset for TEA for voltages from −80 to 100 mV. Voltage dependence of block was determined at negative voltages by fitting Eq. 3 to the data in B up to −20 mV. (C) The parameters obtained from the fit are plotted as a function of the size of the blocker. KD at 0 mV (in units of M; top): TEA, 6.65 × 10−3 ± 3.10−3 (n = 4); TPrA, 2.31 × 10−3 ± 0.11 × 10−3 (n = 4); TBA, 21.3 × 10−5 ± 1.7 × 10−5 (n = 6); TPA, 2.2 × 10−5 ± 10−5 (n = 5). The values of Z (bottom) are (in units of eo): TEA, 1.32 ± 0.097; TPrA, 1.4 ± 0.02; TBA, 0.98 ± 0.02; TPA, 0.61 ± 0.06. The affinity of the channel for the blockers increases with blocker size. Group data are presented as mean ± SEM.
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Related In: Results  -  Collection

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fig3: Voltage dependence and steady-state block at negative voltages. (A) Dose-response curves for the different QAs measured at a voltage of 100 mV. The solid lines represent fits to the Hill equation. The parameters are: TEA, KD = 8.7 ± 0.7 mM, s = 0.77 ± 0.08 (n = 5); TPrA, KD = 940 ± 60 μM, s = 0.79 ± 0.04 (n = 6); TBA, KD = 327 ± 25 μM, s = 0.88 ± 0.02 (n = 9); TPA, KD = 36 ± 8 μM, s = 1.15 ± 0.12 (n = 3). All recordings were performed in the presence of 4 μM capsaicin. (B) The apparent dissociation constant, KD, derived from data as in A at different voltages. The inset shows a complete dataset for TEA for voltages from −80 to 100 mV. Voltage dependence of block was determined at negative voltages by fitting Eq. 3 to the data in B up to −20 mV. (C) The parameters obtained from the fit are plotted as a function of the size of the blocker. KD at 0 mV (in units of M; top): TEA, 6.65 × 10−3 ± 3.10−3 (n = 4); TPrA, 2.31 × 10−3 ± 0.11 × 10−3 (n = 4); TBA, 21.3 × 10−5 ± 1.7 × 10−5 (n = 6); TPA, 2.2 × 10−5 ± 10−5 (n = 5). The values of Z (bottom) are (in units of eo): TEA, 1.32 ± 0.097; TPrA, 1.4 ± 0.02; TBA, 0.98 ± 0.02; TPA, 0.61 ± 0.06. The affinity of the channel for the blockers increases with blocker size. Group data are presented as mean ± SEM.
Mentions: Fig. 3 A shows the dose-response curves for the various QAs obtained at 100 mV. Blockade was dose dependent with the apparent dissociation constant, KD, decreasing with blocker size, indicating an increase in affinity. The steepness of the Hill equation used to fit the data is close to one, suggesting that only a molecule of blocker can bind to the channel at a time (Fig. 3 A). A plot of the apparent dissociation constant, KD, versus voltage indicates that block is clearly voltage dependent; however, contrary to the expectation from a Woodhull-type model (Woodhull, 1973), the value of KD for all blockers reaches an asymptotic value at positive potentials (Fig. 3, B and inset). This apparent relief of block has been explained in other types of ion channels by several different mechanisms, including a permeant blocker mechanism (Guo and Lu, 2000), diffusion limitation of the on-rate at positive voltages (Blaustein and Finkelstein, 1990b), and permeant–ion interactions with the blocker in the conduction pathway (Heginbotham and Kutluay, 2004). For TBA, we have previously shown that relief of block can be explained by the latter mechanism (Oseguera et al., 2007), and it is very likely that the same is true for the rest of QA blockers used here. At negative voltages, the KD does behave as an exponential function of voltage, and we can estimate the valence of the blocking reaction at voltages more negative than 0 mV by fitting Eq. 3 to the data:(3)\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_{D}=K_{D}(0){\mathrm{exp}}(-Z_{app}V/kT),\end{equation*}\end{document}where KD(0) is the value of KD at zero mV and Zapp is the apparent charge associated with the blocking reaction. These parameters are shown in Fig. 3 C for each blocker. Similar to observations in voltage-dependent potassium channels (Choi et al., 1993), the apparent affinity of the blocker increases with the size and hydrophobicity of the QA derivative (Fig. 3 C). It can also be appreciated that the voltage dependence of TEA, TPrA, and TBA, represented by the value of Zapp (Fig. 3 C), is higher than or near a value of one. A similar observation of high apparent valences of blockers in potassium-permeable channels has been shown to be due to the coupling of permeant ion movement with blocker occupancy (French and Shoukimas, 1985; Spassova and Lu, 1998), and we have shown previously that in the case of TBA, a large fraction of this voltage dependence is due to movement of Na+ ions in the selectivity filter of the TRPV1 channel, which is coupled to blocker occupancy, and not due to the blocker traversing a large fraction of the transmembrane voltage (Oseguera et al., 2007).

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