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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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Channel-closing kinetics in the presence of TPrA, TBA, and TPA. (A) Representative traces obtained in the absence (black trace) or presence (gray traces) of 2 and 10 mM TPrA at −100 mV. Tail current kinetics is altered by the presence of the blocker. Channel closure is slowed and the tail currents now display a hook, characteristic of a blocker that interferes with the channel's activation gate. The dotted line represents fits to Eq. 4. (B) Tail currents obtained in the absence (black trace) or presence (gray traces) of 250 μM and 2.5 mM TBA at a voltage of −100 mV. The same behavior as for TPrA is observed, with slowing of the tail currents and the appearance of a hook at the beginning of the repolarization. Dotted lines are fits to Eq. 4. (C) Tail currents obtained in the absence (black trace) and presence (gray trace) of 250 and 500 μM TPA at −100 mV. Channel closure kinetics is slower in the presence of TPA than in its absence, just as for TPrA and TBA. (D) The rate k−1 from fits of Eq. 4 to data as in A was plotted against voltage as an index of the channel's closing rate. The straight lines are fits of equation:\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_{-1}=k_{-1}(0){\mathrm{exp}}(-z_{-1}V/kT).\end{equation*}\end{document}The symbols and parameters of the fit are (n = 5): No TPrA, k−1(0) = 179.86 s−1, z−1 = 0.12 eo (solid circles); 900 μM TPrA, k−1(0) = 164.1 s−1, z−1 = 0.093 eo (solid diamonds); 2 mM TPrA, k−1(0) = 130.3 s−1, z−1 = 0.09 eo (solid squares); 10 mM TPrA, k−1(0) = 128.02 s−1, z−1 = 0.045 eo (solid triangles). (E) As with TPrA, k−1 is plotted as an index of the closing rate as a function of voltage in the presence of TBA. The fit parameters and symbols are (n = 4): No TBA, k−1(0) = 148.01 s−1, z−1 = 0.09 eo (solid circles); 250 μM TBA, k−1(0) = 96.4, z−1 = 0.08 eo (solid diamonds); 500 μM TBA, k−1(0) = 51.2, z−1 = 0.16 eo (solid squares); 2.5 mM TBA, k−1(0) = 60.2, z−1 = 0.13 eo (solid triangles). Group data are presented as mean ± SEM.
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fig9: Channel-closing kinetics in the presence of TPrA, TBA, and TPA. (A) Representative traces obtained in the absence (black trace) or presence (gray traces) of 2 and 10 mM TPrA at −100 mV. Tail current kinetics is altered by the presence of the blocker. Channel closure is slowed and the tail currents now display a hook, characteristic of a blocker that interferes with the channel's activation gate. The dotted line represents fits to Eq. 4. (B) Tail currents obtained in the absence (black trace) or presence (gray traces) of 250 μM and 2.5 mM TBA at a voltage of −100 mV. The same behavior as for TPrA is observed, with slowing of the tail currents and the appearance of a hook at the beginning of the repolarization. Dotted lines are fits to Eq. 4. (C) Tail currents obtained in the absence (black trace) and presence (gray trace) of 250 and 500 μM TPA at −100 mV. Channel closure kinetics is slower in the presence of TPA than in its absence, just as for TPrA and TBA. (D) The rate k−1 from fits of Eq. 4 to data as in A was plotted against voltage as an index of the channel's closing rate. The straight lines are fits of equation:\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_{-1}=k_{-1}(0){\mathrm{exp}}(-z_{-1}V/kT).\end{equation*}\end{document}The symbols and parameters of the fit are (n = 5): No TPrA, k−1(0) = 179.86 s−1, z−1 = 0.12 eo (solid circles); 900 μM TPrA, k−1(0) = 164.1 s−1, z−1 = 0.093 eo (solid diamonds); 2 mM TPrA, k−1(0) = 130.3 s−1, z−1 = 0.09 eo (solid squares); 10 mM TPrA, k−1(0) = 128.02 s−1, z−1 = 0.045 eo (solid triangles). (E) As with TPrA, k−1 is plotted as an index of the closing rate as a function of voltage in the presence of TBA. The fit parameters and symbols are (n = 4): No TBA, k−1(0) = 148.01 s−1, z−1 = 0.09 eo (solid circles); 250 μM TBA, k−1(0) = 96.4, z−1 = 0.08 eo (solid diamonds); 500 μM TBA, k−1(0) = 51.2, z−1 = 0.16 eo (solid squares); 2.5 mM TBA, k−1(0) = 60.2, z−1 = 0.13 eo (solid triangles). Group data are presented as mean ± SEM.

Mentions: The kinetics of channel closure in the presence of a blocker molecule can reflect some aspects of the nature of the gating mechanism. It has been shown that if the blocker is able to only reach its binding site when the channel is open, the time course of channel closure should be slowed down because the blocker needs to leave the open channel before the activation gate can close (Armstrong, 1971; Choi et al., 1993; Li and Aldrich, 2004). To understand the relationship between blocker occupancy and gating, we examined channel closure kinetics with tail current protocols, both in unblocked channels and when channels were blocked by the fast blockers, TEA and TPrA, and by the slow blockers, TBA and TPA. The voltage dependence of deactivation of TRPV1 channels is small (z−1, ∼0.1 eo; Figs. 8 B and 9, D and E) when compared with Kv channels, and as a result the channels cannot be completely closed upon repolarization to the voltages we used; nevertheless, tail currents can be reliably recorded at negative voltages.


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)

Channel-closing kinetics in the presence of TPrA, TBA, and TPA. (A) Representative traces obtained in the absence (black trace) or presence (gray traces) of 2 and 10 mM TPrA at −100 mV. Tail current kinetics is altered by the presence of the blocker. Channel closure is slowed and the tail currents now display a hook, characteristic of a blocker that interferes with the channel's activation gate. The dotted line represents fits to Eq. 4. (B) Tail currents obtained in the absence (black trace) or presence (gray traces) of 250 μM and 2.5 mM TBA at a voltage of −100 mV. The same behavior as for TPrA is observed, with slowing of the tail currents and the appearance of a hook at the beginning of the repolarization. Dotted lines are fits to Eq. 4. (C) Tail currents obtained in the absence (black trace) and presence (gray trace) of 250 and 500 μM TPA at −100 mV. Channel closure kinetics is slower in the presence of TPA than in its absence, just as for TPrA and TBA. (D) The rate k−1 from fits of Eq. 4 to data as in A was plotted against voltage as an index of the channel's closing rate. The straight lines are fits of equation:\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_{-1}=k_{-1}(0){\mathrm{exp}}(-z_{-1}V/kT).\end{equation*}\end{document}The symbols and parameters of the fit are (n = 5): No TPrA, k−1(0) = 179.86 s−1, z−1 = 0.12 eo (solid circles); 900 μM TPrA, k−1(0) = 164.1 s−1, z−1 = 0.093 eo (solid diamonds); 2 mM TPrA, k−1(0) = 130.3 s−1, z−1 = 0.09 eo (solid squares); 10 mM TPrA, k−1(0) = 128.02 s−1, z−1 = 0.045 eo (solid triangles). (E) As with TPrA, k−1 is plotted as an index of the closing rate as a function of voltage in the presence of TBA. The fit parameters and symbols are (n = 4): No TBA, k−1(0) = 148.01 s−1, z−1 = 0.09 eo (solid circles); 250 μM TBA, k−1(0) = 96.4, z−1 = 0.08 eo (solid diamonds); 500 μM TBA, k−1(0) = 51.2, z−1 = 0.16 eo (solid squares); 2.5 mM TBA, k−1(0) = 60.2, z−1 = 0.13 eo (solid triangles). Group data are presented as mean ± SEM.
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fig9: Channel-closing kinetics in the presence of TPrA, TBA, and TPA. (A) Representative traces obtained in the absence (black trace) or presence (gray traces) of 2 and 10 mM TPrA at −100 mV. Tail current kinetics is altered by the presence of the blocker. Channel closure is slowed and the tail currents now display a hook, characteristic of a blocker that interferes with the channel's activation gate. The dotted line represents fits to Eq. 4. (B) Tail currents obtained in the absence (black trace) or presence (gray traces) of 250 μM and 2.5 mM TBA at a voltage of −100 mV. The same behavior as for TPrA is observed, with slowing of the tail currents and the appearance of a hook at the beginning of the repolarization. Dotted lines are fits to Eq. 4. (C) Tail currents obtained in the absence (black trace) and presence (gray trace) of 250 and 500 μM TPA at −100 mV. Channel closure kinetics is slower in the presence of TPA than in its absence, just as for TPrA and TBA. (D) The rate k−1 from fits of Eq. 4 to data as in A was plotted against voltage as an index of the channel's closing rate. The straight lines are fits of equation:\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_{-1}=k_{-1}(0){\mathrm{exp}}(-z_{-1}V/kT).\end{equation*}\end{document}The symbols and parameters of the fit are (n = 5): No TPrA, k−1(0) = 179.86 s−1, z−1 = 0.12 eo (solid circles); 900 μM TPrA, k−1(0) = 164.1 s−1, z−1 = 0.093 eo (solid diamonds); 2 mM TPrA, k−1(0) = 130.3 s−1, z−1 = 0.09 eo (solid squares); 10 mM TPrA, k−1(0) = 128.02 s−1, z−1 = 0.045 eo (solid triangles). (E) As with TPrA, k−1 is plotted as an index of the closing rate as a function of voltage in the presence of TBA. The fit parameters and symbols are (n = 4): No TBA, k−1(0) = 148.01 s−1, z−1 = 0.09 eo (solid circles); 250 μM TBA, k−1(0) = 96.4, z−1 = 0.08 eo (solid diamonds); 500 μM TBA, k−1(0) = 51.2, z−1 = 0.16 eo (solid squares); 2.5 mM TBA, k−1(0) = 60.2, z−1 = 0.13 eo (solid triangles). Group data are presented as mean ± SEM.
Mentions: The kinetics of channel closure in the presence of a blocker molecule can reflect some aspects of the nature of the gating mechanism. It has been shown that if the blocker is able to only reach its binding site when the channel is open, the time course of channel closure should be slowed down because the blocker needs to leave the open channel before the activation gate can close (Armstrong, 1971; Choi et al., 1993; Li and Aldrich, 2004). To understand the relationship between blocker occupancy and gating, we examined channel closure kinetics with tail current protocols, both in unblocked channels and when channels were blocked by the fast blockers, TEA and TPrA, and by the slow blockers, TBA and TPA. The voltage dependence of deactivation of TRPV1 channels is small (z−1, ∼0.1 eo; Figs. 8 B and 9, D and E) when compared with Kv channels, and as a result the channels cannot be completely closed upon repolarization to the voltages we used; nevertheless, tail currents can be reliably recorded at negative voltages.

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