Limits...
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.

Show MeSH

Related in: MedlinePlus

Channel-closing kinetics in the presence of TEA. (A) Representative tail currents obtained at −180 mV after a prepulse of 100 ms at 60 mV in the absence (thick trace) or presence of 20, 40, and 80 mM TEA (gray traces). Dotted lines are fits to a single exponential function. (B) Channel-closing rate as a function of voltage obtained from fits to an exponential as in A. The straight lines are fits to the 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*}{\mathrm{k}}_{1}{\mathrm{(V)}}={\mathrm{k}}_{1}(0){\mathrm{exp}}({\mathrm{z}}_{1}{\mathrm{V/kT}}).\end{equation*}\end{document}Symbols and parameters of the fit are (n = 4): No TEA k1(0) = 196.49 s−1, z1 = 0.073 eo (filled circles); 20 mM TEA k1(0) = 364.9 s−1, z1 = 0.04 eo (filled triangles); 40 mM TEA k1(0) = 323.9 s−1, z1 = 0.083 eo (filled diamonds); 80 mM TEA k1(0) = 457.2 s−1, z1 = 0.086 eo (filled squares). The closing rate increases with blocker concentration, indicating that TEA speeds up channel closure. (C) Representative traces of single-channel openings in the absence or the presence of the indicated concentration of TEA. The dotted lines indicate the current amplitude in the absence of TEA. (D) Burst length distributions obtained from traces as in C. Burst length was measured and compiled in logarithmically binned histograms in the absence or presence of TEA. The black lines are fits with a single exponential function of time with the following time constants: No TEA, 3.17 ms; 2.5 mM TEA, 1.2 ms; 5 mM TEA, 0.8 ms.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2571972&req=5

fig8: Channel-closing kinetics in the presence of TEA. (A) Representative tail currents obtained at −180 mV after a prepulse of 100 ms at 60 mV in the absence (thick trace) or presence of 20, 40, and 80 mM TEA (gray traces). Dotted lines are fits to a single exponential function. (B) Channel-closing rate as a function of voltage obtained from fits to an exponential as in A. The straight lines are fits to the 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*}{\mathrm{k}}_{1}{\mathrm{(V)}}={\mathrm{k}}_{1}(0){\mathrm{exp}}({\mathrm{z}}_{1}{\mathrm{V/kT}}).\end{equation*}\end{document}Symbols and parameters of the fit are (n = 4): No TEA k1(0) = 196.49 s−1, z1 = 0.073 eo (filled circles); 20 mM TEA k1(0) = 364.9 s−1, z1 = 0.04 eo (filled triangles); 40 mM TEA k1(0) = 323.9 s−1, z1 = 0.083 eo (filled diamonds); 80 mM TEA k1(0) = 457.2 s−1, z1 = 0.086 eo (filled squares). The closing rate increases with blocker concentration, indicating that TEA speeds up channel closure. (C) Representative traces of single-channel openings in the absence or the presence of the indicated concentration of TEA. The dotted lines indicate the current amplitude in the absence of TEA. (D) Burst length distributions obtained from traces as in C. Burst length was measured and compiled in logarithmically binned histograms in the absence or presence of TEA. The black lines are fits with a single exponential function of time with the following time constants: No TEA, 3.17 ms; 2.5 mM TEA, 1.2 ms; 5 mM TEA, 0.8 ms.

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 TEA. (A) Representative tail currents obtained at −180 mV after a prepulse of 100 ms at 60 mV in the absence (thick trace) or presence of 20, 40, and 80 mM TEA (gray traces). Dotted lines are fits to a single exponential function. (B) Channel-closing rate as a function of voltage obtained from fits to an exponential as in A. The straight lines are fits to the 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*}{\mathrm{k}}_{1}{\mathrm{(V)}}={\mathrm{k}}_{1}(0){\mathrm{exp}}({\mathrm{z}}_{1}{\mathrm{V/kT}}).\end{equation*}\end{document}Symbols and parameters of the fit are (n = 4): No TEA k1(0) = 196.49 s−1, z1 = 0.073 eo (filled circles); 20 mM TEA k1(0) = 364.9 s−1, z1 = 0.04 eo (filled triangles); 40 mM TEA k1(0) = 323.9 s−1, z1 = 0.083 eo (filled diamonds); 80 mM TEA k1(0) = 457.2 s−1, z1 = 0.086 eo (filled squares). The closing rate increases with blocker concentration, indicating that TEA speeds up channel closure. (C) Representative traces of single-channel openings in the absence or the presence of the indicated concentration of TEA. The dotted lines indicate the current amplitude in the absence of TEA. (D) Burst length distributions obtained from traces as in C. Burst length was measured and compiled in logarithmically binned histograms in the absence or presence of TEA. The black lines are fits with a single exponential function of time with the following time constants: No TEA, 3.17 ms; 2.5 mM TEA, 1.2 ms; 5 mM TEA, 0.8 ms.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571972&req=5

fig8: Channel-closing kinetics in the presence of TEA. (A) Representative tail currents obtained at −180 mV after a prepulse of 100 ms at 60 mV in the absence (thick trace) or presence of 20, 40, and 80 mM TEA (gray traces). Dotted lines are fits to a single exponential function. (B) Channel-closing rate as a function of voltage obtained from fits to an exponential as in A. The straight lines are fits to the 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*}{\mathrm{k}}_{1}{\mathrm{(V)}}={\mathrm{k}}_{1}(0){\mathrm{exp}}({\mathrm{z}}_{1}{\mathrm{V/kT}}).\end{equation*}\end{document}Symbols and parameters of the fit are (n = 4): No TEA k1(0) = 196.49 s−1, z1 = 0.073 eo (filled circles); 20 mM TEA k1(0) = 364.9 s−1, z1 = 0.04 eo (filled triangles); 40 mM TEA k1(0) = 323.9 s−1, z1 = 0.083 eo (filled diamonds); 80 mM TEA k1(0) = 457.2 s−1, z1 = 0.086 eo (filled squares). The closing rate increases with blocker concentration, indicating that TEA speeds up channel closure. (C) Representative traces of single-channel openings in the absence or the presence of the indicated concentration of TEA. The dotted lines indicate the current amplitude in the absence of TEA. (D) Burst length distributions obtained from traces as in C. Burst length was measured and compiled in logarithmically binned histograms in the absence or presence of TEA. The black lines are fits with a single exponential function of time with the following time constants: No TEA, 3.17 ms; 2.5 mM TEA, 1.2 ms; 5 mM TEA, 0.8 ms.
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