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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 TPA. (A) Single-channel traces in the absence (top trace) and presence of 80 μM TPA (bottom trace). (B) Closed time histograms from multiple traces as in A fitted with three exponential components. (C; top) Macroscopic kinetics of block during depolarizing voltage pulses ranging from 40 to 160 mV in the presence of 40 μM TPA. The onset of block can be seen as an exponential decay of the initial current, as expected for a slower blocker. (Bottom) Blocker dissociation observed during tail current experiments. Traces were obtained in the presence of 80 μM TPA at −20, −40, −60, −80, and −100 mV. Blocker dissociation can be seen as an exponential increase in the current. The off-rates were obtained from fits to an exponential (solid lines). (D) The on- and off-rates obtained from traces as in C, plotted as a function of voltage. Both rates are voltage dependent and have the following values estimated from an exponential fit to the data: koff(0 mV) = 10.73 s−1 (n = 3); zoff = 0.45 eo; kon(0 mV) = 1.03 × 105 M−1s−1 (n = 5); zon = 0.68 eo. Group data are presented as mean ± SEM.
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fig7: Kinetics of block by TPA. (A) Single-channel traces in the absence (top trace) and presence of 80 μM TPA (bottom trace). (B) Closed time histograms from multiple traces as in A fitted with three exponential components. (C; top) Macroscopic kinetics of block during depolarizing voltage pulses ranging from 40 to 160 mV in the presence of 40 μM TPA. The onset of block can be seen as an exponential decay of the initial current, as expected for a slower blocker. (Bottom) Blocker dissociation observed during tail current experiments. Traces were obtained in the presence of 80 μM TPA at −20, −40, −60, −80, and −100 mV. Blocker dissociation can be seen as an exponential increase in the current. The off-rates were obtained from fits to an exponential (solid lines). (D) The on- and off-rates obtained from traces as in C, plotted as a function of voltage. Both rates are voltage dependent and have the following values estimated from an exponential fit to the data: koff(0 mV) = 10.73 s−1 (n = 3); zoff = 0.45 eo; kon(0 mV) = 1.03 × 105 M−1s−1 (n = 5); zon = 0.68 eo. Group data are presented as mean ± SEM.

Mentions: It has been shown that the kinetics of TRPV1 block by TBA are sufficiently slow to be observed in macroscopic current recordings as an exponential relaxation of current during depolarizing pulses (Oseguera et al., 2007). We observed that TPA is also a slower blocker than TEA and TPrA. In single-channel recordings, TPA does not affect the single-channel conductance, and blocking events can be well resolved (Fig. 7 A). When dwell-time histograms are compiled, blockade is observed as an increase in the number of long-duration events in the closed-time histogram (Fig. 7 B). The voltage-dependent blocking kinetics of TPA can also be observed in macroscopic recordings as a current relaxation during positive voltage steps (Fig. 7 C). This relaxation can be fitted with an exponential function (Fig. 7 C, top). During repolarizing voltage pulses, the channel seems to no longer be able to deactivate and instead the tail current increases with a rate that is voltage dependent. This behavior of the tail current reflects the time course of TPA leaving its binding site in the channel, and an estimate of this rate can be obtained from the inverse of the time constant of an exponential function fitted to the tail current relaxation (Fig. 7 C, bottom). The summarized data for on- and off-rate constants of block by TPA, obtained from macroscopic current recordings, is presented in Fig. 7 D. Both rates are exponential functions of voltage.


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 TPA. (A) Single-channel traces in the absence (top trace) and presence of 80 μM TPA (bottom trace). (B) Closed time histograms from multiple traces as in A fitted with three exponential components. (C; top) Macroscopic kinetics of block during depolarizing voltage pulses ranging from 40 to 160 mV in the presence of 40 μM TPA. The onset of block can be seen as an exponential decay of the initial current, as expected for a slower blocker. (Bottom) Blocker dissociation observed during tail current experiments. Traces were obtained in the presence of 80 μM TPA at −20, −40, −60, −80, and −100 mV. Blocker dissociation can be seen as an exponential increase in the current. The off-rates were obtained from fits to an exponential (solid lines). (D) The on- and off-rates obtained from traces as in C, plotted as a function of voltage. Both rates are voltage dependent and have the following values estimated from an exponential fit to the data: koff(0 mV) = 10.73 s−1 (n = 3); zoff = 0.45 eo; kon(0 mV) = 1.03 × 105 M−1s−1 (n = 5); zon = 0.68 eo. Group data are presented as mean ± SEM.
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fig7: Kinetics of block by TPA. (A) Single-channel traces in the absence (top trace) and presence of 80 μM TPA (bottom trace). (B) Closed time histograms from multiple traces as in A fitted with three exponential components. (C; top) Macroscopic kinetics of block during depolarizing voltage pulses ranging from 40 to 160 mV in the presence of 40 μM TPA. The onset of block can be seen as an exponential decay of the initial current, as expected for a slower blocker. (Bottom) Blocker dissociation observed during tail current experiments. Traces were obtained in the presence of 80 μM TPA at −20, −40, −60, −80, and −100 mV. Blocker dissociation can be seen as an exponential increase in the current. The off-rates were obtained from fits to an exponential (solid lines). (D) The on- and off-rates obtained from traces as in C, plotted as a function of voltage. Both rates are voltage dependent and have the following values estimated from an exponential fit to the data: koff(0 mV) = 10.73 s−1 (n = 3); zoff = 0.45 eo; kon(0 mV) = 1.03 × 105 M−1s−1 (n = 5); zon = 0.68 eo. Group data are presented as mean ± SEM.
Mentions: It has been shown that the kinetics of TRPV1 block by TBA are sufficiently slow to be observed in macroscopic current recordings as an exponential relaxation of current during depolarizing pulses (Oseguera et al., 2007). We observed that TPA is also a slower blocker than TEA and TPrA. In single-channel recordings, TPA does not affect the single-channel conductance, and blocking events can be well resolved (Fig. 7 A). When dwell-time histograms are compiled, blockade is observed as an increase in the number of long-duration events in the closed-time histogram (Fig. 7 B). The voltage-dependent blocking kinetics of TPA can also be observed in macroscopic recordings as a current relaxation during positive voltage steps (Fig. 7 C). This relaxation can be fitted with an exponential function (Fig. 7 C, top). During repolarizing voltage pulses, the channel seems to no longer be able to deactivate and instead the tail current increases with a rate that is voltage dependent. This behavior of the tail current reflects the time course of TPA leaving its binding site in the channel, and an estimate of this rate can be obtained from the inverse of the time constant of an exponential function fitted to the tail current relaxation (Fig. 7 C, bottom). The summarized data for on- and off-rate constants of block by TPA, obtained from macroscopic current recordings, is presented in Fig. 7 D. Both rates are exponential functions of voltage.

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