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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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Dependence of the on- and off-rates of QA blockers on their size. The blocker dissociation (A) or association rates (B) are plotted as a function of their ionic radii. The continuous lines are drawn as a visual guide and have no theoretical significance. The decrease of the rates as a function of size is monotonic and there is not a clear cut-off size. Data are mean ± SEM of five to six experiments. (C–E) Cartoon depicting the possible ways in which the QAs may interact with the channel pore. (C) Cartoon of the open-channel pore with a blocker the size of TEA (right) or TPrA (left). (D) A model in which all QAs are only able to access the blocking site when the channel is in the open state, but TEA can reside inside the closed channel (left), whereas larger QAs cannot (right). (E) Alternative model in which the TEA molecule can block the channel in a state-independent manner, whereas the rest of the QAs can only access the blocking site when the channel is open, and hence interact with channel gating. In any way, TEA can reside in its blocking site when the channel is closed, whereas the other QAs do not.
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fig10: Dependence of the on- and off-rates of QA blockers on their size. The blocker dissociation (A) or association rates (B) are plotted as a function of their ionic radii. The continuous lines are drawn as a visual guide and have no theoretical significance. The decrease of the rates as a function of size is monotonic and there is not a clear cut-off size. Data are mean ± SEM of five to six experiments. (C–E) Cartoon depicting the possible ways in which the QAs may interact with the channel pore. (C) Cartoon of the open-channel pore with a blocker the size of TEA (right) or TPrA (left). (D) A model in which all QAs are only able to access the blocking site when the channel is in the open state, but TEA can reside inside the closed channel (left), whereas larger QAs cannot (right). (E) Alternative model in which the TEA molecule can block the channel in a state-independent manner, whereas the rest of the QAs can only access the blocking site when the channel is open, and hence interact with channel gating. In any way, TEA can reside in its blocking site when the channel is closed, whereas the other QAs do not.

Mentions: What do these experiments tell us about the probable structure of the inner pore of TRPV1 channels? In principle, probing the channel with varying sizes of blocker molecules should provide us with a way to size the pore in an analogous manner to what was achieved for Na+ channels (Hille, 1971). We observed that both the association and dissociation rate constants decrease in a monotonic fashion as a function of the blocker size (Fig. 10, A and B). Based on the shape of this relationship, it could be argued that the inner pore of an open channel has an admission cutoff size between the size of TPrA and TBA, with 9 and 10 Å, respectively, which corresponds to the region where the association and dissociation rates have their largest reduction. This does not mean that larger organic compounds cannot enter the inner pore. In fact, TPA is able to block TRPV1, and we have observed that QAs as large as tetraoctylammonium (14 Å) can also block this channel (not depicted). This approximate size of the inner pore of open TRPV1 is consistent with the size of the inner pore of the open MthK potassium channel, which is 12 Å (Jiang et al., 2002).


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)

Dependence of the on- and off-rates of QA blockers on their size. The blocker dissociation (A) or association rates (B) are plotted as a function of their ionic radii. The continuous lines are drawn as a visual guide and have no theoretical significance. The decrease of the rates as a function of size is monotonic and there is not a clear cut-off size. Data are mean ± SEM of five to six experiments. (C–E) Cartoon depicting the possible ways in which the QAs may interact with the channel pore. (C) Cartoon of the open-channel pore with a blocker the size of TEA (right) or TPrA (left). (D) A model in which all QAs are only able to access the blocking site when the channel is in the open state, but TEA can reside inside the closed channel (left), whereas larger QAs cannot (right). (E) Alternative model in which the TEA molecule can block the channel in a state-independent manner, whereas the rest of the QAs can only access the blocking site when the channel is open, and hence interact with channel gating. In any way, TEA can reside in its blocking site when the channel is closed, whereas the other QAs do not.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: Dependence of the on- and off-rates of QA blockers on their size. The blocker dissociation (A) or association rates (B) are plotted as a function of their ionic radii. The continuous lines are drawn as a visual guide and have no theoretical significance. The decrease of the rates as a function of size is monotonic and there is not a clear cut-off size. Data are mean ± SEM of five to six experiments. (C–E) Cartoon depicting the possible ways in which the QAs may interact with the channel pore. (C) Cartoon of the open-channel pore with a blocker the size of TEA (right) or TPrA (left). (D) A model in which all QAs are only able to access the blocking site when the channel is in the open state, but TEA can reside inside the closed channel (left), whereas larger QAs cannot (right). (E) Alternative model in which the TEA molecule can block the channel in a state-independent manner, whereas the rest of the QAs can only access the blocking site when the channel is open, and hence interact with channel gating. In any way, TEA can reside in its blocking site when the channel is closed, whereas the other QAs do not.
Mentions: What do these experiments tell us about the probable structure of the inner pore of TRPV1 channels? In principle, probing the channel with varying sizes of blocker molecules should provide us with a way to size the pore in an analogous manner to what was achieved for Na+ channels (Hille, 1971). We observed that both the association and dissociation rate constants decrease in a monotonic fashion as a function of the blocker size (Fig. 10, A and B). Based on the shape of this relationship, it could be argued that the inner pore of an open channel has an admission cutoff size between the size of TPrA and TBA, with 9 and 10 Å, respectively, which corresponds to the region where the association and dissociation rates have their largest reduction. This does not mean that larger organic compounds cannot enter the inner pore. In fact, TPA is able to block TRPV1, and we have observed that QAs as large as tetraoctylammonium (14 Å) can also block this channel (not depicted). This approximate size of the inner pore of open TRPV1 is consistent with the size of the inner pore of the open MthK potassium channel, which is 12 Å (Jiang et al., 2002).

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