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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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Block of TRPV1 currents by intracellular QAs. Current traces without blockers (A) and with blockers (B). The currents were elicited by stepping membrane voltage from a 0-mV holding potential to −120 mV, and then to various test potentials from −150 to 150 mV in 10-mV increments. For clarity, only the traces at every 20 mV are shown. All current traces in B were obtained after applying the corresponding QA to the patch in A and corrected for leak current in the absence of agonist. The blocker concentrations used are: 10 mM TEA, 0.9 mM TPrA, 250 μM TBA, and 40 μM TPA. The dotted lines identify the zero current levels. (C) Steady-state current to voltage relations obtained from the traces in A (closed symbols) and B (open symbols).
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fig2: Block of TRPV1 currents by intracellular QAs. Current traces without blockers (A) and with blockers (B). The currents were elicited by stepping membrane voltage from a 0-mV holding potential to −120 mV, and then to various test potentials from −150 to 150 mV in 10-mV increments. For clarity, only the traces at every 20 mV are shown. All current traces in B were obtained after applying the corresponding QA to the patch in A and corrected for leak current in the absence of agonist. The blocker concentrations used are: 10 mM TEA, 0.9 mM TPrA, 250 μM TBA, and 40 μM TPA. The dotted lines identify the zero current levels. (C) Steady-state current to voltage relations obtained from the traces in A (closed symbols) and B (open symbols).

Mentions: Intracellular application of all four QAs readily blocked currents through TRPV1. Fig. 2 A shows macroscopic TRPV1 currents activated by voltage pulses in the presence of saturating 4 μM capsaicin recorded from inside-out patches in the absence of blocker. The addition of QAs effectively induced current block (Fig. 2 B). Fig. 2 C shows the steady-state current to voltage relations obtained from the traces in A and B. The outward rectifying character of the TRPV1 channel can be clearly seen and block seems to be voltage dependent, with QAs blocking with higher affinity at positive membrane potentials. The reversal potential lies near zero mV, as expected under isometric sodium conditions.


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)

Block of TRPV1 currents by intracellular QAs. Current traces without blockers (A) and with blockers (B). The currents were elicited by stepping membrane voltage from a 0-mV holding potential to −120 mV, and then to various test potentials from −150 to 150 mV in 10-mV increments. For clarity, only the traces at every 20 mV are shown. All current traces in B were obtained after applying the corresponding QA to the patch in A and corrected for leak current in the absence of agonist. The blocker concentrations used are: 10 mM TEA, 0.9 mM TPrA, 250 μM TBA, and 40 μM TPA. The dotted lines identify the zero current levels. (C) Steady-state current to voltage relations obtained from the traces in A (closed symbols) and B (open symbols).
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Block of TRPV1 currents by intracellular QAs. Current traces without blockers (A) and with blockers (B). The currents were elicited by stepping membrane voltage from a 0-mV holding potential to −120 mV, and then to various test potentials from −150 to 150 mV in 10-mV increments. For clarity, only the traces at every 20 mV are shown. All current traces in B were obtained after applying the corresponding QA to the patch in A and corrected for leak current in the absence of agonist. The blocker concentrations used are: 10 mM TEA, 0.9 mM TPrA, 250 μM TBA, and 40 μM TPA. The dotted lines identify the zero current levels. (C) Steady-state current to voltage relations obtained from the traces in A (closed symbols) and B (open symbols).
Mentions: Intracellular application of all four QAs readily blocked currents through TRPV1. Fig. 2 A shows macroscopic TRPV1 currents activated by voltage pulses in the presence of saturating 4 μM capsaicin recorded from inside-out patches in the absence of blocker. The addition of QAs effectively induced current block (Fig. 2 B). Fig. 2 C shows the steady-state current to voltage relations obtained from the traces in A and B. The outward rectifying character of the TRPV1 channel can be clearly seen and block seems to be voltage dependent, with QAs blocking with higher affinity at positive membrane potentials. The reversal potential lies near zero mV, as expected under isometric sodium conditions.

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