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

Kinetics of TPrA block. (A) Single-channel openings in the absence (top-most trace) and presence of 0.9, 2, and 10 mM TPrA obtained at 60 mV. The current amplitude decreases as the TPrA concentration is increased, as expected for a fast blocker. The dashed line represents the closed-channel current level, and the dotted line is the mean open-channel level in the absence of TPrA. (B) Normalized amplitude histograms obtained from traces as in A. Solid lines are fits to the β distribution with parameters: 0.9 mM, β = 8,571.7 s−1, α = 7,315.9 s−1; 2 mM, β = 8,563 s−1, α = 12,193 s−1; 10 mM, β = 11,198 s−1, α = 49,061 s−1. (C) Blocking rate constants obtained from the fits to the β distribution as in B in three different patches at several voltages and blocker concentrations. The on-rate increases with voltage and reaches a plateau. The off-rate decreases with increasing voltages but starts to increase at voltages more positive than 60 mV. The solid line is a fit to a double exponential as in Fig. 4. The values of kon and koff at 0 mV were estimated from an exponential fit to the data between −40 and 60 mV, and the parameters of the fit are: kon(0) = 8.6 × 105 M−1 s−1; z = 0.69 eo; koff(0) = 1.23 × 104 s−1; z = −0.19 eo. Group data are presented as mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Kinetics of TPrA block. (A) Single-channel openings in the absence (top-most trace) and presence of 0.9, 2, and 10 mM TPrA obtained at 60 mV. The current amplitude decreases as the TPrA concentration is increased, as expected for a fast blocker. The dashed line represents the closed-channel current level, and the dotted line is the mean open-channel level in the absence of TPrA. (B) Normalized amplitude histograms obtained from traces as in A. Solid lines are fits to the β distribution with parameters: 0.9 mM, β = 8,571.7 s−1, α = 7,315.9 s−1; 2 mM, β = 8,563 s−1, α = 12,193 s−1; 10 mM, β = 11,198 s−1, α = 49,061 s−1. (C) Blocking rate constants obtained from the fits to the β distribution as in B in three different patches at several voltages and blocker concentrations. The on-rate increases with voltage and reaches a plateau. The off-rate decreases with increasing voltages but starts to increase at voltages more positive than 60 mV. The solid line is a fit to a double exponential as in Fig. 4. The values of kon and koff at 0 mV were estimated from an exponential fit to the data between −40 and 60 mV, and the parameters of the fit are: kon(0) = 8.6 × 105 M−1 s−1; z = 0.69 eo; koff(0) = 1.23 × 104 s−1; z = −0.19 eo. Group data are presented as mean ± SEM.

Mentions: Just as in the case of TEA, blockade by TPrA is exceedingly fast, and the individual blocking events are not well resolved at our recording bandwidth (Fig. 5). For this reason, we also applied the β distribution approach to estimate the rate constants of TPrA block of TRPV1. Fig. 5 A shows single-channel traces in the absence and presence of TPrA at the concentrations indicated in Fig. 5 B. All-point amplitude histograms derived from single-channel openings can be well described by the β distribution (Eq. 2), and on- and off-rate constants can be derived (Fig. 5, B and C). As with TEA, the on-rate constant for TPrA block saturates at positive potentials, and this happens at a value near 5 × 106 M−1s−1, well below the estimated diffusion-limited on-rate of 1.7 × 108 M−1s−1 for TPrA (9 Å diameter), which was calculated as for TEA. The voltage dependence of the on-rate constant is exponential at voltages below 40 mV, with a valence of 0.7 eo. In this same range of voltages, the off-rate constant increases with hyperpolarization with a valence of −0.19 eo, but, as for TEA block, the off-rate constant also increases at voltages more positive than 40 mV, instead of monotonically decreasing, and this behavior accounts for the marked relief of block at positive potentials that is also observed with TPrA.


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 TPrA block. (A) Single-channel openings in the absence (top-most trace) and presence of 0.9, 2, and 10 mM TPrA obtained at 60 mV. The current amplitude decreases as the TPrA concentration is increased, as expected for a fast blocker. The dashed line represents the closed-channel current level, and the dotted line is the mean open-channel level in the absence of TPrA. (B) Normalized amplitude histograms obtained from traces as in A. Solid lines are fits to the β distribution with parameters: 0.9 mM, β = 8,571.7 s−1, α = 7,315.9 s−1; 2 mM, β = 8,563 s−1, α = 12,193 s−1; 10 mM, β = 11,198 s−1, α = 49,061 s−1. (C) Blocking rate constants obtained from the fits to the β distribution as in B in three different patches at several voltages and blocker concentrations. The on-rate increases with voltage and reaches a plateau. The off-rate decreases with increasing voltages but starts to increase at voltages more positive than 60 mV. The solid line is a fit to a double exponential as in Fig. 4. The values of kon and koff at 0 mV were estimated from an exponential fit to the data between −40 and 60 mV, and the parameters of the fit are: kon(0) = 8.6 × 105 M−1 s−1; z = 0.69 eo; koff(0) = 1.23 × 104 s−1; z = −0.19 eo. Group data are presented as mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Kinetics of TPrA block. (A) Single-channel openings in the absence (top-most trace) and presence of 0.9, 2, and 10 mM TPrA obtained at 60 mV. The current amplitude decreases as the TPrA concentration is increased, as expected for a fast blocker. The dashed line represents the closed-channel current level, and the dotted line is the mean open-channel level in the absence of TPrA. (B) Normalized amplitude histograms obtained from traces as in A. Solid lines are fits to the β distribution with parameters: 0.9 mM, β = 8,571.7 s−1, α = 7,315.9 s−1; 2 mM, β = 8,563 s−1, α = 12,193 s−1; 10 mM, β = 11,198 s−1, α = 49,061 s−1. (C) Blocking rate constants obtained from the fits to the β distribution as in B in three different patches at several voltages and blocker concentrations. The on-rate increases with voltage and reaches a plateau. The off-rate decreases with increasing voltages but starts to increase at voltages more positive than 60 mV. The solid line is a fit to a double exponential as in Fig. 4. The values of kon and koff at 0 mV were estimated from an exponential fit to the data between −40 and 60 mV, and the parameters of the fit are: kon(0) = 8.6 × 105 M−1 s−1; z = 0.69 eo; koff(0) = 1.23 × 104 s−1; z = −0.19 eo. Group data are presented as mean ± SEM.
Mentions: Just as in the case of TEA, blockade by TPrA is exceedingly fast, and the individual blocking events are not well resolved at our recording bandwidth (Fig. 5). For this reason, we also applied the β distribution approach to estimate the rate constants of TPrA block of TRPV1. Fig. 5 A shows single-channel traces in the absence and presence of TPrA at the concentrations indicated in Fig. 5 B. All-point amplitude histograms derived from single-channel openings can be well described by the β distribution (Eq. 2), and on- and off-rate constants can be derived (Fig. 5, B and C). As with TEA, the on-rate constant for TPrA block saturates at positive potentials, and this happens at a value near 5 × 106 M−1s−1, well below the estimated diffusion-limited on-rate of 1.7 × 108 M−1s−1 for TPrA (9 Å diameter), which was calculated as for TEA. The voltage dependence of the on-rate constant is exponential at voltages below 40 mV, with a valence of 0.7 eo. In this same range of voltages, the off-rate constant increases with hyperpolarization with a valence of −0.19 eo, but, as for TEA block, the off-rate constant also increases at voltages more positive than 40 mV, instead of monotonically decreasing, and this behavior accounts for the marked relief of block at positive potentials that is also observed with TPrA.

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