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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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Analysis of a fast two-state process with amplitude histograms and the β distribution. (A) Monte-Carlo simulation of the process with blocking rate β and unblocking rate α = 51,000 s−1. The simulation is set so that the amplitudes of the states in the scheme are O (open) = 1 and B (blocked) = 0. (B) The simulated data in A after filtering with a Gaussian filter with corner frequency, fc, of 2500 Hz. The data becomes smeared and the amplitude is reduced. (C) Amplitude histogram compiled from all the points from the trace in B (gray trace). The black trace is the fit of the β distribution to the data (Eq. 2) with parameters β = 51,200 s−1, α = 50,950 s−1, and τ = 0.418/fc.
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fig1: Analysis of a fast two-state process with amplitude histograms and the β distribution. (A) Monte-Carlo simulation of the process with blocking rate β and unblocking rate α = 51,000 s−1. The simulation is set so that the amplitudes of the states in the scheme are O (open) = 1 and B (blocked) = 0. (B) The simulated data in A after filtering with a Gaussian filter with corner frequency, fc, of 2500 Hz. The data becomes smeared and the amplitude is reduced. (C) Amplitude histogram compiled from all the points from the trace in B (gray trace). The black trace is the fit of the β distribution to the data (Eq. 2) with parameters β = 51,200 s−1, α = 50,950 s−1, and τ = 0.418/fc.

Mentions: In these equations, τ is the single-pole filter time constant, α is the blocker dissociation rate constant, β is the association rate constant, and B(a,b) is the β function, used as a normalization factor. We simulated a noiseless two-state Markov process with a Monte-Carlo algorithm and filtered the data at 2,500 Hz, which is the filter cutoff frequency used in our experiments. The simulation was performed with the two states, identified as blocked and open, having amplitudes of 0 and 1, respectively (Fig. 1 A). Data were simulated with rates α and β = 51,000 s−1. The same data after Gaussian filtering and its corresponding amplitude distribution is shown in Fig. 1 (B and C). To fit Eq. 2 to the amplitude distribution, the single-pole filter time constant, τ, is corrected by a factor ε/fc, where fc is the Gaussian corner frequency. The term ε can be determined from fits of Eq. 2 to amplitude distributions derived from simulated data, by holding α and β = 51,000 s−1. We determined a value for ε = 0.418 and thus τ = 0.418/ fc. This value of τ provided the best fits to the amplitude distribution of simulated data over a wide range of values for α and β. All-points histograms collected from individual openings or bursts of openings were scaled in such a way that zero corresponds to the closed channel current level and one is the open-channel amplitude in the absence of blocker. The closed events were subtracted from the histograms to obtain only the open event distribution. Histograms were then normalized so that the area under the curve would be equal to one. To fit the amplitude histograms, β distributions were convolved with a Gaussian function that represents the added noise observed when the channel was fully closed. All data analysis was performed with programs written in Igor Pro (Wavemetrics Inc.).


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)

Analysis of a fast two-state process with amplitude histograms and the β distribution. (A) Monte-Carlo simulation of the process with blocking rate β and unblocking rate α = 51,000 s−1. The simulation is set so that the amplitudes of the states in the scheme are O (open) = 1 and B (blocked) = 0. (B) The simulated data in A after filtering with a Gaussian filter with corner frequency, fc, of 2500 Hz. The data becomes smeared and the amplitude is reduced. (C) Amplitude histogram compiled from all the points from the trace in B (gray trace). The black trace is the fit of the β distribution to the data (Eq. 2) with parameters β = 51,200 s−1, α = 50,950 s−1, and τ = 0.418/fc.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Analysis of a fast two-state process with amplitude histograms and the β distribution. (A) Monte-Carlo simulation of the process with blocking rate β and unblocking rate α = 51,000 s−1. The simulation is set so that the amplitudes of the states in the scheme are O (open) = 1 and B (blocked) = 0. (B) The simulated data in A after filtering with a Gaussian filter with corner frequency, fc, of 2500 Hz. The data becomes smeared and the amplitude is reduced. (C) Amplitude histogram compiled from all the points from the trace in B (gray trace). The black trace is the fit of the β distribution to the data (Eq. 2) with parameters β = 51,200 s−1, α = 50,950 s−1, and τ = 0.418/fc.
Mentions: In these equations, τ is the single-pole filter time constant, α is the blocker dissociation rate constant, β is the association rate constant, and B(a,b) is the β function, used as a normalization factor. We simulated a noiseless two-state Markov process with a Monte-Carlo algorithm and filtered the data at 2,500 Hz, which is the filter cutoff frequency used in our experiments. The simulation was performed with the two states, identified as blocked and open, having amplitudes of 0 and 1, respectively (Fig. 1 A). Data were simulated with rates α and β = 51,000 s−1. The same data after Gaussian filtering and its corresponding amplitude distribution is shown in Fig. 1 (B and C). To fit Eq. 2 to the amplitude distribution, the single-pole filter time constant, τ, is corrected by a factor ε/fc, where fc is the Gaussian corner frequency. The term ε can be determined from fits of Eq. 2 to amplitude distributions derived from simulated data, by holding α and β = 51,000 s−1. We determined a value for ε = 0.418 and thus τ = 0.418/ fc. This value of τ provided the best fits to the amplitude distribution of simulated data over a wide range of values for α and β. All-points histograms collected from individual openings or bursts of openings were scaled in such a way that zero corresponds to the closed channel current level and one is the open-channel amplitude in the absence of blocker. The closed events were subtracted from the histograms to obtain only the open event distribution. Histograms were then normalized so that the area under the curve would be equal to one. To fit the amplitude histograms, β distributions were convolved with a Gaussian function that represents the added noise observed when the channel was fully closed. All data analysis was performed with programs written in Igor Pro (Wavemetrics Inc.).

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