Voltage is a partial activator of rat thermosensitive TRP channels

Abstract

TRPV1 and TRPM8 are sensory nerve ion channels activated by heating and cooling, respectively. A variety of physical and chemical stimuli activate these receptors in a synergistic manner but the underlying mechanisms are unclear. Both channels are voltage sensitive, and temperature and ligands modulate this voltage dependence. Thus, a voltage-sensing mechanism has become an attractive model to explain the generalized gating of these and other thermo-sensitive TRP channels. We show here using whole-cell and single channel measurements that voltage produces only a partial activation of TRPV1 and TRPM8. At room temperature (20-25 degrees C) membrane depolarization evokes responses that saturate at approximately 50-60% of the maximum open probability. Furthermore, high concentrations of capsaicin (10 microm), resiniferatoxin (5 microm) and menthol (6 mm) reveal voltage-independent gating. Similarly, other modes of TRPV1 regulation including heat, protein kinase C-dependent phosphorylation, and protons enhance both the efficacy and sensitivity of voltage activation. In contrast, the TRPV1 antagonist capsazepine produces the opposite effects. These data can be explained by an allosteric model in which voltage, temperature, agonists and inverse agonists are independently coupled, either positively or negatively, to channel gating. Thus, voltage acts separately but in concert with other stimuli to regulate channel activation, and, therefore, a voltage-sensitive mechanism is unlikely to represent a final, gating mechanism for these channels.

Figure 1. Capsaicin and protons enhance the sensitivity and efficacy of voltage activation for TRPV1

A, representative current traces in response to a family of voltage steps (voltage protocols included above current traces, capsaicin is abbreviated cap). B, the representative Boltzmann fits to the conductances obtained from steady state currents at the end of the pulse. C, the mean shift in the V_1/2 from control conditions at 25°C (n = 5). D, the mean fractional G_max normalized to 10 μm capsaicin at +200 mV (n = 5, *P = 0.01, **P = 0.007).

Figure 2. Capsaicin increases the maximal, voltage-evoked activity of TRPV1 in cell-attached patches

A, representative traces of single channel activity in cell-attached patches. Activity was evoked by a 50 ms voltage pulse from 0 mV to 200 mV in the absence (left) or presence (right) of 1 μm capsaicin. Note that the bath contained a high [K⁺] concentration in order to nullify cell membrane potential. B, the ensemble of 13 consecutive traces under control conditions (left) or 1 μm capsaicin (right). C, all-points histograms from selected data segments at +200 mV. The smooth lines are the best fits to Gaussian functions. D, single channel P_o versus voltage before and after 50 nm capsaicin. Activity was evoked by 40 mV voltage steps between −230 mV and +250 mV. The smooth line is the best fit to a Boltzmann function (parameters are given in text). Data are means of 5 cells and 5–7 trials per cell.

Figure 3. Saturating concentrations of capsaicin and RTX uncover voltage-independent gating

A, representative current traces in response to a family of voltage steps (voltage protocols included above current traces) in the absence and presence of 10 μm capsaicin. B, the representative Boltzmann fits to the conductances obtained from steady state currents at the end of the pulse. C, representative current traces in response to a family of voltage steps in the absence and presence of 5 μm RTX. D, boltzmann fits to the conductances obtained from steady state currents at the end of the pulse. E, summary of the V_1/2 values for control and 10 μm capsaicin at 25°C (n = 9, **P = 0.0005). F, summary of the voltage-independent component (G_min, n = 15 for capsaicin and n = 5 for RTX). G, summary of the mean fractional G_max normalized to 10 μm capsaicin or 5 μm RTX (n = 5).

Figure 4. Voltage partially activates TRPM8

A, representative current traces in response to a family of voltage steps (voltage protocols included above current traces) in the absence and presence of 0.8 mm menthol. B, the representative Boltzmann fits to the conductances obtained from steady state currents at the end of the pulse. C, representative current traces in response to a family of voltage steps in the absence and presence of 6 mm menthol. D, Boltzmann fits to the conductances obtained from steady state currents at the end of the pulse. E, summary of the V_1/2 values for control, 0.8 mm menthol, and 6 mm menthol at 25°C (n = 5, **P = 0.003, ***P = 0.0002). F, summary of the voltage-independent fractional response (n = 5, **P = 0.002). G, summary of the mean fractional G_max normalized to 6 mm or 0.8 mm menthol (n = 5).

Figure 5. Heat enhances the sensitivity and efficacy of voltage activation for TRPV1

A, representative current traces in response to a family of voltage steps at 25°C and 32°C in the absence or presence of protons (pH 6.0). B, Boltzmann fits to the conductances obtained from steady state currents at the end of the pulse. C, summary of the V_1/2 values at the given temperatures (n = 4–5). D, summary of the fractional G_max normalized to pH 6.0 (n = 4–5). *P < 0.05 **P < 0.005. Data were compared to 20°C.

Figure 6. PKC-dependent phosphorylation enhances sensitivity and efficacy of voltage activation for TRPV1

A, representative current traces in response to a family of voltage steps on cells pretreated with PDBu (500 nm) in the absence or presence of protons (pH 6.0). B, the representative Boltzmann fits to the conductances obtained from steady state currents at the end of the pulse. C, summary of the shifts in the V_1/2 from control conditions (cells not treated with PDBu) at 20°C (n = 5) or at 25°C when comparing the S502A/S800A mutant (n = 4). D, summary of the fractional G_max normalized to pH 6.0 (n = 5). **P < 0.005.

Figure 7. Capsazepine inhibits voltage-dependent activation of TRPV1

A, representative current traces in response to a family of voltage steps in the absence or presence of various concentrations of capsazepine (CPZ). B, the representative Boltzmann fits to the conductances obtained from steady state currents at the end of the pulse. C, the Hill plot for the reduction of the G_max by various concentrations of capsazepine (maximal inhibition = 0.53 ± 0.02, IC₅₀ = 0.456 ± 0.029 μm, Hill coefficent (h) = 2.38 ± 0.36). D, the Hill plot for the shifts in V_1/2 values by various concentrations of capsazepine (maximal shift = 72.0 ± 2.0 mV, EC₅₀ = 0.292 ± 0.023 μm, h = 1.49 ± 0.15). For C and D the data points are the mean of 6–8 separate experiments.

Figure 8. Capsazepine inhibits voltage-dependent activation after PKC-dependent phosphorylation

A, representative current traces in response to a family of voltage steps in cells pretreated with PDBu (500 nm) in the absence or presence of capsazepine (5 μm). B, the representative Boltzmann fits to the conductances obtained from steady state currents at the end of the pulse.

Figure 9. Allosteric models for gating of thermo TRP channels

A, an 8-state channel schematic diagram for allosteric modulation between voltage and temperature activation. See text for details. B, average G/G_max versus voltage plots with simultaneous fits to the 8-state model for TRPV1 (n = 4–6). C, a 16-state channel schematic diagram for allosteric modulation between voltage, temperature and ligand activation. See text for details. D, average G/G_max versus voltage plots with simultaneous fits to the 16-state model for TRPV1 in the presence or absence of capsaicin (25°C, n = 4). E, average G/G_max versus voltage plots with simultaneous fits to the 16-state model for TRPM8 in the presence or absence of menthol (25°C, n = 4). F, average G/G_max versus voltage plots with simultaneous fits to the 16-state model for TRPV1 in the presence or absence of capsazepine (25°C, n = 4).