Contribution of TRPM8 channels to cold transduction in primary sensory neurons and peripheral nerve terminals

Abstract

Transient receptor potential melastatin 8 (TRPM8) is the best molecular candidate for innocuous cold detection by peripheral thermoreceptor terminals. To dissect out the contribution of this cold- and menthol-gated, nonselective cation channel to cold transduction, we identified BCTC [N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide] as a potent and full blocker of recombinant TRPM8 channels. In cold-sensitive trigeminal ganglion neurons of mice and guinea pig, responses to menthol were abolished by BCTC. In contrast, the effect of BCTC on cold-evoked responses was variable but showed a good correlation with the presence or lack of menthol sensitivity in the same neuron, suggesting a specific blocking action of BCTC on TRPM8 channels. The biophysical properties of native cold-gated currents (I(cold)), and the currents blocked by BCTC were nearly identical, consistent with a role of this channel in cold sensing at the soma. The temperature activation threshold of native TRPM8 channels was significantly warmer than those reported in previous expression studies. The effect of BCTC on native I(cold) was characterized by a dose-dependent shift in the temperature threshold of activation. The role of TRPM8 in transduction was further investigated in the guinea pig cornea, a peripheral territory densely innervated with cold thermoreceptors. All cold-sensitive terminals were activated by menthol, suggesting the functional expression of TRPM8 channels in their membrane. However, the spontaneous activity and firing pattern characteristic of cold thermoreceptors was totally immune to TRPM8 channel blockade with BCTC or SKF96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride). Cold-evoked responses in corneal terminals were also essentially unaffected by these drugs, whereas responses to menthol were completely abolished. The minor impairment in the ability to transduce cold stimuli by peripheral corneal thermoreceptors during TRPM8 blockade unveils an overlapping functional role for various thermosensitive mechanisms in these nerve terminals.

Figure 1.

BCTC blocks TRPM8-mediated responses to cold and menthol in transfected HEK293 cells.A, Ratiometric [Ca²⁺]_i response of a TRPM8⁺ HEK293 cell (black trace) to 100 μm menthol in control solution, in the presence of 3 μm BCTC, and after washout of BCTC. In the same field, a TRPM8-negative (TRPM8⁻) cell (blue trace) did not respond to menthol.B, Time course of current development at +80 and −80 mV in a TRPM8⁺ HEK293 cell, recorded at 33°C, by the application of 100 μm menthol. In the continuous presence of menthol, 3 μm BCTC produced a full suppression of the current. Currents were evoked by a 1-s-duration −100/+100 mV voltage ramp delivered every 5 s. The negative current axis is expanded fivefold to show the very small inward currents.C, Whole-cell rampI–V relationship at 33°C in control solution (black trace), during 100 μm menthol (blue trace), and during 100 μm menthol plus 3 μm BCTC (red trace).D, Relative block of calcium response (open squares) and menthol-evoked currents (filled circles) to cooling pulses as a function of BCTC concentration (n = 5–10 cells for calcium data and 2–5 cells for current data). Solid lines correspond to dose–response fits to the Hill equation, in black for menthol data (EC₅₀ of 647 nm and a Hill coefficient of 1.9), and in red for current data (EC₅₀ of 475 nm and Hill coefficient of 1.1).E, Ratiometric [Ca²⁺]_i response in a TRPM8⁺ HEK293 cell (same as inA) during a cooling stimulus in control solution and in the presence of 3 μm BCTC. Note the lack of response in a TRPM8⁻ cell (blue trace) recorded simultaneously.F, Time course of current at +80 and −80 mV in a TRPM8⁺ HEK293 cell during a cooling stimulus and effect of 0.6 and 3 μm BCTC. Same voltage protocol as inB. The negative current axis is expanded twofold.G,I–V relationship of currents at 35°C (black trace), during cooling at 18°C (blue trace), and during cooling in the presence of 0.6 (gray trace) and 3 μm BCTC (red trace).H, Normalized block of cold-evoked calcium signals (open squares) and currents (filled circles) in TRPM8⁺ HEK293 cells by different concentrations of BCTC. The smooth curves are fits of the Hill equation to the data with an EC₅₀ of 685 nm and a Hill coefficient of 1.9 for calcium data (black line;n = 5–10 cells) and an EC₅₀ of 621 nm andN = 1.8 for current data at +80 mV (red line;n = 2–8 cells).

Figure 2.

BCTC blocks menthol- and cold-induced [Ca²⁺]_i responses in neonatal mice trigeminal sensory neurons.A, Transmitted (left) and pseudocolor ratiometric [Ca²⁺]_i images showing the effects of BCTC on menthol-evoked [Ca²⁺]_i signals in cultured trigeminal neurons. The fluorescence images correspond with the time points marked in red inB. Scale bar, 15 μm.B, Ratiometric [Ca²⁺]_i response of a CS and a CI trigeminal sensory neuron to 100 μm menthol in control solution, in the presence of 3 μm BCTC, and after washout of BCTC.C, Mean evoked [Ca²⁺]_i elevation by menthol, by menthol in the presence of 3 μm BCTC, and after wash of BCTC.D, Ratiometric [Ca²⁺]_i responses to cooling in three CS trigeminal sensory neurons, recorded simultaneously, in control solution, in the presence of 3 μm BCTC, and after washout. A response to 100 μm menthol in the same three neurons is also shown. The time gap equals 550 s.E, Mean evoked [Ca²⁺]_i elevation by cooling in control solution, in the presence of 1 and 3 μm BCTC, and after wash in mouse CS trigeminal neurons.

Figure 3.

BCTC reduces firing and shifts temperature threshold in cold-sensitive trigeminal ganglion neurons.A, Transmitted (left) and pseudocolor ratiometric [Ca²⁺]_i images showing the effects of BCTC on cold-evoked [Ca²⁺]_i signals in a CS trigeminal neuron. Note also the response to menthol. A patch pipette has been positioned in close apposition to the CS cell before initiating the sequence of cell-attach recordings. The fluorescence images correspond with the time points marked in red inB.B, Simultaneous recording of action currents (top trace), [Ca²⁺]_i signals (middle), and bath temperature (bottom) during four consecutive cooling ramps. The two insets at the bottom show the action currents and the temperature change on an expanded timescale, in control (left) and 1 μm BCTC (right). The temperature threshold is marked by a pink arrowhead. The black arrow marks, on the control trace, the temperature threshold in 1 μm BCTC. Note the decline in action current amplitude with low temperature.C, Scatter plot of thresholds for action potential and [Ca²⁺]_i signals in response to cooling in 11 neurons (each neuron has been color coded) recorded in control solution (circles) and 1 μm (triangles) and 3 μm (stars) BCTC. The dotted line represents the unity line.

Figure 4.

BCTC shifts temperature threshold in mouse cold-sensitive trigeminal ganglion neurons in a dose-dependent manner.A, Dot plot summarizing the effect of 1 and 3 μm BCTC on cold-evoked temperature threshold in 88 cold-sensitive TG neurons. Threshold was estimated from the ratiometric [Ca²⁺]_i responses to cooling. The 88 neurons have been plotted according to initial temperature threshold in control solution (black circles) from lowest to highest threshold. The threshold measured in 1 and 3 μm BCTC is represented by cyan triangles and magenta circles, respectively. Those neurons fully inhibited by 3 μm BCTC during a cooling ramp to 20–18°C are represented by a red star. The green circles mark the threshold in 3 μm BCTC for those neurons whose temperature threshold were not augmented by BCTC: 100 μm menthol was tested in three of these neurons (asterisk) and had no effect.B, Diagram representing the percentage of CS neurons fully inhibited by 3 μm BCTC of the total population with responses to cooling affected by BCTC (n = 81). Neurons have been grouped according to initial temperature threshold. The horizontal error bars in four groups overlap with symbol size.C, Bar histogram summarizing the effect of 3 μm BCTC on cold-evoked [Ca²⁺]_i responses in the three subpopulations of neurons grouped according to blocking effect of BCTC.

Figure 5.

BCTC blocks currents induced by cooling and menthol in cold-sensitive trigeminal ganglion neurons.A, Simultaneous recording of membrane current (top trace) and bath temperature (bottom trace) during application of three consecutive cooling ramps to a CS neuron (V_hold of −60 mV). The spike-like currents are the responses to voltage ramps (−100 to +100 mV). Application of 3 μm BCTC fully blockedI_cold. The dotted line represents the 0 holding current.B, Current–voltage relationship of the cold-sensitive (blue trace) and BCTC-sensitive (red trace) current obtained during the voltage ramps. To derive the cold-sensitive current, the ramp current at 35°C (black dot) was subtracted from the current at 20°C (blue dot). To derive the BCTC-sensitive current, the ramp current at 20°C in BCTC (red dot) was subtracted from the current at 20°C in control solution (blue dot).C, Bar histogram summarizing the block ofI_cold by 1 and 3 μm BCTC.D, Current–temperature relationships for a different neuron in control (blue trace) and in the presence of 1 μm (black trace) and 3 μm (red trace) BCTC. Note the marked shift in temperature threshold.E, Simultaneous recording of membrane current (top trace) and bath temperature (bottom trace) during thermal and chemical (500 μm menthol) activation of a CS neuron (V_hold of −60 mV).F, Bar histogram summarizing the block ofI_menthol by 3 μm BCTC.

Figure 6.

BCTC blocks cold-induced generator potentials in cold-sensitive trigeminal ganglion neurons.A, Simultaneous recording of membrane potential (top trace), bath temperature (middle trace), and membrane current (bottom trace) during three consecutive cooling ramps to a cold-sensitive TG neuron recorded in current-clamp mode (I_hold of 0 pA). Application of 3 μm BCTC produced a reversible reduction in the cold-induced depolarization without affecting the voltage response to a 500 pA ramp. The dotted line represents the initial resting membrane potential.B, Bar histogram of normalized responses to cooling and toI-evoked firing in control solution (black bars) and in 3 μm BCTC (stripped bars).C, Simultaneous recording of [Ca²⁺]_i (top trace) and bath temperature (bottom trace) during two consecutive cooling steps to a cold-sensitive TG neuron. BCTC at 3 μm produced a large reduction in the cold-induced response with only minor effects on the depolarization-induced response produced by a 30 mm elevation in extracellular K⁺.D, Bar histogram of normalized [Ca²⁺]_i responses to cooling and to elevated K⁺ in control solution (black bars) and in 3 μm BCTC (striped bars). The reduction in BCTC is significant in both cases (p < 0.0001 for cold-evoked responses andp < 0.001 for K⁺-evoked responses).

Figure 7.

BCTC blocks menthol- and cold-induced [Ca²⁺]_i responses exclusively in the menthol-sensitive subpopulation of guinea pig cold-sensitive trigeminal sensory neurons.A, Transmitted (left) and pseudocolor ratiometric [Ca²⁺]_i images showing the response to low temperature and 100 μm menthol and the effects of BCTC on menthol-evoked [Ca²⁺]_i signals in a CS trigeminal neuron. Note the lack of response in the CI neuron. The fluorescence images correspond with the time points marked in red inB.B, Time course of ratiometric [Ca²⁺]_i responses of a CS and a CI trigeminal sensory neuron (same experiment as inA). The recording starts with a cooling ramp to identify the CS neurons in the imaging field.C, Mean evoked [Ca²⁺]_i elevation by menthol, by menthol in the presence of 3 μm BCTC, and after wash of BCTC.D, Ratiometric [Ca²⁺]_i responses to cooling in a CS trigeminal sensory neurons in control solution, in the presence of 3 μm BCTC, and after washout. After a brief interruption, a fourth cooling ramp was applied in the presence of 100 μm menthol.E, Ratiometric [Ca²⁺]_i responses to cooling in a CS trigeminal sensory neurons in control solution, in the presence of 3 μm BCTC, and after washout. Note the lack of effect of BCTC and the lack of response to 100 μm menthol.

Figure 8.

Lack of effect of BCTC on spontaneous and cold-evoked activity in corneal sensory endings.A, Activity in a corneal CS receptor during two consecutive cooling cycles, in control solution, and during perfusion with 10 μm BCTC. The bottom trace shows the temperature of the bathing solution recorded close to the corneal surface. The top trace shows the effect of changing temperature on the frequency of NTIs. The gray vertical bars mark the cold threshold, projected as a dotted line on the temperature scale. Note the lack of effect of 10 μm BCTC on spontaneous and cold-evoked activity.B, Each trace represents a 1 s original record of the terminal shown inA, at the baseline temperature (35°C), and at the peak of the cold response (25°C). Note the complete lack of effect of BCTC, on neither the mean firing frequency nor the pattern of discharge.

Figure 9.

Effect of BCTC on menthol-evoked NTI activity in corneal cold thermoreceptor nerve endings.A, Application of 10 μm BCTC blocks the enhancement in NTI activity produced by 100 μm menthol.B, Dose–response curve of menthol effects on the frequency of ongoing NTI activity in corneal cold-sensitive receptors. Data have been fitted to the Hill equation (EC₅₀ of 20 μm; Hill coefficient of 0.9).C, Illustrative example of the effect of 10 μm BCTC on menthol-evoked NTI activity in a CS receptor. In the presence of BCTC, application of menthol did not augment ongoing NTI activity. After BCTC removal, the spontaneous NTI discharge nearly doubled, and the cold-evoked activity also increased.D, Summary of menthol (100 μm) and BCTC (10 μm) effects on cold-evoked responses (n = 17). Results have been normalized to those obtained in control solution. Only the response in menthol is significantly different from the rest (ANOVA test, *p < 0.05).

Figure 10.

Effect of SKF96365 on cold and menthol-evoked NTI activity in corneal cold thermoreceptor nerve endings.A, Activity in a corneal CS receptor during two consecutive cooling cycles, in control solution, and during perfusion with 20 μm SKF96365. The bottom trace shows the temperature of the bathing solution recorded close to the corneal surface. The top trace shows the effect of changing temperature on the frequency of NTIs. Note the lack of effect of 20 μm SKF96365 on spontaneous and cold-evoked activity.B, Summary histogram of effects of 20 μm SKF96365 on cold-evoked activity (n = 10). For each terminal, cold-evoked activity has been normalized to the mean frequency at static temperature (32–34°C) before application of SKF96365. The drug did not produce a significant inhibition of the cold-evoked response.C, Application of 20 μm SKF96365 blocks reversibly the enhancement in NTI activity produced by 100 μm menthol at a baseline temperature of 34°C.D, Summary histogram of effects of 20 μm SKF96365 on menthol-evoked activity (n = 10). For each terminal, menthol-evoked activity has been normalized to the mean frequency at static temperature (32–34°C) in control solution, before application of SKF96365. The drug produce a highly significant inhibition of the menthol-evoked response (**p < 0.001).