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. 2009 Jan 16;284(3):1570-82.
doi: 10.1074/jbc.M807270200. Epub 2008 Nov 18.

Activity of the neuronal cold sensor TRPM8 is regulated by phospholipase C via the phospholipid phosphoinositol 4,5-bisphosphate

Richard L Daniels  1 , Yoshio Takashima, David D McKemy
Affiliations

Activity of the neuronal cold sensor TRPM8 is regulated by phospholipase C via the phospholipid phosphoinositol 4,5-bisphosphate

Richard L Daniels et al. J Biol Chem. .

Abstract

Cold temperatures robustly activate a small cohort of somatosensory nerves, yet during a prolonged cold stimulus their activity will decrease, or adapt, over time. This process allows for the discrimination of subtle changes in temperature. At the molecular level, cold is detected by transient receptor potential melastatin 8 (TRPM8), a nonselective cation channel expressed on a subset of peripheral afferent fibers. We and others have reported that TRPM8 channels also adapt in a calcium-dependent manner when activated by the cooling compound menthol. Additionally, TRPM8 activity is sensitive to the phospholipid phosphoinositol 4,5-bisphosphate (PIP2), a substrate for the enzyme phospholipase C (PLC). These results suggest an adaptation model whereby TRPM8-mediated Ca2+ influx activates PLC, thereby decreasing PIP2 levels and resulting in reduced TRPM8 activity. Here we tested this model using pharmacological activation of PLC and by manipulating PIP2 levels independent of both PLC and Ca2+. PLC activation leads to adaptation-like reductions in cold- or menthol-evoked TRPM8 currents in both heterologous and native cells. Moreover, PLC-independent reductions in PIP2 had a similar effect on cold- and menthol-evoked currents. Mechanistically, either form of adaptation does not alter temperature sensitivity of TRPM8 but does lead to a change in channel gating. Our results show that adaptation is a shift in voltage dependence toward more positive potentials, reversing the trend toward negative potentials caused by agonist. These data suggest that PLC activity not only mediates adaptation to thermal stimuli, but likely underlies a more general mechanism that establishes the temperature sensitivity of somatosensory neurons.

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Figures

FIGURE 1.
FIGURE 1.
Adaptation of cold-evoked TRPM8 currents is dependent upon calcium and temperature. A, in two-electrode voltage clamp recordings from rTRPM8-expressing Xenopus oocytes, cooling of nominally Ca2+-free bath solutions to 15 °C evokes robust inward currents (holding potential of -60 mV) that are sustained for the duration of the cold stimulus (5 min). Warming of the perfusate to room temperature inhibited these currents, but a second cold ramp returned currents to previous levels (n = 5). B, in the presence of physiological (2 mm) Ca2+, a cold ramp to 15 °C also produces robust inward currents, yet these decrease over time, and subsequent cold stimuli evoke smaller, adapted currents (n = 4). C, TRPM8 adaptation is dependent upon temperature. In the presence of 2 mm external Ca2+, currents adapt more readily to moderately cool temperatures (15 °C) than strong cold pulses (6 °C) in the presence of 2 mm Ca2+ (n = 3–6). D, before BAPTA injection, cold-evoked inward currents adapt in the presence of 2 mm external Ca2+. After BAPTA injection (50 μl of a 100 mm solution), cold-evoked currents do not adapt (n = 4). E, prolonged cold stimulus (15 °C) in the presence of 2 mm Ca2+ produces robust and adapting inward currents, which remain at adapted current magnitudes (dashed line) until the bath solution is brought to physiological temperatures (>30 °C).
FIGURE 2.
FIGURE 2.
Ca2+ acts as a channel blocker of TRPM8. A, representative whole-cell voltage clamp recordings of TRPM8-expressing HEK293T cells show reduced menthol-evoked currents at both positive and negative membrane potentials as intracellular Ca2+ is increased. B, in the presence of 200 μm menthol, rapid solution exchange (<2 s) from nominally Ca2+-free to 2 mm Ca2+ blocks TRPM8 currents measured during membrane voltage ramps from -80 to +80 mV. Of note, this divalent block was rapidly reversible as well. C, current-voltage relations from time points indicated in B. D, mean remaining currents are reduced by 41.3 ± 8.7% at positive potentials and 75.5 ± 4.9 at negative potentials (n = 5).
FIGURE 3.
FIGURE 3.
Direct pharmacological activation of PLC by m-3M3FBS reduces menthol-evoked TRPM8 currents. A, representative confocal images of menthol-evoked translocation of a PLCδ1-PH-RFP domain fusion protein reporter in the presence of 2 mm external Ca2+. HEK293T cells were co-transfected with rTRPM8 and PLCδ-PH-RFP constructs, and RFP fluorescence was monitored before and after addition of 200 μm menthol to the bath solution (data representative of 10 independent experiments). Cells are shown in negative contrast. B, RFP fluorescence increased in the cytosol (circles) but remained constant in the nucleus (boxes) when the cells were exposed to 200 μm menthol. Data values are arbitrary fluorescence units (F) normalized to basal fluorescence (Fo). C, representative whole-cell voltage clamp recording from an rTRPM8-expressing HEK293T cell. Menthol-evoked currents (200 μm) were rapidly reduced upon bath co-application of 5 μm m-3M3FBS at both positive and negative membrane potentials. D, representative current-voltage relationships for menthol-evoked responses before (a), during (b), and after PLC activation (c). Data corresponds to the points in the recording indicated in C. E, reduction of TRPM8 currents by m-3M3FBS is dose-dependent. Each dosage was tested on 3–6 cells, and bars represent the means ± S.E.
FIGURE 4.
FIGURE 4.
A, direct pharmacological activation of PLC reduces cold-evoked TRPM8 currents in heterologous cells. A cold ramp (from 32 to 19 °C) evoked robust membrane currents (at +80 mV) that were reduced upon application of 2.5 μm m-3M3FBS. In this cell, a subsequent cold pulse was reduced compared with the initial values. B, current-voltage relations obtained from the points indicated in A. C, representative whole-cell voltage clamp recording of TRPM8 cold currents evoked by successive cold ramps from 32 to 17 °C (holding potential (h.p.) +80 mV). When 2.5 μm m-3M3FBS was applied between the 2nd and 3rd cold pulses, cold-evoked TRPM8 currents were reduced but returned to their previous amplitudes with time. D, representative current-voltage relations of cold-evoked responses taken from the points indicated in the recording in C. E, current to temperature relationship of TRPM8 responses before and after activation of PLC by m-3M3FBS. Cold-evoked TRPM8 currents (in the presence of 50 μm menthol) were normalized to a maximal saturating response at 14 °C (n = 6). F, current to temperature relationship of cold-evoked TRPM8 responses across five successive temperature pulses. Data presented are the average normalized temperature response for each of the five consecutive cold pulses, and 2.5 μm m-3M3FBS was applied between the 2nd and 3rd cold pulses (n = 6).
FIGURE 5.
FIGURE 5.
PLC-independent depletion of plasmalemmal PIP2 reduces menthol-evoked TRPM8 currents. A, representative images of HEK293T cells expressing rTRPM8 and Lyn-PH-PP-GFP. A, panel i, GFP fluorescence marks the cells expressing both constructs and have reduced PIP2 levels. Pseudocolored images of the 340/380 nm Fura-2 ratio show low basal Ca2+ before application of 200 μm menthol (A, panel ii). Green arrowheads mark GFP+ cells expressing Lyn-PH-PP-GFP in which menthol evoked a small increase in R values, and red arrowheads mark GFP-negative cells in which menthol evoked a robust change in intracellular Ca2+ (A, panel iii). B, averaged changes in the Fura-2 ratio of control TRPM8-expressing cells (black boxes, n = 20 cells) versus those co-expressing Lyn-PH-PP-GFP (blue circles, n = 25 cells). C, average peak ratio values (1st menthol application) of individual cells and data are averaged responses from four independent experiments and 15–25 cells per experiment. D, representative whole-cell voltage clamp recording (holding potential (h.p.) +80 mV) from a cell transfected with rTRPM8, FKBP-Inp54p, and Lyn11-FRB. Menthol-evoked (200 μm) TRPM8 currents were diminished following application of the dimerizing agent rapamycin that translocates Inp54p to the membrane. E, whole-cell voltage clamp recording from a cell transfected with rTRPM8 and FKBP-Inp54p but not the membrane tethered component Lyn11-FRB. Repeated menthol-evoked (200 μm) TRPM8 currents did not diminish upon application of the dimerizer rapamycin. F, summary data of the reductions in menthol-evoked TRPM8 currents using the rapamycin (Rap), Inp54p translocation system (n = 5 cells for each condition). G, menthol dose-response relationship before and after rapamycin-induced Inp54p translocation and reduction of TRPM8 currents (n = 3–9 cells per menthol concentration).
FIGURE 6.
FIGURE 6.
PLC-independent depletion of plasmalemmal PIP2 reduces cold-evoked TRPM8 currents but does not alter temperature sensitivity. A, representative images of HEK293T cells expressing rTRPM8 and Lyn-PH-PP-GFP. Left panel, GFP fluorescence marks the cells expressing both constructs. Middle and right panels, pseudocolored images of the 340/380 nm Fura-2 ratio (R) show low basal Ca2+ when the perfusate is held at ∼33 °C, but increased R values when perfusate temperature is reduced to 17 °C. Arrowheads mark GFP+ cells expressing Lyn-PH-PP-GFP. B, averaged changes in the Fura-2 ratio taken from A of control TRPM8-expressing cells (black lines, n = 11 cells) versus those co-expressing Lyn-PH-PP-GFP (blue lines, n = 13 cells). C, peak R values for each condition. Data are represented as the average peak R values at 17 °C and are from seven independent experiments that averaged 11–25 cells per experiment. D, normalized Ca2+ responses to the 1st cold ramp shown in B for TRPM8-expressing cells and TRPM8 and Lyn-PH-PP-GFP co-expressing cells. The base-line R values were first subtracted and then each trace was normalized to the peak R value at 17 °C. E, representative whole-cell voltage clamp recording (holding potential (h.p.) +80 mV) from a cell transfected with rTRPM8, FKBP-Inp54p, and Lyn11-FRB. Cold-evoked TRPM8 currents were diminished following application of the dimerizing agent rapamycin (rap). F, representative current-voltage relationships for before and after Inp54p translocation to a cold stimulus (15 °C). Data are taken from the time points indicated in E. G, current to temperature relationship of TRPM8 responses before and after translocation of Inp54p to the plasma membrane by the dimerizer rapamycin. Cold-evoked TRPM8 currents (in the presence of 50 μm menthol) were normalized to a maximal saturating response at 14 °C (n = 6). There was a slight significant difference in the normalized current amplitudes at 24 and 22 °C (p < 0.05, asterisks). H, current to temperature relationships were plotted and fit with a sigmoidal relationship, and the temperature at 20 (Y20), 50 (Y50), and 80% (Y80) of the peak currents at 14 °C were calculated from the curve. At 20% of the peak current, there was a significant difference (p < 0.05) in the temperatures before and after Inp54p translocation, but not at 50 and 80%.
FIGURE 7.
FIGURE 7.
Adaptation of neuronal TRPM8 currents is Ca2+-dependent and mimicked by PLC activation. A, fluorescent photomicrograph of cultured TG TRPM8+ neurons visualized by GFP expression. B, representative RT-PCR DNA bands for expression of PLCδ isozymes in TRPM8 neurons in vivo. All three isozymes are present in whole TG tissue samples, whereas PLCδ3 and PLCδ4 predominate in TRPM8 neurons (n = 3 experiments). C, whole-cell current clamp recordings from a TRPM8-expressing neuron in which trains of action potentials were elicited by two successive cold pulses (n = 5). D, whole-cell menthol-evoked (200 μm) currents from TRPM8-expressing neurons do not adapt to repeated stimuli in the absence of external Ca2+ and with 5 mm EGTA in the recording pipette (n = 5). E, in the presence of physiological (2 mm) calcium and weak intracellular Ca2+ buffering (0.5 mm EGTA), whole-cell menthol-evoked neuronal currents adapt over time and do not recover fully on subsequent menthol exposures when the cell is held at 22 °C (n = 6). F, menthol-evoked currents in TRPM8 TG neurons decrease or adapt upon bath application of 5 μm m-3M3FBS (n = 7). G, current-voltage relations at the points indicated in F. H, average residual TRPM8 currents in neurons after application of 5 μm m-3M3FBS for 3 min. at both positive and negative potentials. m-3M3FBS reduces currents to 66.7 ± 17.9 and 31.2 ± 14.3% (n = 7) at positive and negative membrane potentials, respectively.
FIGURE 8.
FIGURE 8.
PLC activation and PIP2 depletion shifts the voltage dependence of TRPM8 channel gating. A, representative whole-cell TRPM8 current traces in response to the indicated voltage protocol. Traces show activity before and after 1 mm menthol application, and after application of 5 μm m-3M3FBS (while still in the presence of 1 mm menthol). B, steady-state activation curves. The normalized conductance (g/gmax) was determined as explained under “Experimental Procedures.” Lines represent Boltzmann functions fitted to the data (n = 6). C, average voltages of half-maximal g/gmax (V½) obtained by fitting data to a Boltzmann function as described (n = 6). D, representative whole-cell TRPM8 current traces in response to the indicated voltage protocol. Traces show TRPM8 activation by 1 mm menthol, before and after Inp54p translocation induced by 1 μm rapamycin (rap). E, steady-state activation curves. Lines represent Boltzmann functions fitted to the data (n = 12). F, average voltage of half-maximal g/gmax (V½), obtained by fitting data to a Boltzmann function (n = 12).
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