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. 2011 Oct 17;589(Pt 24):6007–6027. doi: 10.1113/jphysiol.2011.220228

Decrease in phosphatidylinositol 4,5-bisphosphate levels mediates desensitization of the cold sensor TRPM8 channels

Yevgen Yudin 1, Viktor Lukacs 1, Chike Cao 1, Tibor Rohacs 1
PMCID: PMC3286682  PMID: 22005680

Abstract

Non-technical summary

The transient receptor potential melastatin 8 (TRPM8) ion channel is a physiological sensor of environmental cold temperatures. This channel is also activated by menthol, which is responsible for the cooling sensation evoked by this compound. It is well known that we adapt to moderately cold temperatures, i.e. the same temperature feels less cold over time, and the cooling effects of menthol also wear off with time, presumably due to the calcium-dependent desensitization of TRPM8 activity. The membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) is known to be required for TRPM8 activity. Our data support a model for desensitization, in which calcium influx through TRPM8 activates a phospholipase C enzyme, which breaks down PIP2, leading to decreased channel activity.

Abstract

The activity of the cold- and menthol-activated transient receptor potential melastatin 8 (TRPM8) channels diminishes over time in the presence of extracellular Ca2+, a phenomenon referred to as desensitization or adaptation. Here we show that activation of TRPM8 by cold or menthol evokes a decrease in cellular phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] levels. The decrease in PtdIns(4,5)P2 levels was accompanied by increased inositol 1,4,5 trisphosphate (InsP3) production, and was inhibited by loading the cells with the Ca2+ chelator BAPTA-AM, showing that it was the consequence of the activation of phospholipase C (PLC) by increased intracellular Ca2+ concentrations. PtdIns(4,5)P2 hydrolysis showed excellent temporal correlation with current desensitization in simultaneous patch clamp and fluorescence-based PtdIns(4,5)P2 level measurements. Intracellular dialysis of PtdIns(4,5)P2 inhibited desensitization both in native neuronal and recombinant TRPM8 channels. PtdIns(4)P, the precursor of PtdIns(4,5)P2, did not inhibit desensitization, consistent with its minimal effect in excised patches. Omission of MgATP from the intracellular solution accelerated desensitization, and MgATP reactivated TRPM8 channels in excised patches in a phosphatidylinositol 4-kinase (PI4K)-dependent manner. PLC-independent depletion of PtdIns(4,5)P2 using a voltage-sensitive phosphatase (ci-VSP) inhibited TRPM8 currents, and omission of ATP from the intracellular solution inhibited recovery from this inhibition. Inhibitors of PKC had no effect on the kinetics of desensitization. We conclude that Ca2+ influx through TRPM8 activates a Ca2+-sensitive PLC isoform, and the resulting depletion of PtdIns(4,5)P2 plays a major role in desensitization of both cold and menthol responses.

Introduction

The transient receptor potential melastatin 8 (TRPM8) ion channel is a member of the TRP channel super-family (Voets et al. 2005), it is activated by cold temperatures, and exogenous ‘cooling agents’ such as menthol and icilin (McKemy et al. 2002; Peier et al. 2002). TRPM8 is mainly expressed in sensory neurons, cell bodies of which are located in the dorsal root ganglia (DRG) and trigeminal ganglia (TG). Deletion of the TRPM8 gene in mice results in impaired cold sensitivity (Bautista et al. 2007; Colburn et al. 2007; Dhaka et al. 2007), signifying that these channels are one of the major sensors of environmental cold temperatures in vivo. The TRPM8 molecule itself is likely to be the cold sensor, because it retains cold sensitivity both in excised patches (Reid et al. 2002; Voets et al. 2004; Rohacs et al. 2005) and in planar lipid bilayers (Zakharian et al. 2009, 2010), as long as PtdIns(4,5)P2 is present (see later).

It is well known that we quickly adapt to moderately cold temperatures, i.e. the same temperature feels less cold over time. Accordingly, exposure of cold-sensitive nerve fibres to constant cooling temperatures results in diminished activity over time (Darian-Smith et al. 1973). TRPM8 channels also show adaptation, i.e. their activity diminishes over time when exposed either to cold or menthol (Reid & Flonta, 2001; McKemy et al. 2002; Daniels et al. 2008). This phenomenon is also called desensitization, and it depends on the presence of extracellular Ca2+. Desensitization refers to the lack of responsiveness after long-term or repeated stimulation, whereas in adaptation, responses can be evoked by subsequent stronger stimuli (Yao & Qin, 2009). Our experiments here do not differentiate between these two phenomena, as we used a single, saturating menthol concentration, and the limited solubility of this compound makes it difficult to apply significantly higher concentrations. Similarly, when working with cold, we applied a single sustained cold pulse. Thus, here we use desensitization and adaptation interchangeably. Even though cold is the physiological stimulus of TRPM8, most experiments addressing the mechanism of desensitization so far have been performed using menthol as a stimulus.

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), commonly referred to as PIP2, is a membrane phospholipid with a multitude of biological functions. Its hydrolysis by PLC enzymes results in the formation of the two classical second messengers InsP3 and diacylglycerol (DAG). PtdIns(4,5)P2 is required for the activity of a large number and variety of ion channels (Gamper & Shapiro, 2007; Logothetis & Nilius, 2007; Suh & Hille, 2008), including many TRP channels (Hardie, 2003; Nilius et al. 2008; Rohacs, 2009).

TRPM8 activity in the plasma membrane depends on the presence of PtdIns(4,5)P2 (Liu & Qin, 2005; Rohacs et al. 2005; Varnai et al. 2006; Zakharian et al. 2010). We have proposed that the desensitization of menthol-induced currents is caused by degradation of PtdIns(4,5)P2, resulting from the activation of calcium-sensitive PLC isoforms by Ca2+ influx through the channels (Rohacs et al. 2005). An alternative model for the desensitization of TRPM8 with the involvement of protein kinase C (PKC) has also been suggested (Abe et al. 2006). This model is also feasible, because PKC is expected to be activated downstream of PLC activation, and most tools used so far do not differentiate well between these two alternative hypotheses (see Discussion).

Most work on TRPM8 channels so far have been performed in expression systems, because of the difficulty locating the TRPM8-expressing neurons, due to the small proportion (5–10%) of cells expressing these channels. Two mouse models have recently been developed in which the cells that express TRPM8 channels are labelled with green fluorescent protein (GFP) (Takashima et al. 2007; Dhaka et al. 2008), allowing thorough electrophysiological studies of TRPM8 in native sensory neurons.

The goal of this study was to elucidate the mechanism of desensitization of TRPM8 in response to both cold and menthol, and to differentiate between the two alternative models of PtdIns(4,5)P2 depletion and PKC activation. We demonstrate that both cold and menthol deplete PtdIns(4,5)P2 in TRPM8-expressing cells, and the reduction of PtdIns(4,5)P2 levels shows excellent temporal correlation with current desensitization in parallel patch clamp and fluorescence measurements for both stimuli. We also show that inclusion of PtdIns(4,5)P2 in the patch pipette in whole-cell patch clamp experiments significantly diminish desensitization of both native and recombinant TRPM8 channels. Intracellular MgATP also inhibited desensitization and supported activity in excised patches in a phosphatidylinositol 4-kinase (PI4K)-dependent manner. PLC-independent depletion of PtdIns(4,5)P2 using a voltage-sensitive phosphatase inhibited TRPM8 currents. On the other hand, we found no change in desensitization levels in the presence of PKC inhibitors. Our data support the role of PtdIns(4,5)P2 depletion by PLC in both cold- and menthol-induced desensitization of TRPM8 channels.

Methods

Preparation of DRG neurons

All animal procedures were approved by the Institutional Animal Care and Use Committee. The DRG isolation procedure was slightly modified from the protocol described in Malin et al. (2007). Briefly, adult mice (2–3 months) expressing the green fluorescent protein (GFP) driven by the promoter of TRPM8 (Takashima et al. 2007) were killed by inhalation of 100% CO2, followed by decapitation. DRGs were removed from all cervical, thoracic and lumbar segments and treated in calcium- and magnesium-free Hank's balanced salt solution (Invitrogen) with a collagenase IA–dispase solution (1.0 and 5.0 mg ml−1, respectively; Sigma and Gibco) for 25–30 min at 37°C. After incubation, tissue fragments were gently triturated with a fire-polished glass pipette until single-cell suspensions were obtained. The dissociated cells were plated on round coverslips pre-coated with poly-l-lysine and laminin, allowed to adhere for 2 h and cultured in a medium containing: 90% Dulbecco's modified Eagle's medium (DMEM/F-12, Invitrogen) and 10% fetal calf serum supplemented with 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin. The majority of experiments on the neurons were performed 4–36 h after plating.

HEK cell cultures and gene expression

Human embryonic kidney 293 (HEK293) cells were obtained from the American Type Culture Collection and were grown in minimal essential medium (MEM) media (Invitrogen) containing 10% (v/v) fetal calf serum, 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin. Cells were transiently cotransfected with the rat TRPM8 cloned in the pCDNA3 vector (McKemy et al. 2002) and pEYFPN1 using the Effectene transfection reagent (Qiagen). Then, 24 h post-transfection, cells were trypsinized and replated on poly-l-lysine-coated round coverslips; 24–48 h post-transfection, yellow fluorescent protein (YFP)-positive cells were selected for electrophysiological recordings. Cultured HEK cells and DRG neurons were kept in a humidity-controlled incubator with 5% CO2 at 37oC.

Whole-cell patch clamp recordings

Whole-cell voltage clamp recordings of menthol-evoked currents were performed on GFP-expressing DRG neurons and YFP-expressing HEK cells. The standard extracellular solution used in all experiments contained (in mm): 137 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 Hepes and 10 glucose, pH 7.4 (adjusted with NaOH). The pipette (intracellular) solution for HEK whole-cell recordings contained (in mm): 140 potassium gluconate, 0.2 or 5 EGTA, 1 MgCl2, 2 Na-ATP and 10 Hepes, pH 7.3 (adjusted with KOH). The pipette solution for DRG whole-cell recordings contained (in mm): 140 potassium gluconate, 0.2 EGTA, 0.5 Mg, 5 Na-ATP and 10 Hepes, pH 7.3 (adjusted with KOH). Pipette solutions were supplemented with various concentrations of dioctanoyl (DiC8), PtdIns(4,5)P2 and PtdIns(4)P. We noticed that high free Mg2+ concentrations in the pipette solutions precipitated PtdIns(4,5)P2, consistent with earlier reports (Flanagan et al. 1997). Thus, for most measurements, ATP was added in excess of Mg2+ to keep the free concentration of Mg2+ low. Patch pipettes were pulled from borosilicate glass capillaries (1.5 mm outer diameter, WPI) on a P-97 pipette puller (Sutter Instrument Company) and pipette resistance was 2–4 MΩ. After formation of gigaohm-resistance seals, the whole-cell configuration was established, and currents were measured at a holding potential of −60 mV using an Axopatch 200B amplifier (Axon Instruments). For experiments where the effects of phosphoinositides were tested, we waited 3–5 min before recording after the establishment of the whole-cell configuration to allow time for these compounds to diffuse into the cell from the patch pipette. Currents were low-pass filtered at 2 kHz, and digitized using a Digidata 1322A unit (Axon Instruments). No series resistance compensation was performed. Solutions were exchanged by gravity-fed tubes, switching between solutions with a valve system (ALA Scientific). All recordings were performed at room temperature (22–24°C). Cold stimulation was performed with a custom-made system with a temperature probe positioned in close proximity to the measured cell. The electrical signal from the temperature probe (Warner Instruments) was fed into the Digidata 1322A and pCLAMP as an input signal. Data were collected and analysed with pCLAMP, and further analysed and plotted with Origin 8.0 (Microcal Software Inc., Northampton, MA, USA).

Drugs were added from the following stock solutions: (–)-menthol (Sigma) in ethanol; phorbol 12-myristate 13-acetate (PMA) and bisindolylmaleimide-VII (BIM) (Axxora, USA) in DMSO, protein kinase C fragment 19–31 amide (Sigma) in water. DiC8 phosphoinositides (Cayman Chemicals) were dissolved in water and aliquots of the stock solution were kept at –70°C. All dilutions were prepared on the day of the experiment.

Perforated patch clamp measurements

The pipette solution for perforated patch recordings contained (in mm): 120 potassium gluconate, 10 KCl, 10 NaCl, 1 EGTA, 1 MgCl2 and 10 Hepes, pH 7.3 (adjusted with KOH). Amphotericin B was added to the pipette solutions at the beginning of the experiments from a freshly made DMSO stock solution, to a final concentration of 300 μg ml−1. Patch pipette resistances were 1.5–2 MΩ; the recording chamber was perfused during seal formation to remove amphotericin B leaking from the patch pipette and avoid damaging the cell. After formation of gigaohm-resistance seals, the action of amphotericin B was visible as a continuous decrease in serial resistance and it required about 20–40 min to develop. Recordings were started when the serial resistance attained stable values of about 40–50 MΩ; currents were measured at a holding potential of −60 mV.

Fluorescence measurements

Fluorescence resonance energy transfer (FRET) in cells transfected with the cyan fluorescent protein (CFP)- and YFP-tagged PLCδ4 PH domains (Lee et al. 2004) or IRIS-1 (Matsu-ura et al. 2006) was detected as described earlier (Lukacs et al. 2007). Briefly, measurements were performed using a photomultiplier-based dual-emission system mounted on an IX-71 (Olympus) inverted microscope, equipped with a DeltaRAM excitation light source (Photon Technology International (PTI), Birmingham, NJ, USA). Excitation wavelength was 430 nm; emission was detected parallel at 480 and 535 nm using two interference filters and a dichroic mirror to separate the two emission wavelengths. Data were collected with the Clampex program, and analysed using Clampfit both for experiments with and without parallel patch clamp measurements. Calcium imaging was performed in cells loaded with 1.2 μm fura-2 for 60 min using a dual-excitation imaging system (PTI); excitation wavelengths were 340 and 380 nm, and emission was detected at 510 nm, with a Roper Cool-Snap digital CCD camera.

Xenopus oocyte electrophysiology

Measurements were conducted on oocytes extracted from Xenopus laevis frogs using collagenase digestion, as described earlier (Lukacs et al. 2007). Oocytes were maintained in a solution containing (in mm): 87.5 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2 and 5 Hepes, pH 7.4. Expression of TRPM8 was achieved by microinjection of cRNA made from a linearized pGEMSH vector containing TRPM8, using the mMessage Machine kit from Ambion. Excised inside-out patch clamp experiments were performed with borosilicate glass pipettes (World Precision Instruments) of 0.8–1.2 MΩ resistance. After establishing gigaohm-resistance seals on devitellinized surfaces of Xenopus oocytes, the inside-out configuration was established, and currents were measured using an Axopatch 200B amplifier (Axon Instruments). The pipette solution contained (in mm): 96 NaCl, 2 KCl, 1 MgCl2, 0.5 menthol and 5 Hepes, pH adjusted to 7.4; the perfusion solution contained (in mm): 96 KCl, 5 EGTA and 10 Hepes, pH adjusted to 7.4. For two-electrode voltage clamp, the oocytes were perfused with the same solution as used for the pipette solution for excised patch measurements. For excised patch measurements we used a ramp protocol from –100 to +100 mV (0.25 mV ms−1) applied every second. The voltage protocol for two-electrode voltage clamp experiments is shown in the legend to Fig. 9.

Figure 9. Dephosphorylation of PtdIns(4,5)P2 by a voltage-sensitive phosphatase inhibits TRPM8 channels.

Figure 9

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A–C, measurements in Xenopus oocytes. Oocytes expressing TRPM8 and either the wild-type (A) or the phosphatase-inactive mutant (B) of ci-VSP were used for the measurements. For the control periods, currents were measured at –60 mV; for the depolarizing steps, indicated by the horizontal bars, the voltage protocol shown in the inset was used. Currents for these periods were measured at –60 mV, 250 ms after stepping back to –60 mV. C shows the summary; we have plotted the currents at the end of the pulsing protocols at various voltages, normalized to the current immediately before the pulsation started, for wild-type ci-VSP, the C366S and the G365A mutants. D–F, measurements in HEK cells in a Ca2+-free extracellular solution containing 2 mm EGTA, and high intracellular Ca2+ buffering 4 mm (EGTA) to minimize desensitization. The seals were formed in an extracellular solution containing 2 mm Ca2+, and the perfusion with the Ca2+-free solution started 1 min before the measurement. The intracellular solution contained 2 mm ATP and 2 mm Mg2+. In these measurements constant holding at –60 mV was used, then a long depolarizing step to + 100 mV was applied (grey shaded area), and currents were evaluated immediately after stepping back to –60 mV (n = 5–6). G, the effect of depolarization in ci-VSP-expressing cells where the intracellular pipette solution did not contain MgATP. H, comparison of recovery from depolarization-induced inhibition between cells dialysed with 2 mm MgATP and cells where the intracellular solution did not contain any ATP (n = 4–5). In both conditions, we compared the recovery after ∼10 min of establishing the whole-cell configuration.

Statistics

Data were expressed as mean ± SEM, and statistical significance was calculated using Student's t test or analysis of variance. In all figures, statistical significance is labelled the following way: *P < 0.05, **P < 0.01 and ***P < 0.005.

Results

Cold and menthol activate PLC and reduce PtdIns(4,5)P2 levels in TRPM8-expressing cells

Activation of TRPM8 both by menthol and cold depends on the presence of PtdIns(4,5)P2 (Rohacs et al. 2005; Daniels et al. 2008; Zakharian et al. 2010). It has been proposed that activation of TRPM8 by menthol activates a calcium-sensitive PLC isoform, leading to a decrease in PtdIns(4,5)P2 levels (Rohacs et al. 2005; Daniels et al. 2008), and this would lead to current desensitization. Here we set out to examine whether activation of TRPM8 by its physiological stimulus, cold, leads to PtdIns(4,5)P2 depletion. We used a FRET-based technique (van der Wal et al. 2001; Rohacs et al. 2005) to detect PtdIns(4,5)P2 hydrolysis in TRPM8-expressing cells. HEK cells were transfected with the CFP- and YFP-tagged PH domains of PLCδ4 (Lee et al. 2004). In resting cells these constructs localize to the plasma membrane, through binding of the PH domains to PtdIns(4,5)P2, and undergo FRET, due to their close proximity. By detecting fluorescence at 480 and 535 nm simultaneously, with excitation wavelength of 430 nm, changes in FRET can be monitored in a ratiometric manner. Upon hydrolysis of PtdIns(4,5)P2, the fluorescent constructs move from the plasma membrane to the cytoplasm, their distance increases, which manifests as a decrease in the FRET ratio. We used the PLCδ4 PH domain instead of the more traditionally used PLCδ1 PH domain, because it binds PtdIns(4,5)P2 with similar specificity as the PLCδ1 PH domain, but its affinity for InsP3 is reported to be lower than that of the PLCδ1 (Lee et al. 2004), and other studies found that it does not bind InsP3 (Nagano et al. 1999). For the translocation of the PLCδ1 PH domain it is thought that its binding to InsP3 contributes to its translocation, the extent of which is debated (van der Wal et al. 2001; Xu et al. 2003). For the PLCδ4 PH domain, the contribution of InsP3 to its translocation is unlikely, since 1 μm InsP3 did not displace its binding to PtdIns(4,5)P2-containing vesicles (Lee et al. 2004) whereas the same concentration resulted in a 50% displacement of the PLCδ1 PH domain (Lee et al. 2004). InsP3 levels are thought to reach low micromolar levels in stimulated cells (Luzzi et al. 1998; Xu et al. 2003).

Figure 1A shows the effect of repeated applications of menthol on individual fluorescence traces and on the fluorescence ratio in TRPM8-expressing cells. Fluorescence at the CFP emission wavelength (480 nm) increased, whereas it showed a decrease at the emission wavelength of YFP (535 nm) in response to menthol, as expected if FRET decreased (Fig. 1A). No significant changes in response to menthol were observed in cells not expressing TRPM8 (Fig. 1C and D). The ratio of the two wavelengths can be used to estimate changes in FRET and to cancel out non-specific changes in the individual fluorescence traces.

Figure 1. Cold and menthol induce PtdIns(4,5)P2 hydrolysis and InsP3 production in TRPM8-expressing cells.

Figure 1

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Fluorescence measurements were performed in HEK293 cells transfected with the CFP- and YFP-tagged PLCδ4 PH domains and TRPM8 (A and B) or pCDNA3 (C) as described in the Methods section. A, upper traces, fluorescence traces detected at 480 and 535 nm; excitation wavelength was 430 nm. Lower trace, ratio of the two individual traces. The consecutive applications of 500 μm menthol are indicated by the horizontal bars. B, similar measurement using cold as a stimulus; upper trace shows temperature, lower trace shows the fluorescence ratio 535/480 nm. C, similar measurement on cells not expressing TRPM8. D, summary of the changes evoked by cold or menthol, in TRPM8-expressing and control cells (n = 7–12). E and F, HEK cells were transfected with the InsP3 reporter IRIS-1, and the human muscarinic M1 receptor (E) or TRPM8 (F). The ratio of 480/535 nm is shown; applications of 15 μm carbachol (CCh) and 500 μm menthol are indicated by the horizontal bars. IRIS-1 responds to InsP3 formation with a decrease in FRET; the traces shown are reverted i.e. we show the 480/535 ratio, to visually reflect increased InsP3 formation. All fluorescence ratio traces are normalized to the ratio level before the application of the stimulus.

When TRPM8-expressing cells were exposed to cold temperatures, the FRET ratio also decreased (Fig. 1B). The same temperature decrease induced only a minimal, statistically non-significant, drop in the 535/480 ratio in cells not expressing TRPM8 (Fig. 1C and D). In the control cells, cold induced a quick non-specific increase in fluorescence in both detection wavelengths, but this was largely canceled out in the ratio (data not shown). In the TRPM8-expressing cells, we also detected a quick parallel increase at both wavelengths in response to cold, which was canceled out by the ratio, and preceded the antiparallel changes that manifested as a decrease in the 535/480 ratio (not shown).

Our data so far show that activation of TRPM8 by either cold or menthol decreases cellular PtdIns(4,5)P2 levels. This can principally happen either by activation of PLC, where InsP3 and DAG are formed, or by dephosphorylation where the end product is either PtdIns(4)P or PtdIns. To demonstrate that the decrease in PtdIns(4,5)P2 levels proceeds through the activation of PLC, we measured InsP3 levels in response to menthol, using IRIS-1, a FRET-based InsP3 sensor, constructed from the ligand-binding domain of the InsP3 receptor and two fluorescent proteins, CFP and venus (Matsu-ura et al. 2006). As a positive control, we transfected HEK cells with the human muscarinic M1 receptor and IRIS-1, and stimulated the cells with carbachol. Figure 1E shows that, when stimulated with 15 μm carbachol, these cells responded with increased InsP3 levels. In cells co-transfected with TRPM8 and IRIS-1, menthol increased InsP3 levels with very similar kinetics and amplitude to the signals elicited by carbachol in M1-transfected cells. Menthol induced an 8.05 ± 1.6% relative change in the 480/535 nm ratio (n = 5), while carbachol evoked a 6.75 ± 0.55% change (n = 6). Note that IRIS-1 responds to binding of InsP3 with a decrease in FRET; in the figure we reversed the change to positive, i.e. plotted the 480/535 ratio, to visually reflect increased InsP3 levels. These data demonstrate InsP3 production in response to menthol, and thus confirm that the decrease in PtdIns(4,5)P2 levels is due to the activation of PLC.

TRPM8 is a plasma membrane ion channel, but similarly to many other channels, a significant fraction of it can be found in the endoplasmic reticulum, where its activation can lead to Ca2+ release from the internal stores (Tsuzuki et al. 2004). To investigate the role of intracellular Ca2+ release versus Ca2+ influx through TRPM8 in inducing PLC activation, we have performed additional experiments in the presence and absence of extracellular Ca2+, as well as loading the cells with the intracellular Ca2+ chelator BAPTA-AM. In Ca2+ imaging experiments (Fig. 2A) in the absence of extracellular Ca2+, menthol induced a small and transient Ca2+ response, presumably due to Ca2+ release through TRPM8 from the ER, which was followed by a much larger increase upon restoration of extracellular Ca2+ to 2 mm due to Ca2+ influx through the channel. BAPTA-AM loading eliminated the menthol-induced Ca2+ signal in the absence of Ca2+, and strongly reduced it in the presence of Ca2+ (Fig. 2B). Next we examined the effect of menthol on PtdIns(4,5)P2 hydrolysis using a similar protocol. In control TRPM8-expressing cells, in the absence of extracellular Ca2+, menthol induced a small decrease in the FRET signal of the PLCδ PH domains, which was followed by a much larger decrease upon restoring extracellular Ca2+ to 2 mm (Fig. 2C). When the cells were loaded with BAPTA-AM, the FRET decrease induced by Ca2+ was completely eliminated (Fig. 2D and E), showing that Ca2+ influx through the channel is required for PLC activation. BAPTA-AM loading also eliminated cold-induced PtdIns(4,5)P2 hydrolysis (Fig. 2F–H); these measurements were performed in the presence of 2 mm extracellular Ca2+. Interestingly the small decrease in FRET in response to menthol in the absence of extracellular Ca2+ persisted even in BAPTA-AM loaded cells, (Fig. 2D and E), despite the fact that the Ca2+ signal was eliminated under those conditions (Fig. 2B).

Figure 2. BAPTA-AM loading inhibits both calcium signals and PtdIns(4,5)P2 hydrolysis in TRPM8-expressing cells.

Figure 2

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A and B, cytoplasmic calcium measurements in TRPM8-expressing cells loaded with fura-2. The cells were perfused with an extracellular solution containing no added calcium and 0.2 mm EGTA, then 500 μm menthol was applied; finally the extracellular calcium was increased to 2 mm, in the continuous presence of menthol, as indicated by the horizontal lines. A, control cells; B, cells loaded with 30 μm BAPTA-AM for 60 min in the presence of 0.02% pluronic F127. The traces show mean ± SEM for n = 56–58 cells. C–E, FRET measurements on HEK cells transfected with the CFP- and YFP-tagged PLCδ4 PH domains and TRPM8 under similar experimental conditions. E, summary of the data (n = 8–9). F–H, cold-induced FRET measurements in control and BAPTA-AM-loaded cells (n = 6–7).

To confirm the role of Ca2+ influx, we also performed experiments with the R1008Q mutant of TRPM8 (Rohacs et al. 2005) which displays no detectable inward current upon activation by menthol. Menthol did not induce any Ca2+ signal in fura-2-loaded cells expressing TRPM8-R1008Q in the absence of extracellular Ca2+, and restoration of extracellular Ca2+ to 2 mm induced only a slow minimal increase in cytoplasmic Ca2+, showing that activation of this mutant induces minimal or no Ca2+ influx (Fig. 3A). In cells expressing TRPM8-R1008Q, menthol did not induce any PtdIns(4,5)P2 hydrolysis in the absence of extracellular Ca2+, and restoration of Ca2+ did not induce any decrease in FRET either (Fig. 3B and C). These data confirm the role of Ca2+ influx through TRPM8 in activating PLC.

Figure 3. Menthol does not increase cytoplasmic Ca2+ and does not induce PtdIns(4,5)P2 hydrolysis in cells expressing the TRPM8-R1008Q mutant.

Figure 3

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A, calcium imaging measurement in HEK293 cells expressing the TRPM8-R1008Q mutant, using the same protocol as in Fig. 2; mean ± SEM is shown for n = 40 cells. The dashed trace is the Ca2+ response of the wild-type TRPM8 from Fig. 2 for comparison. B, representative FRET measurement for n = 6 cells expressing the TRPM8-R1008Q mutant, and the CFP- and YFP-tagged PH domains of PLCδ4, using the same protocol as in Fig. 2. The dashed trace shows the response of the wild-type TRPM8 for comparison from Fig. 2. C, summary of the FRET data; the response of wild-type TRPM8 is shown for comparison. Inset: representative current traces recorded with a ramp protocol from –100 to +100 mV from a HEK cell expressing the TRPM8-R1008Q mutant in the absence (bottom trace) and presence (top trace) of 500 μm menthol.

Next we performed parallel whole-cell patch clamp and PtdIns(4,5)P2 hydrolysis (FRET) measurements in the same cells to see if the decrease in PtdIns(4,5)P2 levels correlated with current desensitization. Figure 4A shows a representative trace for these measurements using menthol as a stimulus in the presence of extracellular Ca2+. In response to a 1 min application of 500 μm menthol, the initial fast increase of inward current was followed by a relatively rapid decline to a lower level, in about 10–20 s (desensitization). The increase in current always preceded the drop in FRET ratio, as expected if PtdIns(4,5)P2 hydrolysis is a consequence of TRPM8 activation. The rapid decline in current levels showed a remarkable correlation with the decrease in FRET ratio. This rapid decline was followed by a more variable phase both in current amplitude and in FRET ratio. On average, currents further declined very slowly, in this second phase but the FRET ratio seemed to recover slightly (see Fig. 4C for average traces with SEM for nine measurements).

Figure 4. PtdIns(4,5)P2 hydrolysis correlates with current desensitization.

Figure 4

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A and B are representative current and FRET measurements in response to 500 μm menthol (A) or a drop in temperature (B). The FRET traces (grey) represent the 535/480 nm fluorescence ratio; the black traces show currents measured at –60 mV, and the dashed trace in B shows the temperature. C and D show average traces for current, FRET and temperature; the grey shaded areas show SEM for n = 8–9 measurements. Currents were normalized to the peak current evoked by menthol, whereas the FRET traces were normalized to the highest point preceding the application of the stimulus. The absolute current levels for menthol were 1.263 ± 0.214 nA (n = 9), and for cold, they were 1.153 ± 0.51 nA (n = 8).

For cold activation, we found that in whole-cell patch clamp experiments the cold-induced FRET decrease was less reproducible than using menthol as a stimulus. Thus, we performed parallel measurement with cold in the more physiological perforated patch configuration and found a reproducible decrease in FRET ratio. Cold-induced currents showed marked desensitization, and the decrease in current showed excellent temporal correlation with the decrease of FRET ratio (Fig. 4B and D).

Intracellular PtdIns(4,5)P2 dialysis inhibits desensitization of TRPM8

Having established that cold and menthol stimulation of TRPM8 leads to PtdIns(4,5)P2 depletion, we proceeded to test if interfering with PtdIns(4,5)P2 depletion reduces desensitization. First, we tested the effect of supplying excess PtdIns(4,5)P2 through the whole-cell patch pipette in native TRPM8 channels in adult mouse DRG neurons (Fig. 5). These measurements were performed in neurons isolated from a mouse strain expressing GFP, driven by the promoter of TRPM8, labelling the sparse cells that express these channels (Takashima et al. 2007). Three consecutive pulses of menthol (500 μm) were applied and currents were measured at –60 mV holding potential; we plotted the currents at the beginning and at the end of each menthol application. We have noticed that TRPM8 currents desensitized faster and more completely in smaller neurons than in larger ones. Menthol-induced current densities were higher in small neurons compared to large ones (Fig. 5E), which may explain this difference. We have tested the effect of dioctanoyl PtdIns(4,5)P2, which can support TRPM8 activity in excised patches (Rohacs et al. 2005; Brauchi et al. 2007). For data analysis the neurons were divided into two subgroups based on size estimated by whole-cell capacitance. Dialysis of PtdIns(4,5)P2 through the whole-cell patch pipette markedly inhibited desensitization in smaller neurons (10–20 pF) (Fig. 5A–C). In larger neurons (20–45 pF), where menthol-induced currents desensitized slower and less completely, PtdIns(4,5)P2 had a less pronounced, yet statistically significant effect (Fig. 5D).

Figure 5. Intracellular dialysis of PtdIns(4,5)P2 inhibits desensitization of native TRPM8 channels.

Figure 5

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Menthol-induced currents were measured at –60 mV in GFP+ DRG neurons as described in the Methods. The subsequent applications of 500 μm menthol are indicated by the horizontal bars. A, current measurement in a small neuron; B, similar measurement in a small neuron dialysed with 100 μm PtdIns(4,5)P2 through the whole-cell patch pipette. For both groups, patch pipettes contained 5 mm ATP with 0.5 mm Mg2+ and 0.2 mm EGTA. C and D show normalized current statistics for groups of small and large neurons, respectively; n = 10–16 for each of the 4 groups. Currents were measured at the time points marked from 1 to 6 in A. E, current densities for control and PtdIns(4,5)P2-dialysed small and large neurons.

Next we examined the specificity of the effect of PtdIns(4,5)P2 in HEK cells transiently expressing TRPM8 channels (Fig. 6). We have tested the effect of intracellular dialysis of two dioctanoyl phosphoinositides, PtdIns(4,5)P2 and PtdIns(4)P. We have shown earlier that PtdIns(4)P is much less active than PtdIns(4,5)P2 in activating TRPM8 in excised patches (Rohacs et al. 2005) and in planar lipid bilayers (Zakharian et al. 2010). Consistent with these earlier observations, PtdIns(4,5)P2 (Fig. 6B), but not PtdIns(4)P (Fig. 6C), inhibited desensitization of menthol-induced TRPM8 currents.

Figure 6. Intracellular dialysis of PtdIns(4,5)P2, but not PtdIns(4)P, inhibits TRPM8 desensitization.

Figure 6

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Menthol-induced currents were measured at –60 mV in TRPM8-transfected HEK293 cells, as described in the Methods. The applications of 500 μm menthol are indicated by the horizontal bars. A–C, representative current traces; A, control cells; B, a cell was dialysed with 50 μm PtdIns(4,5)P2; C, a cell dialysed with 50 μm PtdIns(4)P. For all groups the patch pipette contained 2 mm ATP with 1 mm Mg2+ and 0.2 mm EGTA. D, normalized currents for the 3 groups measured at the time points marked 1 to 6 in A. Mean ± SEM are shown; n = 16 for the control group, n = 12 for PtdIns(4)P and n = 13 for the PtdIns(4,5)P2 group.

The effects of intracellular MgATP

Intracellular hydrolysable ATP is necessary for the activity of the lipid kinases responsible for the synthesis of PtdIns(4,5)P2 (Hilgemann, 1997). The two major groups of these enzymes are PI4Ks and phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks). If the mechanism of desensitization is the loss of PtdIns(4,5)P2, omission of MgATP from the whole-cell pipette is anticipated to accelerate desensitization (Suh & Hille, 2002). On the other hand, protein phosphorylation also requires hydrolysable ATP; thus, if the mechanism of desensitization is PKC-induced phosphorylation, omission of MgATP from the patch pipette is expected to inhibit desensitization. Thus, we have performed whole-cell patch clamp experiments with and without MgATP in the patch pipette in HEK cells expressing TRPM8. Omission of MgATP from the patch pipette augmented desensitization (Fig. 7A and B), whereas inclusion of PtdIns(4,5)P2 in addition to MgATP inhibited it (Fig. 7C). The effect of omitting ATP from the patch pipette took time to develop; it was not apparent at the end of the first menthol pulse, only marginal at the second, and reached substantial levels at the third menthol pulse (see Fig. 7D for statistics).

Figure 7. Intracellular MgATP inhibits desensitization of TRPM8.

Figure 7

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A–C, menthol-induced currents were measured at –60 mV in TRPM8-expressing HEK293 cells, as described in the Methods. The applications of 500 μm menthol are indicated by the horizontal bars. A–C, representative current traces. The pipette solution in the control group (B) contained 1 mm Mg2+, 2 mm ATP and 5 mm EGTA; in trace A we omitted ATP and in trace C, the ATP-containing pipette solution was supplemented with 50 μm PtdIns(4,5)P2. D, summary for currents measured at the time points marked from 1 to 6, in B (n = 12–22). E, Ca2+ dependence of menthol-induced current desensitization in the 3 different intracellular buffering conditions in the presence of 2 mm external Ca2+.

These measurements were performed at higher intracellular Ca2+ buffering (5 mm EGTA), where desensitization is less pronounced, thus, it is presumably easier to see acceleration of desensitization. For our earlier measurements (Figs 4 and 6) we used low intracellular Ca2+ buffering (0.2 mm EGTA). See Fig. 7E for comparison of desensitization kinetics at various intracellular Ca2+ buffering conditions.

Intracellular ATP has been suggested to directly regulate several TRP channels (Lishko et al. 2007; Al Ansary et al. 2010); thus, to test its effects directly on the channels, we reconstituted the effect of MgATP in excised patches. Figure 8A shows that TRPM8 currents decrease in inside-out patches after excision into the ATP-free bath solution, due to the decrease of endogenous PtdIns(4,5)P2 levels in the patch membrane, most probably caused by lipid phosphatases (Huang et al. 1998). Current activity could be restored with the application of DiC8 PtdIns(4,5)P2, consistent with the dependence of TRPM8 activity on this lipid. When MgATP was applied, we observed a slow increase in current amplitude (Fig. 8A), which was essentially eliminated by the PI4K inhibitor LY294002 (300 μm) (Fig. 8B). LY294002 is a specific phosphoinositide 3-kinase (PI3K) inhibitor at lower concentrations (10 μm), and inhibits both PI3Ks and type III PI4Ks at 300 μm. At 10 μm LY294002 had no effect on MgATP-induced recovery of TRPM8 currents in excised patches (Fig. 8D). These data are in agreement with our recently published data obtained with TRPV6 (Zakharian et al. 2011), and show that MgATP reactivates TRPM8 channels by supplying substrate for LY294002-sensitive PI4K enzymes (presumably type III, see (Balla & Balla, 2006).

Figure 8. MgATP supports TRPM8 channel activity in excised patches in a PI4K-dependent manner.

Figure 8

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Excised patch measurements using TRPM8-expressing Xenopus oocytes were performed as described in the Methods. Currents were measured with a ramp protocol, from –100 to +100 mV, once every second and the currents at –100, 0 and +100 mV were plotted. Patches were excised (i/o) into an ATP-free solution, and 25 μm DiC8 PtdIns(4,5)P2, 2 mm ATP with 2 mm Mg2+ and LY294002 were applied as indicated by the horizontal bars. The free concentration of Mg2+ in the MgATP was calculated to be 300 μm using the MaxChelator program. For controls, 0.3% DMSO was applied and the concentration of the solvent was kept constant for the two different concentrations of LY294002. A–C are representative measurements; D is the summary at +100 mV, normalized to the current level right after patch excision. The currents plotted for MgATP were measured immediately after the washout of MgATP, to remove the inhibitory effect of Mg2+.

The activating effect of MgATP was usually preceded by a fast and transient current inhibition (Fig. 8A), which persisted even in patches treated with 300 μm LY294002 (Fig. 8B). This is most probably due to inhibition of TRPM8 activity by Mg2+ in the MgATP solution. Consistent with this idea, after the washout of MgATP, current run-down was always preceded by a transient increase in current amplitude, presumably because of a release from the inhibition by Mg2+. This inhibition could be due to direct effect of Mg2+ on TRPM8, or could also be caused by electrostatic shielding of PtdIns(4,5)P2 by Mg2+ (Suh & Hille, 2007). The free concentration of Mg2+, however, in our MgATP solutions was ∼300 μm, which is somewhat lower than the concentrations that were required by Suh & Hille (2007) to exert electrostatic interactions with PtdIns(4,5)P2.

Dephosphorylation of PtdIns(4,5)P2 inhibits TRPM8 currents

PLC activation decreases PtdIns(4,5)P2 levels, but it also induces other downstream signalling events, such as PKC activation and formation of InsP3. To show that a decrease in PtdIns(4,5)P2 concentration is sufficient to inhibit TRPM8 activity, we have used the voltage-sensitive phosphatase from Ciona intestinalis (ci-VSP) (Iwasaki et al. 2008). Upon depolarization, ci-VSP removes the 5-phosphate from PtdIns(4,5)P2, thus converting it to PtdIns(4)P, without the formation of either InsP3 or DAG. Figure 9A shows that depolarizing pulses inhibited channel activity measured at –60 mV in Xenopus oocytes expressing TRPM8 and wild-type ci-VSP. Depolarization did not inhibit TRPM8 activity when co-expressed with the phosphatase-deficient mutant C363S (Fig. 9B). The G365A mutation diminishes the activity of the phosphatase towards PtdIns(4,5)P2; accordingly, depolarization was less effective in inhibiting TRPM8 activity when it was co-expressed with this mutant, compared to wild-type ci-VSP (Fig. 9C). We also performed experiments with ci-VSP in mammalian (HEK) cells; Fig. 9D–F shows that depolarization inhibited TRPM8 activity when co-expressed with the wild-type, but not with the C363S mutant. Current activity recovered within ∼10 s (T50 = 4.3 ± 0.47 s, n = 5) after the depolarizing pulse, consistent with earlier results with KCNQ channels (Falkenburger et al. 2010). When the intracellular solution contained MgATP, currents consistently recovered after repeated depolarizations, with similar time courses. When we omitted ATP from the intracellular solution, the recovery from inhibition became slower and less complete with each subsequent depolarization (not shown), and eventually no recovery was observed. Figure 9G and H compares the recovery from depolarization-induced inhibition of TRPM8 ∼10 min after the establishment of the whole-cell configuration, where essentially no recovery was observed in ATP-free intracellular conditions, regardless of whether previous depolarizations were applied or not.

The role of PKC

We have shown that menthol and cold induces activation of PLC in TRPM8-expressing cells. Hydrolysis of PtdIns(4,5)P2 by PLC results in formation of DAG; thus, it is expected to activate PKC. Protein kinases require hydrolysable ATP, thus our data showing that omission of MgATP from the patch pipette accelerated desensitization argues against the role of PKC in this phenomenon. Note, however, that in contrast to type III PI4 kinases, which have very low affinity for ATP (Balla & Balla, 2006), the affinity of protein kinases for ATP generally is thought to be very high (O'Rourke, 1993; Hilgemann, 1997). Thus, it is possible that due to incomplete dialysis of endogenous nucleotides through the ATP free patch pipette, there is still enough ATP left in the cells to support protein kinases, but low ATP levels limit the activity of PI4K. Thus, we performed additional experiments with various activators and inhibitors of PKC.

The idea that PKC is involved in desensitization of menthol-induced TRPM8 currents is based on the inhibitory effects of various phorbol esters on menthol-induced TRPM8 currents. We have reproduced the inhibitory effect of PMA in the absence of extracellular Ca2+ (Fig. 10A–C) but we observed no change in desensitization levels in the presence of extracellular Ca2+ (Fig. 10D). Interestingly, menthol-induced currents still diminished somewhat even in the nominal absence of extracellular Ca2+ (Fig. 10C). We also observed no inhibition by 100 μm OAG (a cell-permeable DAG analogue), consistent with earlier reports (data not shown) (Rohacs et al. 2005; Liu & Qin, 2005).

Figure 10. The effects of PMA on TRPM8 channel activity.

Figure 10

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A and B, representative current traces (measured at –60 mV) in HEK cell expressing TRPM8 channel in response to applications of 500 μm menthol in a nominally Ca2+-free external solution. The cells were kept in the nominally Ca2+-free solution for 4–5 min before the measurement. The applications of 500 μm menthol and 100 nm PMA are indicated by the horizontal bars. The summary of these measurements is shown in C (n = 5–7). D shows summary for similar measurements in an external solution containing 2 mm Ca2+ (n = 7–9). The patch pipettes contained 2 mm ATP with 1 mm Mg2+ and 0.2 mm EGTA.

To test if PKC is involved in desensitization of menthol-induced currents in the absence of direct pharmacological stimulation of PKC, we have used two different approaches to inhibit PKC. We have pretreated TRPM8-expressing HEK cells with 1 μm bisindolmaleimide, or dialysed a PKC inhibitory peptide (House & Kemp, 1987) through the patch pipette. Neither of these manoeuvers changed the kinetics of desensitization of menthol-induced currents (Fig. 11). The same peptide inhibited bradykinin-induced sensitization of TRPV1 currents (V. Lukacs and T. Rohacs, unpublished observation).

Figure 11. PKC inhibitors do not affect TRPM8 desensitization.

Figure 11

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A and B, representative currents traces (measured at –60 mV) in HEK cell expressing TRPM8 in the presence of 2 mm external Ca2+. The applications of 500 μm menthol are indicated by the horizontal bars. In B, the cells were pretreated with for 10 min with a PKC inhibitor, BIM (1 μm). For all measurements the patch pipette contained 2 mm ATP with 1 mm Mg2+ and 0.2 mm EGTA. For experiments with PKC inhibitor PKC amide 19–31 the peptide (RFARKGALRQKNV) was dissolved in the intracellular solution before experiments, final concentration 5 μm. C, summary for experiments with a BIM and PKC amide 19–31 (n = 11–12).

Discussion

The main goal of the current study was to elucidate the mechanism of desensitization/adaptation of the cold-sensing TRPM8 channels. Both the depletion of PtdIns(4,5)P2 (Rohacs et al. 2005; Daniels et al. 2008) and PKC activation (Premkumar et al. 2005; Abe et al. 2006) have been implied in this process. Based on the data published so far both hypotheses are feasible, because most of the tools used in earlier studies do not differentiate between these two pathways (see later). Furthermore, most studies performed so far examined TRPM8 desensitization in response to menthol, in recombinant systems, and relatively little has been done either in native sensory neurons or using cold as a stimulus. In the present study we have rigorously tested these models, and established the role of PtdIns(4,5)P2 depletion in the desensitization of TRPM8 channels both in response to cold and menthol.

The role of PtdIns(4,5)P2 depletion in desensitization

The activity of TRPM8 depends on PtdIns(4,5)P2 both in excised patches (Liu & Qin, 2005; Rohacs et al. 2005) and in planar lipid bilayers (Zakharian et al. 2010). Activation of TRPM8 by menthol (Rohacs et al. 2005; Daniels et al. 2008) or cold (present study) leads to activation of PLC and reduction of PtdIns(4,5)2 levels. PLC activity, however, also leads to the generation of DAG and Ca2+ signal, both of which may activate PKC. Stimulation of PLC using a chemical activator inhibited both native and expressed TRPM8 channels (Daniels et al. 2008); however, this manoeuver is expected to lead to both depletion of PtdIns(4,5)P2 and activation of PKC.

Establishing causal relationship between PtdIns(4,5)P2 depletion and channel inhibition requires the demonstration that interfering with PtdIns(4,5)P2 depletion, i.e. accelerating or inhibiting it, changes Ca2+-induced desensitization. We have shown earlier that over-expression of PLCδ1 accelerated Ca2+-induced inhibition of menthol-activated TRPM8 currents (Rohacs et al. 2005). Again, increased PLC activity is expected to lead not only to enhanced PtdIns(4,5)P2 depletion, but also increased activation of PKC. It has also been shown that over-expression of PIP5K slowed desensitization of menthol-induced currents (Rohacs et al. 2005), but this effect was relatively modest. This is not surprising because activation of PLC may lead to depletion of both PtdIns(4,5)P2 and its precursor PtdIns(4)P, which is the substrate for PIP5K. Also, long-term over-expression of an enzyme involved in phosphoinositide synthesis may have a multitude of effects on the cell.

In the current study, we have used the most direct tool to establish a causal relationship between PtdIns(4,5)P2 depletion and channel inhibition, the intracellular dialysis of phosphoinositides in whole-cell patch clamp experiments. Inhibition of desensitization by providing excess PtdIns(4,5)P2 is expected if the mechanism of desensitization is PtdIns(4,5)P2 depletion. We have found that PtdIns(4,5)P2 inhibited desensitization of both the recombinant TRPM8 and native channels in sensory neurons. This manoeuver is expected to increase PKC activity, thus enhance desensitization, if PKC is the major mechanism mediating this phenomenon (see discussion later). PtdIns(4)P did not inhibit desensitization, which is consistent with the finding that it is much less efficient in supporting TRPM8 activity than PtdIns(4,5)P2 both in excised patches (Rohacs et al. 2005) and in planar lipid bilayers (Zakharian et al. 2010).

Which PLC isoform is activated by Ca2+ influx? We have shown earlier that over-expression of PLCδ1 accelerated Ca2+-induced inhibition of TRPM8 in Xenopus oocytes (Rohacs et al. 2005). All three PLCδs have been shown to be expressed in mixed TG neuron populations in mice, and PLCδ3 and 4 are expressed in TRPM8-expressing neurons (Daniels et al. 2008); thus, these enzymes are attractive candidates for the PLC isoform activated by Ca2+ influx through TRPM8.

What is the mechanism of PLC activation? Direct activation by increased cytoplasmic Ca2+ in the physiological range has been shown to activate PLCδ, but not PLCβ and PLCγ isoforms (Allen et al. 1997). BAPTA-AM loading eliminated PtdIns(4,5)P2 hydrolysis induced by either cold (Fig. 2G) or by restoring extracellular Ca2+ to 2 mm in the presence of menthol (Fig. 2D). These data convincingly demonstrate that Ca2+ influx through TRPM8 is required for full activation of PLC both when cold or menthol is used as a stimulus (Fig. 2). On the other hand, BAPTA-AM loading did not eliminate the small PtdIns(4,5)P2 decrease induced by menthol in the absence of extracellular Ca2+ (Fig. 2D) despite the fact that the bulk cytoplasmic Ca2+ signal was eliminated by the same treatment (Fig. 2B). This finding shows that there may be other auxiliary factors than the increase in cytoplasmic Ca2+ in activating PLC by TRPM8 activation, even though Ca2+ influx clearly plays the most important role. The small, apparently Ca2+-independent PtdIns(4,5)P2 depletion may also contribute to the finding that in most cases we observed some desensitization of menthol-induced currents even in the absence of extracellular Ca2+ (Fig. 10C) and even in patch clamp experiments where the pipette solution contained 10 mm BAPTA (Fig. 7E). It is also possible that the small FRET decrease in the absence of extracellular Ca2+ was due to menthol-induced sequestration of PItdIns(4,5)P2 by the TRPM8 protein, which is known to increase its binding affinity for PtdIns(4,5)P2 upon menthol stimulation (Rohacs et al. 2005; Zakharian et al. 2010). This is consistent with the finding that the small FRET decrease induced by menthol in the absence of extracellular Ca2+ was not detected with the R1008Q mutant (Fig. 3), which has very low apparent affinity for PtdIns(4,5)P2 (Rohacs et al. 2005).

Is the depletion of PtdIns(4,5)P2 the sole mechanism of desensitization of TRPM8? Collectively, our data firmly establish a major role of PtdIns(4,5)P2 depletion in desensitization of TRPM8. Some of our data, however, are potentially compatible with the involvement of other unidentified factors. (1) Intracellular dialysis of PtdIns(4,5)P2 did not completely eliminate desensitization in most of our measurements. This could also mean, however, that the rate of diffusion of PtdIns(4,5)P2 into the cell is slower than that of its hydrolysis. Diffusion of PtdIns(4,5)P2 to the inside surface of the plasma membrane from the patch pipette can be limited by a number of factors, including precipitation by Mg2+ (Flanagan et al. 1997), adhering to the glass pipette and binding to cytoplasmic proteins. (2) The correlation of current decay with changes in PtdIns(4,5)P2 levels in the second, slower phase of desensitization was opposite to what we expect based on our model (Fig. 4C and D). Note, however, that the FRET probes are probably better indicators of fast changes than slower ones, because of the tendency of drifts of the fluorescence signals due to photo-bleaching and potential diffusion of the fluorescent probes out of the patch pipette.

As we will discuss later, our data do not support a major role of PKC. InsP3, the downstream product of PLC activation, is unlikely to mediate Ca2+-induced inhibition either because it was reported that inclusion of InsP3 in the whole-cell patch pipette (Liu & Qin, 2005) or its injection into Xenopus oocytes in two-electrode voltage clamp experiments (Rohacs et al. 2005), did not inhibit TRPM8 currents. Consistent with these earlier data we found that 10 μm InsP3 did not inhibit TRPM8 channels activated by DiC8 PtdIns(4,5)P2 in excised patches (data not shown). In conclusion, our data firmly establish that depletion of PtdIns(4,5)P2 plays a major role in TRPM8 desensitization, but some contribution of other unidentified mechanisms cannot be completely excluded.

In the TRP channel literature, especially with the capsaicin-activated TRPV1, a distinction is often made between acute desensitization and tachyphylaxis, where the former refers to decrease of current in the presence of continued stimulation and the latter to decrease of the current amplitudes evoked by subsequent repeated stimulations. For assessing tachyphylaxis usually very short pulses (5–10 s) of agonists are applied (Koplas et al. 1997; Liu et al. 2005). Here we apply subsequent pulses of agonist, but the length of the individual stimulations is relatively long, 1 min, thus allowing acute desensitization to develop. This protocol has been used both by us and others before (Liu et al. 2005; Lukacs et al. 2007). Overall, our data suggest that PtdIns(4,5)P2 depletion plays a role in both phenomena. The excellent correlation between PtdIns(4,5)P2 hydrolysis and current decrease during the first stimulation argues that acute desensitization mainly happens through PtdIns(4,5)P2 depletion (Fig. 4). In the experiments where we measured PtdIns(4,5)P2 hydrolysis in response to repeated stimulations, FRET values did not return to basal levels inbetween consecutive menthol and cold pulses (Fig. 1), indicating that PtdIns(4,5)P2 levels did not return to resting values. Consistent with this PtdIns(4,5)P2 dialysis through the patch pipette inhibited the decrease in current values in the second and third stimulations (Figs 5 and 6). The effect of PtdIns(4,5)P2 dialysis on the acute desensitization, i.e. current level at the end of the first menthol pulse, was variable, having a significant effect in small DRG neurons (Fig. 5C, column 2), but no significant effect in HEK cells (Fig. 6D, column 2 and Fig. 7D, column 2). This could, however, be due to the different rate of diffusion of the lipid into the cell in different cell types, or to the different sequestration patterns of the exogenous lipid in various membrane compartments, such as rafts in the different cell types.

The role of lipid kinases

PtdIns(4,5)P2 is generated by two sequential phosphorylation steps from its abundant precursor PtdIns; PI4Ks catalyse the formation of PtdIns(4)P which is further phosphorylated by PIP5Ks on the 5′ position. We show that omission of ATP from the intracellular pipette solution accelerates desensitization (Fig. 7), and also show that MgATP restores TRPM8 channel activity in excised patches after current run-down in a PI4K-dependent manner (Fig. 8); thus, it is likely that intracellular ATP provides substrate for lipid kinases to synthesize PtdIns(4,5)P2.

We also show that converting PtdIns(4,5)P2 to PtdIns(4)P by activating the ci-VSP enzyme inhibits TRPM8 activity. The recovery from this inhibition depended on intracellular MgATP (Fig. 9G and H), again showing that MgATP provides substrate for lipid kinases to synthesize PtdIns(4,5)P2. When intracellular MgATP was provided, the recovery from ci-VSP-mediated inhibition was quite fast (T50 = 4.3 ± 0.47 s) and almost complete. In contrast, recovery between subsequent applications of menthol was only partial, even though the intervals between menthol applications were 60 s. These data are in very good agreement with recent measurements using PtdIns(4,5)P2-sensitive KCNQ channels, a voltage-sensitive 5′ phosphatase (VSP) and muscarinic stimulation to activate PLC (Falkenburger et al. 2010). In that study, recovery after PtdIns(4,5)P2 depletion by the VSP was much faster than after muscarinic stimulation. This difference was attributed to the higher speed of PIP5Ks compared to PI4Ks, because PLC degrades not only PtdIns(4,5)P2, but also PtdIns(4)P, thus requiring both PIP5K and PI4K to recover PtdIns(4,5)P2, while the VSP removes only the 5′ phosphate, thus requiring only PIP5K to re-synthesize PtdIns(4,5)P2.

If PIP5Ks are fast and efficient, why did PtdIns(4)P not inhibit menthol-induced desensitization (Fig. 6)? It is likely that PIP5Ks would convert this compound to PtdIns(4,5)P2, which clearly inhibited desensitization (Figs 5–7). In these experiments, however, we used water-soluble dioctanoyl (DiC8) phosphoinositides. Phosphorylation of DiC8 PtdIns(4)P by PIP5Ks would result in DiC8 PtdIns(4,5)P2. It is quite likely that this compound is continuously formed by PIP5Ks in the cell from the provided PtdIns(4)P, but it will also probably continuously diffuse out of the plasma membrane into the cell interior, and out into the patch pipette, down its concentration gradient. Note the fast off-rate of the activation of TRPM8 in excised patches by DiC8 PtdIns(4,5)P2 after the cessation of the stimulus (Fig. 8).

Adaptation to cold

Cold is the physiological stimulus of TRPM8, yet most measurements addressing the mechanism of desensitization so far have been performed with menthol as a stimulus. This is not surprising since a chemical agonist is easier to apply technically in patch clamp experiments than changing temperature reliably. Given that there are clear differences between menthol, cold and icilin, another chemical agonist of TRPM8, both in activation mechanism (Andersson et al. 2004) and in desensitization (Kuhn et al. 2009), it cannot be excluded that the mechanism of cold- and menthol-induced desensitization is different. A key experiment missing so far was to demonstrate that cold activation of TRPM8 leads to PtdIns(4,5)P2 depletion. Given that cooling may change the entire metabolism of the cell, we felt that this was an important point to demonstrate. We show here that cold activation of TRPM8 leads to reduction in cellular PtdIns(4,5)P2 levels (Fig. 1B), which is eliminated by loading the cells with the intracellular Ca2+ chelator BAPTA-AM (Fig. 2G). The kinetics of the cold-induced decrease of PtdIns(4,5)P2 showed a remarkable correlation with the reduction in current levels (Fig. 4B and D). These data, together with earlier results showing that reduction of PtdIns(4,5)P2 levels both by a PLC-dependent and PLC-independent manner is sufficient to inhibit cold-induced TRPM8 currents (Daniels et al. 2008), give strong support to the idea that cold adaptation of TRPM8 proceeds via PtdIns(4,5)P2 depletion.

It is noteworthy that even though the extent of PtdIns(4,5)P2 depletion induced by cold and menthol were similar (Fig. 4C and D), cold-induced currents desensitized more. What could account for this difference? The following explanation is one possibility. Generally, the effect of the depletion of PtdIns(4,5)P2 will depend on the apparent affinity of the channel for the lipid: channels with high affinity will be inhibited less than low-affinity channels (Suh & Hille, 2008). We have shown earlier that cold-activated TRPM8 channels have lower apparent affinity for PtdIns(4,5)P2 than menthol-activated ones (Rohacs et al. 2005); thus, it is feasible that the same extent of PtdIns(4,5)P2 depletion inhibits cold-induced currents more than those activated by menthol. Of course, there are other possible explanations too, including the difference in the experimental conditions, i.e. perforated patch (cold) versus whole-cell (menthol).

The role of PKC

The major argument for the involvement of PKC in Ca2+-dependent desensitization is the reported inhibition of menthol-induced currents by phorbol esters, chemical activators of PKC. In one study, 5–20 min pretreatment with 100 nm PMA inhibited menthol-induced currents and Ca2+ signals, and the effect of PMA was inhibited by inhibitors of PKC (Abe et al. 2006). The same study showed that menthol-induced currents decrease over time in the presence of extracellular Ca2+, but the effects of PKC inhibitors were not tested on Ca2+-dependent desensitization. Another study showed that incubation with very high concentrations (1 μm) of PDBu, another phorbol ester, inhibited menthol-induced currents, but lower concentrations of the drug were ineffective (Premkumar et al. 2005). The same article reported that bradykinin inhibited menthol-induced Ca2+ signals in sensory neurons, which was inhibited by the PKC inhibitor bisindolmaleimide, but the compound was not tested in the menthol-induced desensitization protocol. An additional study showed that PKC inhibitors (staurosporin and bisindolmaleimide) rendered menthol-induced Ca2+ signals in DRG neurons more sustained in capsaicin-insensitive neurons, but not in capsaicin-sensitive neurons (Sarria & Gu, 2010). Menthol-induced Ca2+ signals in neurons, however, are not necessarily proportional to TRPM8 activity, rather they are a sum of Ca2+ influx through TRPM8 and voltage-gated Ca2+ channels, cytoplasmic calcium buffering and the activity of the various Ca2+ extrusion mechanisms.

Arguing against the role of PKC, a cell-permeable DAG analogue OAG was reported not to inhibit menthol-induced currents of TRPM8 expressed in mammalian cells (Liu & Qin, 2005) and in Xenopus oocytes (Rohacs et al. 2005). Furthermore, Nerve Growth Factor-induced inhibition of TRPM8 currents was reported not to be inhibited by PKC inhibitors (Liu & Qin, 2005). In the current study we reproduced a moderate inhibitory effect of PMA on menthol-induced TRPM8 currents in the absence of extracellular Ca2+ (Fig. 10C). All of our other data, however, are inconsistent with a major role of PKC in menthol-induced Ca2+-dependent desensitization. PMA did not inhibit menthol-induced TRPM8 currents in the presence of extracellular Ca2+ (Fig. 10D). OAG, another activator of PKC, had no effect on menthol-induced TRPM8 currents (not shown), consistent with earlier reports. Most importantly, we found no inhibition of Ca2+-dependent desensitization of TRPM8 by two different PKC inhibitors (Fig. 11). In addition, our finding that dialysis of PtdIns(4,5)P2 through the whole-cell patch pipette (Figs 5–7) inhibits desensitization, also argues against the role of PKC for the following reason. Excess PtdIns(4,5)P2 provides more substrate for PLC; thus, it is expected to enhance PKC activation, thus accelerate desensitization, if PKC is involved in this phenomenon. We have used water-soluble DiC8 PtdIns(4,5)P2, which has been shown to be hydrolysed by PLCδ1 (Rebecchi et al. 1993), which results in the formation of dioctanoylglycerol, which has been shown to activate PKC (Davis et al. 1985). Thus, intracellular dialysis of DiC8 PtdIns(4,5)P2 is expected to increase PKC activity. In conclusion, our data do not support a major role for PKC in Ca2+-dependent menthol-induced desensitization of TRPM8.

Collectively, our data firmly establish a model in which depletion of PtdIns(4,5)P2 plays an important role in both adaptation to cold, and desensitization of menthol-induced currents.

Acknowledgments

The authors are grateful to Linda Zabelka for maintaining the mouse colony, Dr David McKemy (University of Southern California) for providing the TRPM8-GFP mouse line, Dr Yasushi Okamura (Osaka University, Japan) for providing the ci-VSP clone, Dr Tamas Balla (NIH) for providing the CFP and YFP tagged PLCδ4 PH domains, and to Dr Katsuhiko Mikoshiba (RIKEN) for providing the IRIS-1 clone. T.R. was supported by R01NS055159 and R01GM093290 from the National Institutes of Health.

Glossary

Abbreviations

BIM

bisindolylmaleimide

CFP

cyan fluorescent protein

ci-VSP

Ciona intestinalis voltage-sensitive phosphatase

DAG

diacylglycerol

DiC8

dioctanoyl

DRG

dorsal root ganglia

DMEM

Dulbecco's modified Eagles medium

FRET

fluorescence resonance energy transfer

GFP

green fluorescent protein

HEK293

human embryonic kidney 293

InsP3

inositol 1,4,5 trisphosphate

MEM

minimal essential medium

PMA

phorbol 12-myristate 13-acetate

PtdIns(4,5)P2

phosphatidylinositol 4,5-bisphosphate

PtdIns(4)P

phosphatidylinositol 4-phosphate

PI4K

phosphatidylinositol 4-kinase

PIP5K

phosphatidylinositol 4-phosphate 5-kinase

PKC

protein kinase C

TG

trigeminal ganglia

TRP

transient receptor potential

TRPM8

transient receptor potential melastatin 8

YFP

yellow fluorescent protein

Author contributions

T.R. and Y.Y. designed and conceived the experiments; Y.Y., C.C. and V.L. collected the data; Y.Y., C.C., V.L. and T.R. analysed and interpreted the data. T.R. and Y.Y. wrote the article. All authors approved the final version.

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