TRPA1 modulation by Sigma-1 receptor prevents oxaliplatin-induced painful peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy is a frequent, disabling side effect of anticancer drugs. Oxaliplatin, a platinum compound used in the treatment of advanced colorectal cancer, often leads to a form of chemotherapy-induced peripheral neuropathy characterized by mechanical and cold hypersensitivity. Current therapies for chemotherapy-induced peripheral neuropathy are ineffective, often leading to the cessation of treatment. Transient receptor potential ankyrin 1 (TRPA1) is a polymodal, non-selective cation-permeable channel expressed in nociceptors, activated by physical stimuli and cellular stress products. TRPA1 has been linked to the establishment of chemotherapy-induced peripheral neuropathy and other painful neuropathic conditions. Sigma-1 receptor is an endoplasmic reticulum chaperone known to modulate the function of many ion channels and receptors. Sigma-1 receptor antagonist, a highly selective antagonist of Sigma-1 receptor, has shown effectiveness in a phase II clinical trial for oxaliplatin chemotherapy-induced peripheral neuropathy. However, the mechanisms involved in the beneficial effects of Sigma-1 receptor antagonist are little understood. We combined biochemical and biophysical (i.e. intermolecular Förster resonance energy transfer) techniques to demonstrate the interaction between Sigma-1 receptor and human TRPA1. Pharmacological antagonism of Sigma-1R impaired the formation of this molecular complex and the trafficking of functional TRPA1 to the plasma membrane. Using patch-clamp electrophysiological recordings we found that antagonists of Sigma-1 receptor, including Sigma-1 receptor antagonist, exert a marked inhibition on plasma membrane expression and function of human TRPA1 channels. In TRPA1-expressing mouse sensory neurons, Sigma-1 receptor antagonists reduced inward currents and the firing of actions potentials in response to TRPA1 agonists. Finally, in a mouse experimental model of oxaliplatin neuropathy, systemic treatment with a Sigma-1 receptor antagonists prevented the development of painful symptoms by a mechanism involving TRPA1. In summary, the modulation of TRPA1 channels by Sigma-1 receptor antagonists suggests a new strategy for the prevention and treatment of chemotherapy-induced peripheral neuropathy and could inform the development of novel therapeutics for neuropathic pain.

Constitutive Phosphorylation as a Key Regulator of TRPM8 Channel Function.

In mammals, environmental cold sensing conducted by peripheral cold thermoreceptor neurons mostly depends on TRPM8, an ion channel that has evolved to become the main molecular cold transducer. This TRP channel is activated by cold, cooling compounds, such as menthol, voltage, and rises in osmolality. TRPM8 function is regulated by kinase activity that phosphorylates the channel under resting conditions. However, which specific residues, how this post-translational modification modulates TRPM8 activity, and its influence on cold sensing are still poorly understood. By mass spectrometry, we identified four serine residues within the N-terminus (S26, S29, S541, and S542) constitutively phosphorylated in the mouse ortholog. TRPM8 function was examined by Ca2+ imaging and patch-clamp recordings, revealing that treatment with staurosporine, a kinase inhibitor, augmented its cold- and menthol-evoked responses. S29A mutation is sufficient to increase TRPM8 activity, suggesting that phosphorylation of this residue is a central molecular determinant of this negative regulation. Biophysical and total internal reflection fluorescence-based analysis revealed a dual mechanism in the potentiated responses of unphosphorylated TRPM8: a shift in the voltage activation curve toward more negative potentials and an increase in the number of active channels at the plasma membrane. Importantly, basal kinase activity negatively modulates TRPM8 function at cold thermoreceptors from male and female mice, an observation accounted for by mathematical modeling. Overall, our findings suggest that cold temperature detection could be rapidly and reversibly fine-tuned by controlling the TRPM8 basal phosphorylation state, a mechanism that acts as a dynamic molecular brake of this thermo-TRP channel function in primary sensory neurons.SIGNIFICANCE STATEMENT Post-translational modifications are one of the main molecular mechanisms involved in adjusting the sensitivity of sensory ion channels to changing environmental conditions. Here we show, for the first time, that constitutive phosphorylation of the well-conserved serine 29 within the N-terminal domain negatively modulates TRPM8 channel activity, reducing its activation by agonists and decreasing the number of active channels at the plasma membrane. Basal phosphorylation of TRPM8 acts as a key regulator of its function as the main cold-transduction channel, significantly contributing to the net response of primary sensory neurons to temperature reductions. This reversible and dynamic modulatory mechanism opens new opportunities to regulate TRPM8 function in pathologic conditions where this thermo-TRP channel plays a critical role.

Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression.

TRPM8 is a non-selective cation channel expressed in primary sensory neurons and other tissues, including the prostate and urothelium. Its participation in different physiological and pathological processes such as thermoregulation, pain, itch, inflammation and cancer has been widely described, making it a promising target for therapeutic approaches. The detection and quantification of TRPM8 seems crucial for advancing the knowledge of the mechanisms underlying its role in these pathophysiological conditions. Antibody-based techniques are commonly used for protein detection and quantification, although their performance with many ion channels, including TRPM8, is suboptimal. Thus, the search for reliable antibodies is of utmost importance. In this study, we characterized the performance of six TRPM8 commercial antibodies in three immunodetection techniques: Western blot, immunocytochemistry and immunohistochemistry. Different outcomes were obtained for the tested antibodies; two of them proved to be successful in detecting TRPM8 in the three approaches while, in the conditions tested, the other four were acceptable only for specific techniques. Considering our results, we offer some insight into the usefulness of these antibodies for the detection of TRPM8 depending on the methodology of choice.

Piezo2 Mediates Low-Threshold Mechanically Evoked Pain in the Cornea.

Mammalian Piezo2 channels are essential for transduction of innocuous mechanical forces by proprioceptors and cutaneous touch receptors. In contrast, mechanical responses of somatosensory nociceptor neurons evoking pain, remain intact or are only partially reduced in Piezo2-deficient mice. In the eye cornea, comparatively low mechanical forces are detected by polymodal and pure mechanosensory trigeminal ganglion neurons. Their activation always evokes ocular discomfort or pain and protective reflexes, thus being a unique model to study mechanotransduction mechanisms in this particular class of nociceptive neurons. Cultured male and female mouse mechano- and polymodal nociceptor corneal neurons display rapidly, intermediately and slowly adapting mechanically activated currents. Immunostaining of the somas and peripheral axons of corneal neurons responding only to mechanical force (pure mechano-nociceptor) or also exhibiting TRPV1 (transient receptor potential cation channel subfamily V member 1) immunoreactivity (polymodal nociceptor) revealed that they express Piezo2. In sensory-specific Piezo2-deficient mice, the distribution of corneal neurons displaying the three types of mechanically evoked currents is similar to the wild type; however, the proportions of rapidly adapting neurons, and of intermediately and slowly adapting neurons were significantly reduced. Recordings of mechano- and polymodal-nociceptor nerve terminals in the corneal surface of Piezo2 conditional knock-out mice revealed a reduced number of mechano-sensitive terminals and lower frequency of nerve terminal impulse discharges under mechanical stimulation. Eye blinks evoked by von Frey filaments applied on the cornea were lower in Piezo2-deficient mice compared with wild type. Together, our results provide direct evidence that Piezo2 channels support mechanically activated currents of different kinetics in corneal trigeminal neurons and contributes to transduction of mechanical forces by corneal nociceptors.SIGNIFICANCE STATEMENT The cornea is a richly innervated and highly sensitive tissue. Low-threshold mechanical forces activate corneal receptors evoking discomfort or pain. To examine the contribution of Piezo2, a low-threshold mechanically activated channel, to acute ocular pain, we characterized the mechanosensitivity of corneal sensory neurons. By using Piezo2 conditional knock-out mice, we show that Piezo2 channels, present in the cell body and terminals of corneal neurons, are directly involved in acute corneal mechano-nociception. Inhibition of Piezo2 for systemic pain treatment is hindered because of its essential role for mechano-transduction processes in multiple body organs. Still, topical modulation of Piezo2 in the cornea may be useful to selectively relief unpleasant sensations and pain associated with mechanical irritation accompanying many ocular surface disorders.

The Immunosuppressant Macrolide Tacrolimus Activates Cold-Sensing TRPM8 Channels.

TRPM8 is a polymodal, nonselective cation channel activated by cold temperature and cooling agents that plays a critical role in the detection of environmental cold. We found that TRPM8 is a pharmacological target of tacrolimus (FK506), a macrolide immunosuppressant with several clinical uses, including the treatment of organ rejection following transplants, treatment of atopic dermatitis, and dry eye disease. Tacrolimus is an inhibitor of the phosphatase calcineurin, an action shared with cyclosporine. Tacrolimus activates TRPM8 channels in different species, including humans, and sensitizes their response to cold temperature by inducing a leftward shift in the voltage-dependent activation curve. The effects of tacrolimus on purified TRPM8 in lipid bilayers demonstrates conclusively that it has a direct gating effect. Moreover, the lack of effect of cyclosporine rules out the canonical signaling pathway involving the phosphatase calcineurin. Menthol (TRPM8-Y745H)- and icilin (TRPM8-N799A)-insensitive mutants were also activated by tacrolimus, suggesting a different binding site. In cultured mouse DRG neurons, tacrolimus evokes an increase in intracellular calcium almost exclusively in cold-sensitive neurons, and these responses were drastically blunted in Trpm8 KO mice or after the application of TRPM8 antagonists. Cutaneous and corneal cold thermoreceptor endings are also activated by tacrolimus, and tacrolimus solutions trigger blinking and cold-evoked behaviors. Together, our results identify TRPM8 channels in sensory neurons as molecular targets of the immunosuppressant tacrolimus. The actions of tacrolimus on TRPM8 resemble those of menthol but likely involve interactions with other channel residues.SIGNIFICANCE STATEMENT TRPM8 is a polymodal TRP channel involved in cold temperature sensing, thermoregulation, and cold pain. TRPM8 is also involved in the pathophysiology of dry eye disease, and TRPM8 activation has antiallodynic and antipruritic effects, making it a prime therapeutic target in several cutaneous and neural diseases. We report the direct agonist effect of tacrolimus, a potent natural immunosuppressant with multiple clinical applications, on TRPM8 activity. This interaction represents a novel neuroimmune interface. The identification of a clinically approved drug with agonist activity on TRPM8 channels could be used experimentally to probe the function of TRPM8 in humans. Our findings may explain some of the sensory and anti-inflammatory effects described for this drug in the skin and the eye surface.

Deletion of the Cold Thermoreceptor TRPM8 Increases Heat Loss and Food Intake Leading to Reduced Body Temperature and Obesity in Mice.

The coupling of energy homeostasis to thermoregulation is essential to maintain homeothermy in changing external environments. We studied the role of the cold thermoreceptor TRPM8 in this interplay in mice of both sexes. We demonstrate that TRPM8 is required for a precise thermoregulation in response to cold, in fed and fasting. Trpm8-/- mice exhibited a fall of 0.7°C in core body temperature when housed at cold temperatures, and a deep hypothermia (<30°C) during food deprivation. In both situations, TRPM8 deficiency induced an increase in tail heat loss. This, together with the presence of TRPM8-sensory fibers innervating the main tail vessels, unveils a major role of this ion channel in tail vasomotor regulation. Finally, TRPM8 deficiency had a remarkable impact on energy balance. Trpm8-/- mice raised at mild cold temperatures developed late-onset obesity and metabolic dysfunction, with daytime hyperphagia and reduction of fat oxidation as plausible causal factors. In conclusion, TRPM8 fine-tunes eating behavior and fuel utilization during thermoregulatory adjustments to mild cold. Persistent imbalances in these responses result in obesity.SIGNIFICANCE STATEMENT The thermosensitive ion channel TRPM8 is required for a precise thermoregulatory response to cold and fasting, playing an important role in tail vasoconstriction, and therefore heat conservation, as well as in the regulation of ingestive behavior and metabolic fuel selection upon cooling. Indeed, TRPM8-deficient mice, housed in a mild cold environment, displayed an increase in tail heat loss and lower core body temperature, associated with the development of late-onset obesity with glucose and lipid metabolic dysfunction. A persistent diurnal hyperphagia and reduced fat oxidation constitute plausible underlying mechanisms in the background of a deficient thermoregulatory adjustment to mild cold ambient temperatures.

Mammalian cold TRP channels: impact on thermoregulation and energy homeostasis.

Body temperature regulation is a fundamental homeostatic function in homeothermic animals. It is governed by the central nervous system that integrates temperature signals from internal body structures and the skin and provides efferent responses to adjust heat-exchange rates with the environment. Thermoregulation has a major influence on energy balance by regulating food intake as well as heat production and energy expenditure. Surprisingly, although almost 50% of our energy expenditure is dedicated to maintaining homeothermy, very little is yet known about the molecular aspects and the neural wiring involved in the intimate interrelationship between these two critical homeostatic systems. Some non-selective cation channels of the transient receptor potential (TRP) family work as molecular thermal sensors in sensory neurons and other cells. In this review, we discuss recent advances in our understanding of the basic mechanisms responsible for thermoregulation in the cold. We have focused our attention on the role of two cold-activated TRP channels (transient receptor potential melastatin 8 and transient receptor potential ankyrin 1) in body temperature regulation as well as their impact on energy balance and metabolism. A better understanding of the mechanisms coupling thermoregulation to energy homeostasis, including the involvement of thermosensitive TRPs, may uncover additional mechanisms underlying the pathogenesis of obesity and its metabolic consequences in humans, opening new strategies for the diagnosis, treatment, and prevention of this disease.

New Insight in Cold Pain: Role of Ion Channels, Modulation, and Clinical Perspectives.

Cold temperature detection involves the process of sensory transduction in cutaneous primary sensory nerve terminals, which converts thermal stimuli into depolarizations of the membrane. This transformation into electrical signals is followed by the subsequent propagation of action potentials in cold-sensitive afferent nerve fibers. A large array of ion channels shapes this process; however, the precise contribution of specific ion channel subtypes to cold perception and cold pain remains elusive. This review aims at giving an update on our current understanding of the role played by TRPs, leak K+ and voltage-gated Na+ and K+ channels in the transduction of cold by nociceptors and in cold-induced pain.

TRPA1 channels: molecular sentinels of cellular stress and tissue damage.

TRPA1 is a non-selective cation channel expressed in mammalian peripheral pain receptors, with a major role in chemonociception. TRPA1 has also been implicated in noxious cold and mechanical pain sensation. TRPA1 has an ancient origin and plays important functions in lower organisms, including thermotaxis, mechanotransduction and modulation of lifespan. Here we highlight the role of TRPA1 as a multipurpose sensor of harmful signals, including toxic bacterial products and UV light, and as a sensor of stress and tissue damage. Sensing roles span beyond the peripheral nervous system to include major barrier tissues: gut, skin and lung. Tissue injury, environmental irritants and microbial pathogens are danger signals that can threaten the health of organisms. These signals lead to the coordinated activation of the nociceptive and the innate immune system to provide a homeostatic response trying to re-establish physiological conditions including tissue repair. Activation of TRPA1 participates in protective neuroimmune interactions at multiple levels, sensing ROS and bacterial products and triggering the release of neuropeptides. However, an exaggerated response to danger signals is maladaptive and can lead to the development of chronic inflammatory conditions.

Bidirectional modulation of thermal and chemical sensitivity of TRPM8 channels by the initial region of the N-terminal domain.

TRPM8, a nonselective cation channel activated by cold, voltage, and cooling compounds such as menthol, is the principal molecular detector of cold temperatures in primary sensory neurons of the somatosensory system. The N-terminal domain of TRPM8 consists of 693 amino acids, but little is known about its contribution to channel function. Here, we identified two distinct regions within the initial N terminus of TRPM8 that contribute differentially to channel activity and proper folding and assembly. Deletion or substitution of the first 40 residues yielded channels with augmented responses to cold and menthol. The thermal threshold of activation of these mutants was shifted 2 °C to higher temperatures, and the menthol dose-response curve was displaced to lower concentrations. Site-directed mutagenesis screening revealed that single point mutations at positions Ser-26 or Ser-27 by proline caused a comparable increase in the responses to cold and menthol. Electrophysiological analysis of the S27P mutant revealed that the enhanced sensitivity to agonists is related to a leftward shift in the voltage dependence of activation, increasing the probability of channel openings at physiological membrane potentials. In addition, we found that the region encompassing positions 40-60 is a key element in the proper folding and assembly of TRPM8. Different deletions and mutations within this region rendered channels with an impaired function that are retained within the endoplasmic reticulum. Our results suggest a critical contribution of the initial region of the N-terminal domain of TRPM8 to thermal and chemical sensitivity and the proper biogenesis of this polymodal ion channel.

Plasma membranes as heat stress sensors: from lipid-controlled molecular switches to therapeutic applications.

The classic heat shock (stress) response (HSR) was originally attributed to protein denaturation. However, heat shock protein (Hsp) induction occurs in many circumstances where no protein denaturation is observed. Recently considerable evidence has been accumulated to the favor of the "Membrane Sensor Hypothesis" which predicts that the level of Hsps can be changed as a result of alterations to the plasma membrane. This is especially pertinent to mild heat shock, such as occurs in fever. In this condition the sensitivity of many transient receptor potential (TRP) channels is particularly notable. Small temperature stresses can modulate TRP gating significantly and this is influenced by lipids. In addition, stress hormones often modify plasma membrane structure and function and thus initiate a cascade of events, which may affect HSR. The major transactivator heat shock factor-1 integrates the signals originating from the plasma membrane and orchestrates the expression of individual heat shock genes. We describe how these observations can be tested at the molecular level, for example, with the use of membrane perturbers and through computational calculations. An important fact which now starts to be addressed is that membranes are not homogeneous nor do all cells react identically. Lipidomics and cell profiling are beginning to address the above two points. Finally, we observe that a deregulated HSR is found in a large number of important diseases where more detailed knowledge of the molecular mechanisms involved may offer timely opportunities for clinical interventions and new, innovative drug treatments. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

TRPM8.

Transient receptor potential melastatin 8 (TRPM8) was originally cloned from prostate tissue. Shortly thereafter, the protein was identified as a cold- and menthol-activated ion channel in peripheral sensory neurons, where it plays a critical role in cold temperature detection. In this chapter, we review our current understanding of the molecular and biophysical properties, the pharmacology, and the modulation by signaling molecules of this TRP channel. Finally, we examine the physiological role of TRPM8 and its emerging link to various human diseases, including pain, prostate cancer, dry eye disease, and metabolic disorders.

The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

BACKGROUND AND PURPOSE: The mechanistic target of rapamycin (mTOR) signalling pathway is a key regulator of cell growth and metabolism. Its deregulation is implicated in several diseases. The macrolide rapamycin, a specific inhibitor of mTOR, has immunosuppressive, anti-inflammatory and antiproliferative properties. Recently, we identified tacrolimus, another macrolide immunosuppressant, as a novel activator of TRPM8 ion channels, involved in cold temperature sensing, thermoregulation, tearing and cold pain. We hypothesized that rapamycin may also have agonist activity on TRPM8 channels. EXPERIMENTAL APPROACH: Using calcium imaging and electrophysiology in transfected HEK293 cells and wildtype or Trpm8 KO mouse DRG neurons, we characterized rapamycin's effects on TRPM8 channels. We also examined the effects of rapamycin on tearing in mice. KEY RESULTS: Micromolar concentrations of rapamycin activated rat and mouse TRPM8 channels directly and potentiated cold-evoked responses, effects also observed in human TRPM8 channels. In cultured mouse DRG neurons, rapamycin increased intracellular calcium levels almost exclusively in cold-sensitive neurons. Responses were markedly decreased in Trpm8 KO mice or by TRPM8 channel antagonists. Cutaneous cold thermoreceptor endings were also activated by rapamycin. Topical application of rapamycin to the eye surface evokes tearing in mice by a TRPM8-dependent mechanism. CONCLUSION AND IMPLICATIONS: These results identify TRPM8 cationic channels in sensory neurons as novel molecular targets of the immunosuppressant rapamycin. These findings may help explain some of its therapeutic effects after topical application to the skin and the eye surface. Moreover, rapamycin could be used as an experimental tool in the clinic to explore cold thermoreceptors.

N-glycosylation of TRPM8 ion channels modulates temperature sensitivity of cold thermoreceptor neurons.

TRPM8 is a member of the transient receptor potential ion channel superfamily, which is expressed in sensory neurons and is activated by cold and cooling compounds, such as menthol. Activation of TRPM8 by agonists takes place through shifts in its voltage activation curve, allowing channel opening at physiological membrane potentials. Here, we studied the role of the N-glycosylation occurring at the pore loop of TRPM8 on the function of the channel. Using heterologous expression of recombinant channels in HEK293 cells we found that the unglycosylated TRPM8 mutant (N934Q) displays marked functional differences compared with the wild type channel. These differences include a shift in the threshold of temperature activation and a reduced response to menthol and cold stimuli. Biophysical analysis indicated that these modifications are due to a shift in the voltage dependence of TRPM8 activation toward more positive potentials. By using tunicamycin, a drug that prevents N-glycosylation of proteins, we also evaluated the effect of the N-glycosylation on the responses of trigeminal sensory neurons expressing TRPM8. These experiments showed that the lack of N-glycosylation affects the function of native TRPM8 ion channels in a similar way to heterologously expressed ones, causing an important shift of the temperature threshold of cold-sensitive thermoreceptor neurons. Altogether, these results indicate that post-translational modification of TRPM8 is an important mechanism modulating cold thermoreceptor function, explaining the marked differences in temperature sensitivity observed between recombinant and native TRPM8 ion channels.

Sex differences in thermoregulation in mammals: Implications for energy homeostasis.

Thermal homeostasis is a fundamental process in mammals, which allows the maintenance of a constant internal body temperature to ensure an efficient function of cells despite changes in ambient temperature. Increasing evidence has revealed the great impact of thermoregulation on energy homeostasis. Homeothermy requires a fine regulation of food intake, heat production, conservation and dissipation and energy expenditure. A great interest on this field of research has re-emerged following the discovery of thermogenic brown adipose tissue and browning of white fat in adult humans, with a potential clinical relevance on obesity and metabolic comorbidities. However, most of our knowledge comes from male animal models or men, which introduces unwanted biases on the findings. In this review, we discuss how differences in sex-dependent characteristics (anthropometry, body composition, hormonal regulation, and other sexual factors) influence numerous aspects of thermal regulation, which impact on energy homeostasis. Individuals of both sexes should be used in the experimental paradigms, considering the ovarian cycles and sexual hormonal regulation as influential factors in these studies. Only by collecting data in both sexes on molecular, functional, and clinical aspects, we will be able to establish in a rigorous way the real impact of thermoregulation on energy homeostasis, opening new avenues in the understanding and treatment of obesity and metabolic associated diseases.

The cold-sensing ion channel TRPM8 regulates central and peripheral clockwork and the circadian oscillations of body temperature.

AIM: Physiological functions in mammals show circadian oscillations, synchronized by daily cycles of light and temperature. Central and peripheral clocks participate in this regulation. Since the ion channel TRPM8 is a critical cold sensor, we investigated its role in circadian function. METHODS: We used TRPM8 reporter mouse lines and TRPM8-deficient mice. mRNA levels were determined by in situ hybridization or RT-qPCR and protein levels by immunofluorescence. A telemetry system was used to measure core body temperature (Tc). RESULTS: TRPM8 is expressed in the retina, specifically in cholinergic amacrine interneurons and in a subset of melanopsin-positive ganglion cells which project to the central pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. TRPM8-positive fibres were also found innervating choroid and ciliary body vasculature, with a putative function in intraocular temperature, as shown in TRPM8-deficient mice. Interestingly, Trpm8-/- animals displayed increased expression of the clock gene Per2 and vasopressin (AVP) in the SCN, suggesting a regulatory role of TRPM8 on the central oscillator. Since SCN AVP neurons control body temperature, we studied Tc in driven and free-running conditions. TRPM8-deficiency increased the amplitude of Tc oscillations and, under dim constant light, induced a greater phase delay and instability of Tc rhythmicity. Finally, TRPM8-positive fibres innervate peripheral organs, like liver and white adipose tissue. Notably, Trpm8-/- mice displayed a dysregulated expression of Per2 mRNA in these metabolic tissues. CONCLUSION: Our findings support a function of TRPM8 as a temperature sensor involved in the regulation of central and peripheral clocks and the circadian control of Tc.

Role of Ih in the firing pattern of mammalian cold thermoreceptor endings.

Mammalian peripheral cold thermoreceptors respond to cooling of their sensory endings with an increase in firing rate and modification of their discharge pattern. We recently showed that cultured trigeminal cold-sensitive (CS) neurons express a prominent hyperpolarization-activated current (I(h)), mainly carried by HCN1 channels, supporting subthreshold resonance in the soma without participating in the response to acute cooling. However, peripheral pharmacological blockade of I(h), or characterization of HCN1(-/-) mice, reveals a deficit in acute cold detection. Here we investigated the role of I(h) in CS nerve endings, where cold sensory transduction actually takes place. Corneal CS nerve endings in mice show a rhythmic spiking activity at neutral skin temperature that switches to bursting mode when the temperature is lowered. I(h) blockers ZD7288 and ivabradine alter firing patterns of CS nerve endings, lengthening interspike intervals and inducing bursts at neutral skin temperature. We characterized the CS nerve endings from HCN1(-/-) mouse corneas and found that they behave similar to wild type, although with a lower slope in the firing frequency vs. temperature relationship, thus explaining the deficit in cold perception of HCN1(-/-) mice. The firing pattern of nerve endings from HCN1(-/-) mice was also affected by ZD7288, which we attribute to the presence of HCN2 channels in the place of HCN1. Mathematical modeling shows that the firing phenotype of CS nerve endings from HCN1(-/-) mice can be reproduced by replacing HCN1 channels with the slower HCN2 channels rather than by abolishing I(h). We propose that I(h) carried by HCN1 channels helps tune the frequency of the oscillation and the length of bursts underlying regular spiking in cold thermoreceptors, having important implications for neural coding of cold sensation.

Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity.

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell-penetrating, membrane-anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP-p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC(50)<10 µM), without significantly affecting other thermoTRP channels. In contrast, its retrosequence or the corresponding sequences of other TRPV channels did not alter TRPV1 channel activity (IC(50)>100 µM). TRP-p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP-p5 interferes with protein-protein interactions at the level of the TRP domain that are essential for the "conformational" change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage-independent, sequence-specific TRPV1 blockers. Our findings indicate that TRPducin-like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels.

Origins of direction selectivity in the primate retina.

From mouse to primate, there is a striking discontinuity in our current understanding of the neural coding of motion direction. In non-primate mammals, directionally selective cell types and circuits are a signature feature of the retina, situated at the earliest stage of the visual process. In primates, by contrast, direction selectivity is a hallmark of motion processing areas in visual cortex, but has not been found in the retina, despite significant effort. Here we combined functional recordings of light-evoked responses and connectomic reconstruction to identify diverse direction-selective cell types in the macaque monkey retina with distinctive physiological properties and synaptic motifs. This circuitry includes an ON-OFF ganglion cell type, a spiking, ON-OFF polyaxonal amacrine cell and the starburst amacrine cell, all of which show direction selectivity. Moreover, we discovered that macaque starburst cells possess a strong, non-GABAergic, antagonistic surround mediated by input from excitatory bipolar cells that is critical for the generation of radial motion sensitivity in these cells. Our findings open a door to investigation of a precortical circuitry that computes motion direction in the primate visual system.

Pharmacological and functional properties of TRPM8 channels in prostate tumor cells.

Prostate cancer (PC) is a major health problem in adult males. TRPM8, a cationic TRP channel activated by cooling and menthol is upregulated in PC. However, the precise role of TRPM8 in PC is still unclear. Some studies hypothesized that TRPM8-mediated transmembrane Ca(2+) fluxes play a key role in cellular proliferation of PC cells. In contrast, other findings suggest that high TRPM8 levels may reduce the metastatic potential of PC cells. A detailed understanding of the response of TRPM8 channels to pharmacological modulators of their activity is relevant when considering potential therapies, targeting this ion channel to treat PC. We characterized the pharmacological and functional properties of native TRPM8 channels in four human prostate cell lines, PNT1A, LNCaP, DU145, and PC3, commonly used as experimental models of PC. PNT1A is a non-tumoral prostate cell line while the other three correspond to different stages of PC. Here, we show that cold- and agonist-evoked [Ca(2+)](i) responses in PC cells are much less sensitive to well-characterized agonists (menthol and icilin) and antagonists (BCTC, clotrimazole, and DD01050) of TRPM8 channels, compared to TRPM8 channels in other tissues, suggesting a different molecular composition and/or spatial organization. In addition, the forced overexpression of human TRPM8 facilitated the trafficking of TRPM8 channels residing in the endoplasmic reticulum to the plasma membrane, leading to a marked potentiation in the efficacy of the different blockers. These results predict that blockers of canonical TRPM8 channels may be less effective in halting proliferation of PC cells than expected.