RESUMO
Cold thermoreceptor neurons detect temperature drops with highly sensitive molecular machinery concentrated in their peripheral free nerve endings. The main molecular entity responsible for cold transduction in these neurons is the thermo-TRP channel TRPM8. Cold, cooling compounds such as menthol, voltage, and osmolality rises activate this polymodal ion channel. Dysregulation of TRPM8 activity underlies several physiopathological conditions, including painful cold hypersensitivity in response to axonal damage, migraine, dry-eye disease, overactive bladder, and several forms of cancer. Although TRPM8 could be an attractive target for treating these highly prevalent diseases, there is still a need for potent and specific modulators potentially suitable for future clinical trials. This goal requires a complete understanding of the molecular determinants underlying TRPM8 activation by chemical and physical agonists, inhibition by antagonists, and the modulatory mechanisms behind its function to guide future and more successful treatment strategies. This review recapitulates information obtained from different mutagenesis approaches that have allowed the identification of specific amino acids in the cavity comprised of the S1-S4 and TRP domains that determine modulation by chemical ligands. In addition, we summarize different studies revealing specific regions within the N- and C-terminus and the transmembrane domain that contribute to cold-dependent TRPM8 gating. We also highlight the latest milestone in the field: cryo-electron microscopy structures of TRPM8, which have provided a better comprehension of the 21 years of extensive research in this ion channel, shedding light on the molecular bases underlying its modulation, and promoting the future rational design of novel drugs to selectively regulate abnormal TRPM8 activity under pathophysiological conditions.
RESUMO
Non-cell-autonomous mechanisms contribute to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), in which astrocytes release unidentified factors that are toxic to motoneurons (MNs). We report here that mouse and patient iPSC-derived astrocytes with diverse ALS/FTD-linked mutations (SOD1, TARDBP, and C9ORF72) display elevated levels of intracellular inorganic polyphosphate (polyP), a ubiquitous, negatively charged biopolymer. PolyP levels are also increased in astrocyte-conditioned media (ACM) from ALS/FTD astrocytes. ACM-mediated MN death is prevented by degrading or neutralizing polyP in ALS/FTD astrocytes or ACM. Studies further reveal that postmortem familial and sporadic ALS spinal cord sections display enriched polyP staining signals and that ALS cerebrospinal fluid (CSF) exhibits increased polyP concentrations. Our in vitro results establish excessive astrocyte-derived polyP as a critical factor in non-cell-autonomous MN degeneration and a potential therapeutic target for ALS/FTD. The CSF data indicate that polyP might serve as a new biomarker for ALS/FTD.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Esclerose Lateral Amiotrófica/genética , Animais , Astrócitos , Proteína C9orf72/genética , Meios de Cultivo Condicionados/farmacologia , Demência Frontotemporal/genética , Humanos , Camundongos , Neurônios Motores , PolifosfatosRESUMO
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.
Assuntos
Membrana Celular/genética , Membrana Celular/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Animais , Células COS , Chlorocebus aethiops , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/fisiologia , Gânglio Trigeminal/metabolismoRESUMO
TRPM8 is the main molecular entity responsible for cold sensing. This polymodal ion channel is activated by cold, cooling compounds such as menthol, voltage, and rises in osmolality. In corneal cold thermoreceptor neurons (CTNs), TRPM8 expression determines not only their sensitivity to cold, but also their role as neural detectors of ocular surface wetness. Several reports suggest that Protein Kinase C (PKC) activation impacts on TRPM8 function; however, the molecular bases of this functional modulation are still poorly understood. We explored PKC-dependent regulation of TRPM8 using Phorbol 12-Myristate 13-Acetate to activate this kinase. Consistently, recombinant TRPM8 channels, cultured trigeminal neurons, and free nerve endings of corneal CTNs revealed a robust reduction of TRPM8-dependent responses under PKC activation. In corneal CTNs, PKC activation decreased ongoing activity, a key parameter in the role of TRPM8-expressing neurons as humidity detectors, and also the maximal cold-evoked response, which were validated by mathematical modeling. Biophysical analysis indicated that PKC-dependent downregulation of TRPM8 is mainly due to a decreased maximal conductance value, and complementary noise analysis revealed a reduced number of functional channels at the cell surface, providing important clues to understanding the molecular mechanisms of how PKC activity modulates TRPM8 channels in CTNs.
Assuntos
Temperatura Baixa , Neurônios/metabolismo , Proteína Quinase C/metabolismo , Canais de Cátion TRPM/metabolismo , Termorreceptores/metabolismo , Sensação Térmica , Nervo Trigêmeo/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Células Receptoras Sensoriais/metabolismo , Nervo Trigêmeo/citologiaRESUMO
TRPM8 is the main ion channel responsible for cold transduction in the somatosensory system. Nerve terminal availability of TRPM8 determines cold sensitivity, but how axonal secretory organelles control channel delivery remains poorly understood. Here we examine the distribution of TRPM8 and trafficking organelles in cold-sensitive peripheral axons and disrupt trafficking by targeting the ARF-GEF GBF1 pharmacologically or the small GTPase RAB6 by optogenetics. In axons of the sciatic nerve, inhibition of GBF1 interrupts TRPM8 trafficking and increases association with the trans-Golgi network, LAMP1, and Golgi satellites, which distribute profusely along the axonal shaft. Accordingly, both TRPM8-dependent ongoing activity and cold-evoked responses reversibly decline upon GBF1 inhibition in nerve endings of corneal cold thermoreceptors. Inhibition of RAB6, which also associates to Golgi satellites, decreases cold-induced responses in vivo. Our results support a non-conventional axonal trafficking mechanism controlling the availability of TRPM8 in axons and cold sensitivity in the peripheral nervous system.
Assuntos
Axônios/metabolismo , Temperatura Baixa , Organelas/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Axônios/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Masculino , Mentol/farmacologia , Camundongos , Optogenética , Organelas/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Termorreceptores/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Thermal Fluctuations Spectroscopy (TFS) in combination with novel optical-based instrumentation was used to study mechanical properties of cell-cultured neurites with a spatial resolution limited only by the light diffraction. The analysis of thermal fluctuations together with a physical model of cellular elasticity allow us to determine relevant mechanical properties of neurite as axial tension σ, flexural rigidity B , plasma membrane tension γ, membrane bending rigidity K , and cytoskeleton to membrane-coupling ρ bk, whose values are consistent with previously reported values measured using invasive approaches. The value obtained for the membrane-coupling parameter was used to estimate the average number of coupling elements between the plasma membrane and the cytoskeleton that fell in the range of 30 elements per area of the laser spot used to record the fluctuations. Furthermore, to expand the TFS analysis, we investigate the correlation between F-actin linear density and the mechanical features of PC12 neurites. Using a hybrid instrument that combines TFS and a simple fluorescent technique, our results show that the fluctuations are related with the F-actin concentration. These measurements have an advantage of not requiring the application of an external force, allowing as to directly establish a correlation between changes in the mechanical parameters and cytoskeleton-protein concentrations. The sensibility of our method was also tested by the application of TFS technique to PC12 neurite under Paraformaldehyde and Latrunculin-A effect. These results show a dramatic modification in the fluctuations that are consistent with the reported effect of these drugs, confirming the high sensitivity of this technique. Finally, the thermal fluctuation approach was applied to DRG axons to show that its utility is not limited to studies of PC12 neurites, but it is suitable to measure the general characteristic of various neuron-like cells.
RESUMO
In mammals, the main molecular entity involved in innocuous cold transduction is TRPM8. This polymodal ion channel is activated by cold, cooling compounds such as menthol and voltage. Despite its relevance, the molecular determinants involved in its activation by cold remain elusive. In this study we explored the use of TRPM8 orthologs with different cold responses as a strategy to identify new molecular determinants related with their thermosensitivity. We focused on mouse TRPM8 (mTRPM8) and chicken TRPM8 (cTRPM8), which present complementary thermosensitive and chemosensitive phenotypes. Although mTRPM8 displays larger responses to cold than cTRPM8 does, the avian ortholog shows a higher sensitivity to menthol compared with the mouse channel, in both HEK293 cells and primary somatosensory neurons. We took advantage of these differences to build multiple functional chimeras between these orthologs, to identify the regions that account for these discrepancies. Using a combination of calcium imaging and patch clamping, we identified a region encompassing positions 526-556 in the N terminus, whose replacement by the cTRPM8 homolog sequence potentiated its response to agonists. More importantly, we found that the characteristic cold response of these orthologs is due to nonconserved residues located within the pore loop, suggesting that TRPM8 has evolved by increasing the magnitude of its cold response through changes in this region. Our results reveal that these structural domains are critically involved in cold sensitivity and functional modulation of TRPM8, and support the idea that the pore domain is a key molecular determinant in temperature responses of this thermo-transient receptor potential (TRP) channel.
Assuntos
Proteínas Aviárias/metabolismo , Cálcio/metabolismo , Temperatura Baixa , Ativação do Canal Iônico/fisiologia , Canais de Cátion TRPM/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Aviárias/genética , Galinhas , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Mentol/farmacologia , Camundongos , Mutagênese Sítio-Dirigida , Mutação , Domínios Proteicos , Homologia de Sequência , Canais de Cátion TRPM/genéticaRESUMO
In primary sensory neurons of the spinal and trigeminal somatosensory system, cold-sensitivity is strongly dependent on the functional balance between TRPM8 channels, the main molecular entity responsible for the cold-activated excitatory current, and Shaker-like Kv1.1-1.2 potassium channels, the molecular counterpart underlying the excitability brake current IKD. This slow-inactivating outward K+ current reduces the excitability of cold thermoreceptor neurons increasing their thermal threshold, and prevents unspecific activation by cold of neurons of other somatosensory modalities. Here we examine the main biophysical properties of this current in primary sensory neurons, its central role in cold thermotransduction, and its contribution to alterations in cold sensitivity triggered by peripheral nerve damage.
Assuntos
Síndromes Periódicas Associadas à Criopirina/metabolismo , Canal de Potássio Kv1.1/metabolismo , Células Receptoras Sensoriais/metabolismo , Termorreceptores/metabolismo , Animais , Temperatura Baixa , Canais de Cátion TRPM/metabolismoRESUMO
Cold allodynia is a common symptom of neuropathic and inflammatory pain following peripheral nerve injury. The mechanisms underlying this disabling sensory alteration are not entirely understood. In primary somatosensory neurons, cold sensitivity is mainly determined by a functional counterbalance between cold-activated TRPM8 channels and Shaker-like Kv1.1-1.2 channels underlying the excitability brake current IKD Here we studied the role of IKD in damage-triggered painful hypersensitivity to innocuous cold. We found that cold allodynia induced by chronic constriction injury (CCI) of the sciatic nerve in mice, was related to both an increase in the proportion of cold-sensitive neurons (CSNs) in DRGs contributing to the sciatic nerve, and a decrease in their cold temperature threshold. IKD density was reduced in high-threshold CSNs from CCI mice compared with sham animals, with no differences in cold-induced TRPM8-dependent current density. The electrophysiological properties and neurochemical profile of CSNs revealed an increase of nociceptive-like phenotype among neurons from CCI animals compared with sham mice. These results were validated using a mathematical model of CSNs, including IKD and TRPM8, showing that a reduction in IKD current density shifts the thermal threshold to higher temperatures and that the reduction of this current induces cold sensitivity in former cold-insensitive neurons expressing low levels of TRPM8-like current. Together, our results suggest that cold allodynia is largely due to a functional downregulation of IKD in both high-threshold CSNs and in a subpopulation of polymodal nociceptors expressing TRPM8, providing a general molecular and neural mechanism for this sensory alteration.SIGNIFICANCE STATEMENT This paper unveils the critical role of the brake potassium current IKD in damage-triggered cold allodynia. Using a well-known form of nerve injury and combining behavioral analysis, calcium imaging, patch clamping, and pharmacological tools, validated by mathematical modeling, we determined that the functional expression of IKD is reduced in sensory neurons in response to peripheral nerve damage. This downregulation not only enhances cold sensitivity of high-threshold cold thermoreceptors signaling cold discomfort, but it also transforms a subpopulation of polymodal nociceptors signaling pain into neurons activated by mild temperature drops. Our results suggest that cold allodynia is linked to a reduction of IKD in both high-threshold cold thermoreceptors and nociceptors expressing TRPM8, providing a general model for this form of cold-induced pain.
Assuntos
Temperatura Baixa/efeitos adversos , Hiperalgesia/fisiopatologia , Nociceptores/metabolismo , Traumatismos dos Nervos Periféricos/fisiopatologia , Potássio/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Células Cultivadas , Doença Crônica , Simulação por Computador , Hiperalgesia/diagnóstico , Hiperalgesia/etiologia , Ativação do Canal Iônico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Neurológicos , Traumatismos dos Nervos Periféricos/complicações , Traumatismos dos Nervos Periféricos/diagnósticoRESUMO
We reported earlier that TNF-α, a proinflammatory cytokine implicated in many inflammatory disorders causing orofacial pain, increases the activity of Cdk5, a key kinase involved in brain development and function and recently found to be involved in pain signaling. To investigate a potential mechanism underlying inflammatory pain in trigeminal ganglia (TGs), we engineered a transgenic mouse model (TNF) that can conditionally overexpresses TNF-α upon genomic recombination by Cre recombinase. TNF mice were bred with Nav1.8-Cre mouse line that expresses the Cre recombinase in sensory neurons to obtain TNF-α:Nav1.8-Cre (TNF-α cTg) mice. Although TNF-α cTg mice appeared normal without any gross phenotype, they displayed a significant increase in TNF-α levels after activation of NFκB signaling in the TG. IL-6 and MCP-1 levels were also increased along with intense immunostaining for Iba1 and GFAP in TG, indicating the presence of infiltrating macrophages and the activation of satellite glial cells. TNF-α cTg mice displayed increased trigeminal Cdk5 activity, and this increase was associated with elevated levels of phospho-T407-TRPV1 and capsaicin-evocated Ca influx in cultured trigeminal neurons. Remarkably, this effect was prevented by roscovitine, an inhibitor of Cdk5, which suggests that TNF-α overexpression induced sensitization of the TRPV1 channel. Furthermore, TNF-α cTg mice displayed more aversive behavior to noxious thermal stimulation (45°C) of the face in an operant pain assessment device as compared with control mice. In summary, TNF-α overexpression in the sensory neurons of TNF-α cTg mice results in inflammatory sensitization and increased Cdk5 activity; therefore, this mouse model would be valuable for investigating the mechanism of TNF-α involved in orofacial pain.
Assuntos
Cálcio/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPV/metabolismo , Gânglio Trigeminal/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Quimiocina CCL2/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Fator de Necrose Tumoral alfa/genéticaRESUMO
In the absence of simple noninvasive measurements, the knowledge of temporal and spatial variations of axons mechanics remains scarce. By extending thermal fluctuation spectroscopy (TFS) to long protrusions, we determine the transverse amplitude thermal fluctuation spectra that allow direct and simultaneous access to three key mechanics parameters: axial tension, bending flexural rigidity and plasma membrane tension. To test our model, we use PC12 cell protrusions-a well-know biophysical model of axons-in order to simplify the biological system under scope. For instance, axial and plasma membrane tension are found in the range of nano Newton and tens of pico Newtons per micron respectively. Furthermore, our results shows that the TFS technique is capable to distinguish quasi-identical protrusions. Another advantage of our approach is the time resolved nature of the measurements. Indeed, in the case of long term experiments on PC12 protrusions, TFS has revealed large temporal, correlated variations of the protrusion mechanics, displaying extraordinary feedback control over the axial tension in order to maintain a constant tension value.
Assuntos
Membrana Celular/química , Neuritos/fisiologia , Animais , Fenômenos Biomecânicos , Células PC12 , Ratos , Análise Espectral , Temperatura , Fatores de TempoRESUMO
The detection of environmental temperature is critical for the survival of the most diverse organisms. Thermosensitive transient receptor potential (thermoTRP) channels have evolved as a class of ion channels activated by a wide range of temperatures. These molecular thermal sensors are spread through the different TRP channel subfamilies. Among the Melastatin subfamily of TRP channels, the eighth member, TRPM8, is a calcium-permeable cationic ion channel activated by cold, by substances that evoke cold sensation such as menthol, and by voltage. This channel is considered the main molecular entity responsible for the sensitivity to cold of primary sensory neurons of the somatosensory system. Here we present to the readers a summary of some the most relevant biophysical properties, physiological role, and molecular intimacies of this polymodal thermoTRP channel.
Assuntos
Temperatura Baixa , Canais de Cátion TRPM/metabolismo , Sensação Térmica , Animais , Glicosilação , Humanos , Transporte Proteico , Canais de Cátion TRPM/químicaRESUMO
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.
Assuntos
Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas , Temperatura Baixa , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Mentol/farmacologia , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Canais de Cátion TRPM/genéticaRESUMO
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.
Assuntos
Temperatura Baixa , Neurônios/fisiologia , Canais de Cátion TRPM/metabolismo , Termorreceptores/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Glicosilação , Humanos , Camundongos , Processamento de Proteína Pós-Traducional , Canais de Cátion TRPM/fisiologia , Termorreceptores/fisiologiaRESUMO
BACKGROUND: TRPM8 is a non-selective cation channel that belongs to the melastatin subfamily of the transient receptor potential (TRP) ion channels. TRPM8 is activated by voltage, cold and cooling compounds such as menthol. Despite its essential role for cold temperature sensing in mammals, the pharmacology of TRPM8 is still in its infancy. Recently, tyrosine 745 (Y745) was identified as a critical residue for menthol sensitivity of the channel. In this report, we study the effect of mutating this residue on the action of several known TRPM8 antagonists: BCTC, capsazepine, SKF96365, and clotrimazole as well as two new inhibitor candidates, econazole and imidazole. RESULTS: We show that Y745 at the menthol binding site is critical for inhibition mediated by SKF96365 of cold- and voltage-activated TRPM8 currents. In contrast, the inhibition by other antagonists was unaffected by the mutation (BCTC) or only partially reduced (capsazepine, clotrimazole, econazole), suggesting that additional binding sites exist on the TRPM8 channel from where the inhibitors exert their negative modulation. Indeed, a molecular docking model implies that menthol and SKF96365 interact readily with Y745, while BCTC is unable to bind to this residue. CONCLUSION: In summary, we identify structural elements on the TRPM8 channel that are critical for the action of channel antagonists, providing valuable information for the future design of new, specific modulator compounds.
Assuntos
Temperatura Baixa , Ativação do Canal Iônico/fisiologia , Mentol/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Tirosina/metabolismo , Animais , Capsaicina/análogos & derivados , Capsaicina/química , Capsaicina/farmacologia , Clotrimazol/química , Clotrimazol/farmacologia , Humanos , Imidazóis/química , Imidazóis/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Mentol/química , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Fenótipo , Pirazinas/química , Pirazinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Transient receptor potential channels are a family of cation channels involved in diverse cellular functions. Most of these channels are expressed in the nervous system and play a key role in sensory physiology. TRPM8 (transient receptor potential melastatine 8), a member of this family, is activated by cold, cooling substances such menthol and icilin and voltage. Although TRPM8 is a thermosensitive channel highly expressed in cold sensory neurons, the mechanisms underlying its temperature sensitivity are still poorly understood. Here we show that, in sensory neurons, TRPM8 channel is localized in cholesterol-rich specialized membrane domains known as lipid rafts. We also show that, in heterologous expression systems, lipid raft segregation of TRPM8 is favored by glycosylation at the Asn(934) residue of the polypeptide. In electrophysiological and imaging experiments, using cold and menthol as agonists, we also demonstrate that lipid raft association modulates TRPM8 channel activity. We found that menthol- and cold-mediated responses of TRPM8 are potentiated when the lipid raft association of the channel is prevented. In addition, lipid raft disruption shifts the threshold for TRPM8 activation to a warmer temperature. In view of these data, we suggest a role for lipid rafts in the activity and temperature sensitivity of TRPM8. We propose a model wherein different lipid membrane environments affect the cold sensing properties of TRPM8, modulating the response of cold thermoreceptors.
Assuntos
Microdomínios da Membrana/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Linhagem Celular , Fenômenos Eletrofisiológicos , Glicosilação , Humanos , Camundongos , Mutação/genética , Ácido N-Acetilneuramínico , Técnicas de Patch-Clamp , Transporte Proteico , Canais de Cátion TRPM/genéticaRESUMO
A characteristic feature of many vertebrate axons is their wrapping by a lamellar stack of glially derived membranes known as the myelin sheath. Myelin is a cholesterol-rich membrane that allows for rapid saltatory nerve impulse conduction. Axonal neuregulins instruct glial cells on when and how much myelin they should produce. However, how neuregulin regulates myelin sheath development and thickness is unknown. Here we show that neuregulin receptors are activated by drops in plasma membrane cholesterol, suggesting that they can sense sterol levels. In Schwann cells neuregulin-1 increases the transcription of the 3-hydroxy-3-methylglutarylcoenzyme A reductase, the rate-limiting enzyme for cholesterol biosynthesis. Neuregulin activity is mediated by the phosphatidylinositol 3-kinase pathway and a cAMP-response element located on the reductase promoter. We propose that by activating neuregulin receptors, neurons exploit a cholesterol homeostatic mechanism forcing Schwann cells to produce new membranes for the myelin sheath. We also show that a strong phylogenetic correlation exists between myelination and cholesterol biosynthesis, and we propose that the absence of the sterol branch of the mevalonate pathway in invertebrates precluded the myelination of their nervous system.
Assuntos
Colesterol/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/biossíntese , Bainha de Mielina/enzimologia , Proteínas do Tecido Nervoso/farmacologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Animais , Axônios/enzimologia , Células COS , Chlorocebus aethiops , Colesterol/genética , AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Humanos , Ácido Mevalônico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuregulina-1 , Fosfatidilinositol 3-Quinases/metabolismo , Elementos de Resposta/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Transcrição Gênica/fisiologiaRESUMO
Down's syndrome (trisomy 21) brain tissue is considered to be susceptible to oxidative injury, mainly because its increased Cu/Zn-superoxide dismutase (SOD1) activity is not followed by an adaptive rise in hydrogen peroxide metabolizing enzymes. In vitro, trisomic neurons suffer oxidative stress and degenerate. We studied the response of trisomy 21 neuron and astrocyte cultures to hydrogen peroxide injury and found that they were, respectively, more and less vulnerable than their euploid counterparts. Differences were detected 24 h after exposures in the region of 50 microm and 500 microm hydrogen peroxide for neuron and astrocyte cultures, respectively. Cytotoxicity results were paralleled by a decrease in cellular glutathione. In addition, trisomic astrocytes showed a lower basal content of superoxide ion and a higher clearance of hydrogen peroxide from the culture medium. In the presence of hydrogen peroxide, trisomic astrocytes maintained their concentration of intracellular superoxide and hydroperoxides at a lower level than euploid astrocytes. Consistent with these results, trisomic astrocytes in neuron coculture were more neuroprotective than euploid astrocytes against hydrogen peroxide injury. We suggest that SOD1 overexpression has beneficial effects on astrocytes, as it does in other systems with similarly high disposal of hydroperoxides. In addition to a higher enzymatic activity of SOD1, cultures of trisomic astrocytes showed slightly higher glutathione reductase activity than euploid cultures. Thus, trisomy 21 astrocytes showed a greater antioxidant capacity against hydrogen peroxide than euploid astrocytes, and they partially counteracted the oxidative vulnerability of trisomic neurons in culture.
Assuntos
Antioxidantes/metabolismo , Astrócitos/metabolismo , Encéfalo/metabolismo , Síndrome de Down/metabolismo , Estresse Oxidativo/genética , Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Encéfalo/fisiopatologia , Catalase/metabolismo , Células Cultivadas , Cromossomos Humanos Par 21/genética , Relação Dose-Resposta a Droga , Síndrome de Down/genética , Síndrome de Down/fisiopatologia , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Taxa de Depuração Metabólica/efeitos dos fármacos , Taxa de Depuração Metabólica/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Superóxidos/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Trimethyltin (TMT) and triethyltin (TET) caused cell death in cultures of primary human neurons and astrocytes, rat neurons and human neuroblastoma cell lines. Human neurons and astrocytes showed a delayed response to TMT cytotoxicity. After 24h of TMT exposure, LC50 values were 148.1, 335.5 and 609.7 microM for SK-N-MC neuroblastoma cell line, neurons and astrocytes, respectively. Over 5 days of exposure, the cytotoxic potency of TMT increased about 70-fold in human cortical neurons. Rat hippocampal neurons were the most vulnerable cells to TMT cytotoxicity, exhibiting an LC50 value 30-fold lower (1.4 microM) than that of rat cerebellar granule cells (44.28 microM). With the exception of rat hippocampal neurons, TET was more potent than TMT in inducing cell death (LC50 values of 3.5-16.9 microM). Moreover, TET was more effective than TMT in increasing intracellular free Ca2+ concentration in human and rat neurons. This work shows that human fetal neuron and astrocyte cultures are a useful model for studying the neurotoxic effects of these environmental contaminants and, thus, predicting their impact on human health.