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1.
Front Pharmacol ; 14: 1213337, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37388453

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.

2.
Neuron ; 110(10): 1656-1670.e12, 2022 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-35276083

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 , Polifosfatos
3.
J Neurosci ; 41(41): 8475-8493, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34446569

RESUMO

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/metabolismo
4.
Int J Mol Sci ; 21(12)2020 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-32580281

RESUMO

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/citologia
5.
Cell Rep ; 30(13): 4505-4517.e5, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32234483

RESUMO

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/metabolismo
6.
J Neurosci ; 39(41): 8177-8192, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31471469

RESUMO

The cornea is extensively innervated by trigeminal ganglion cold thermoreceptor neurons expressing TRPM8 (transient receptor potential cation channel subfamily M member 8). These neurons respond to cooling, hyperosmolarity and wetness of the corneal surface. Surgical injury of corneal nerve fibers alters tear production and often causes dry eye sensation. The contribution of TRPM8-expressing corneal cold-sensitive neurons (CCSNs) to these symptoms is unclear. Using extracellular recording of CCSNs nerve terminals combined with in vivo confocal tracking of reinnervation, Ca2+ imaging and patch-clamp recordings of fluorescent retrogradely labeled corneal neurons in culture, we analyzed the functional modifications of CCSNs induced by peripheral axonal damage in male mice. After injury, the percentage of CCSNs, the cold- and menthol-evoked intracellular [Ca2+] rises and the TRPM8 current density in CCSNs were larger than in sham animals, with no differences in the brake K+ current IKD Active and passive membrane properties of CCSNs from both groups were alike and corresponded mainly to those of canonical low- and high-threshold cold thermoreceptor neurons. Ongoing firing activity and menthol sensitivity were higher in CCSN terminals of injured mice, an observation accounted for by mathematical modeling. These functional changes developed in parallel with a partial reinnervation of the cornea by TRPM8(+) fibers and with an increase in basal tearing in injured animals compared with sham mice. Our results unveil key TRPM8-dependent functional changes in CCSNs in response to injury, suggesting that increased tearing rate and ocular dryness sensation derived from deep surgical ablation of corneal nerves are due to enhanced functional expression of TRPM8 channels in these injured trigeminal primary sensory neurons.SIGNIFICANCE STATEMENT We unveil a key role of TRPM8 channels in the sensory and autonomic disturbances associated with surgical damage of eye surface nerves. We studied the damage-induced functional alterations of corneal cold-sensitive neurons using confocal tracking of reinnervation, extracellular corneal nerve terminal recordings, tearing measurements in vivo, Ca2+ imaging and patch-clamp recordings of cultured corneal neurons, and mathematical modeling. Corneal nerve ablation upregulates TRPM8 mainly in canonical cold thermoreceptors, enhancing their cold and menthol sensitivity, inducing a rise in the ongoing firing activity of TRPM8(+) nerve endings and an increase in basal tearing. Our results suggest that unpleasant dryness sensations, together with augmented tearing rate after corneal nerve injury, are largely due to upregulation of TRPM8 in cold thermoreceptor neurons.


Assuntos
Axônios/fisiologia , Temperatura Baixa , Córnea/inervação , Córnea/fisiologia , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPM/fisiologia , Sensação Térmica/fisiologia , Animais , Lesões da Córnea/fisiopatologia , Fenômenos Eletrofisiológicos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Neurológicos , Modelos Teóricos , Fibras Nervosas , Técnicas de Patch-Clamp , Lágrimas , Termorreceptores/fisiologia
7.
J Biol Chem ; 293(32): 12454-12471, 2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-29880642

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/genética
8.
Adv Exp Med Biol ; 1015: 265-277, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29080031

RESUMO

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/metabolismo
9.
J Neurosci ; 37(12): 3109-3126, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28179555

RESUMO

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/diagnóstico
10.
11.
Pain ; 157(6): 1346-1362, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26894912

RESUMO

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/genética
12.
PLoS One ; 10(10): e0139314, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26426259

RESUMO

Cold-sensitive nerve terminals (CSNTs) encode steady temperatures with regular, rhythmic temperature-dependent firing patterns that range from irregular tonic firing to regular bursting (static response). During abrupt temperature changes, CSNTs show a dynamic response, transiently increasing their firing frequency as temperature decreases and silencing when the temperature increases (dynamic response). To date, mathematical models that simulate the static response are based on two depolarizing/repolarizing pairs of membrane ionic conductance (slow and fast kinetics). However, these models fail to reproduce the dynamic response of CSNTs to rapid changes in temperature and notoriously they lack a specific cold-activated conductance such as the TRPM8 channel. We developed a model that includes TRPM8 as a temperature-dependent conductance with a calcium-dependent desensitization. We show by computer simulations that it appropriately reproduces the dynamic response of CSNTs from mouse cornea, while preserving their static response behavior. In this model, the TRPM8 conductance is essential to display a dynamic response. In agreement with experimental results, TRPM8 is also needed for the ongoing activity in the absence of stimulus (i.e. neutral skin temperature). Free parameters of the model were adjusted by an evolutionary optimization algorithm, allowing us to find different solutions. We present a family of possible parameters that reproduce the behavior of CSNTs under different temperature protocols. The detection of temperature gradients is associated to a homeostatic mechanism supported by the calcium-dependent desensitization.


Assuntos
Temperatura Baixa , Modelos Teóricos , Canais de Cátion TRPM/metabolismo , Termorreceptores/metabolismo , Sensação Térmica/fisiologia , Animais , Simulação por Computador , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Análise Numérica Assistida por Computador
13.
Plant Cell ; 27(1): 177-88, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25595824

RESUMO

Sensory modalities are essential for navigating through an ever-changing environment. From insects to mammals, transient receptor potential (TRP) channels are known mediators for cellular sensing. Chlamydomonas reinhardtii is a motile single-celled freshwater green alga that is guided by photosensory, mechanosensory, and chemosensory cues. In this type of alga, sensory input is first detected by membrane receptors located in the cell body and then transduced to the beating cilia by membrane depolarization. Although TRP channels seem to be absent in plants, C. reinhardtii possesses genomic sequences encoding TRP proteins. Here, we describe the cloning and characterization of a C. reinhardtii version of a TRP channel sharing key features present in mammalian TRP channels associated with sensory transduction. In silico sequence-structure analysis unveiled the modular design of TRP channels, and electrophysiological experiments conducted on Human Embryonic Kidney-293T cells expressing the Cr-TRP1 clone showed that many of the core functional features of metazoan TRP channels are present in Cr-TRP1, suggesting that basic TRP channel gating characteristics evolved early in the history of eukaryotes.


Assuntos
Chlamydomonas/metabolismo , Canais Iônicos/metabolismo , Mamíferos/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Linhagem Celular , Eletrofisiologia , Humanos
14.
Curr Top Membr ; 74: 293-324, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25366241

RESUMO

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/química
15.
J Biol Chem ; 289(32): 21828-43, 2014 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-24917670

RESUMO

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/genética
16.
J Neurophysiol ; 109(11): 2803-14, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23486205

RESUMO

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by dysfunction and degeneration of motoneurons starting in adulthood. Recent studies using cell or animal models document that astrocytes expressing disease-causing mutations of human superoxide dismutase 1 (hSOD1) contribute to the pathogenesis of ALS by releasing a neurotoxic factor(s). Neither the mechanism by which this neurotoxic factor induces motoneuron death nor its cellular site of action has been elucidated. Here we show that acute exposure of primary wild-type spinal cord cultures to conditioned medium derived from astrocytes expressing mutant SOD1 (ACM-hSOD1(G93A)) increases persistent sodium inward currents (PC(Na)), repetitive firing, and intracellular calcium transients, leading to specific motoneuron death days later. In contrast to TTX, which paradoxically increased twofold the amplitude of calcium transients and killed motoneurons, reduction of hyperexcitability by other specific (mexiletine) and nonspecific (spermidine and riluzole) blockers of voltage-sensitive sodium (Na(v)) channels restored basal calcium transients and prevented motoneuron death induced by ACM-hSOD1(G93A). These findings suggest that riluzole, the only FDA-approved drug with known benefits for ALS patients, acts by inhibiting hyperexcitability. Together, our data document that a critical element mediating the non-cell-autonomous toxicity of ACM-hSOD1(G93A) on motoneurons is increased excitability, an observation with direct implications for therapy of ALS.


Assuntos
Potenciais de Ação , Astrócitos/metabolismo , Neurônios Motores/efeitos dos fármacos , Mutação , Superóxido Dismutase/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Morte Celular , Células Cultivadas , Meios de Cultivo Condicionados/toxicidade , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/fisiologia , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia
17.
J Neurophysiol ; 108(11): 3009-23, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22956791

RESUMO

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.


Assuntos
Potenciais de Ação/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Terminações Nervosas/fisiologia , Canais de Potássio/genética , Termorreceptores/fisiologia , Potenciais de Ação/genética , Animais , Benzazepinas/farmacologia , Fármacos Cardiovasculares/farmacologia , Temperatura Baixa , Córnea/inervação , Canais de Cátion Regulados por Nucleotídeos Cíclicos/antagonistas & inibidores , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/metabolismo , Ivabradina , Camundongos , Modelos Neurológicos , Nervo Óptico/fisiologia , Pirimidinas/farmacologia
18.
J Biol Chem ; 287(22): 18218-29, 2012 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-22493431

RESUMO

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/fisiologia
19.
Physiology (Bethesda) ; 26(4): 273-85, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21841075

RESUMO

Transient receptor potential melastatin 8 (TRPM8), a calcium-permeable cation channel activated by cold, cooling compounds and voltage, is the main molecular entity responsible for detection of cold temperatures in the somatosensory system. Here, we review the biophysical properties, physiological role, and near-membrane trafficking of this exciting polymodal ion channel.


Assuntos
Canais de Cálcio/fisiologia , Canais de Cátion TRPM/fisiologia , Sensação Térmica/fisiologia , Animais , Temperatura Baixa , Humanos , Transdução de Sinais
20.
Nat Med ; 16(12): 1396-9, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21076394

RESUMO

Basal tearing is crucial to maintaining ocular surface wetness. Corneal cold thermoreceptors sense small oscillations in ambient temperature and change their discharge accordingly. Deletion of the cold-transducing ion channel Transient receptor potential cation channel subfamily M member 8 (TRPM8) in mice abrogates cold responsiveness and reduces basal tearing without affecting nociceptor-mediated irritative tearing. Warming of the cornea in humans also decreases tearing rate. These findings indicate that TRPM8-dependent impulse activity in corneal cold receptors contributes to regulating basal tear flow.


Assuntos
Córnea/metabolismo , Síndromes do Olho Seco/etiologia , Fenômenos Fisiológicos Oculares , Canais de Cátion TRPM/metabolismo , Lágrimas/fisiologia , Termorreceptores/metabolismo , 4-Aminopiridina/metabolismo , Animais , Temperatura Baixa , Síndromes do Olho Seco/metabolismo , Mentol/farmacologia , Camundongos , Camundongos Transgênicos , Pirazinas/farmacologia , Piridinas/farmacologia , Superfamília Shaker de Canais de Potássio/metabolismo , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/genética
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