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1.
Cell Res ; 29(2): 95-109, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30607017

RESUMO

Mammals possess a remarkable ability to sense subtle temperature deviations from the thermoneutral skin temperature of ~33 °C, which ensures precise warm sensation. However, the underlying mechanisms remain unclear. Here we show that STIM1, an endoplasmic reticulum (ER) resident transmembrane protein that responds to both ER Ca2+ depletion and heat, mediates temperature-induced Ca2+ influx in skin keratinocytes via coupling to Orai Ca2+ channels in plasma membrane. Behaviorally, the keratinocyte-specific knockout of STIM1 shifts the optimal preference temperature (OPT) of mice from ~32 °C to ~34 °C, resulting in a strikingly reversed preference between 32 °C and 34 °C. Importantly, the thermally inactive STIM1-ΔK knock-in mice show altered OPT and warm preference behaviors as well, demonstrating the requirement of STIM1 thermosensitivity for warm sensation. Furthermore, the wild-type and mutant mice prefer temperatures closer to their respective OPTs, but poorly distinguish temperatures that are equally but oppositely deviated from their OPTs. Mechanistically, keratinocyte STIM1 affects the in vivo warm responses of sensory neurons by likely involving TRPA1 as a downstream transduction channel. Collectively, our data suggest that STIM1 serves as a novel in vivo thermosensor in keratinocytes to define the OPT, which might be utilized as a peripheral reference temperature for precise warm sensation.


Assuntos
Retículo Endoplasmático/metabolismo , Temperatura Alta , Queratinócitos/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Sensação Térmica/fisiologia , Animais , Animais Recém-Nascidos , Comportamento Animal/fisiologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Feminino , Gânglios Espinais/citologia , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína ORAI1/metabolismo , Células Receptoras Sensoriais/metabolismo , Molécula 1 de Interação Estromal/genética
2.
Cell ; 173(3): 762-775.e16, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29677517

RESUMO

Mechanotransduction plays a crucial role in vascular biology. One example of this is the local regulation of vascular resistance via flow-mediated dilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction and a precursor to a wide array of vascular diseases, such as hypertension and atherosclerosis. Yet the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small-diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium and is a critical signaling component in cardiovascular pathophysiology.


Assuntos
Mecanotransdução Celular , Interferência de RNA , Receptores Acoplados a Proteínas G/fisiologia , Animais , Materiais Biocompatíveis , Cálcio/metabolismo , Linhagem Celular Tumoral , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração de Íons de Hidrogênio , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/genética , Resistência ao Cisalhamento , Estresse Mecânico , Resistência Vascular
3.
Nature ; 516(7529): 121-5, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25471886

RESUMO

The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.


Assuntos
Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologia , Pele/inervação , Tato/fisiologia , Animais , Canais Iônicos/genética , Mecanorreceptores/metabolismo , Mecanotransdução Celular/genética , Células de Merkel/fisiologia , Camundongos , Camundongos Knockout , Células Receptoras Sensoriais/fisiologia , Tato/genética
4.
J Med Chem ; 57(12): 5129-40, 2014 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-24884675

RESUMO

A high throughput screening campaign identified 5-(2-chlorophenyl)indazole compound 4 as an antagonist of the transient receptor potential A1 (TRPA1) ion channel with IC50 = 1.23 µM. Hit to lead medicinal chemistry optimization established the SAR around the indazole ring system, demonstrating that a trifluoromethyl group at the 2-position of the phenyl ring in combination with various substituents at the 6-position of the indazole ring greatly contributed to improvements in vitro activity. Further lead optimization resulted in the identification of compound 31, a potent and selective antagonist of TRPA1 in vitro (IC50 = 0.015 µM), which has moderate oral bioavailability in rodents and demonstrates robust activity in vivo in several rodent models of inflammatory pain.


Assuntos
Indazóis/química , Proteínas do Tecido Nervoso/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Administração Oral , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Disponibilidade Biológica , Células CHO , Canais de Cálcio , Cricetulus , Adjuvante de Freund , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Indazóis/farmacocinética , Indazóis/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Mostardeira , Óleos de Plantas , Ratos Wistar , Especificidade da Espécie , Relação Estrutura-Atividade , Canal de Cátion TRPA1 , Canais de Cátion TRPC/antagonistas & inibidores
5.
Nature ; 509(7502): 622-6, 2014 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-24717433

RESUMO

How we sense touch remains fundamentally unknown. The Merkel cell-neurite complex is a gentle touch receptor in the skin that mediates slowly adapting responses of Aß sensory fibres to encode fine details of objects. This mechanoreceptor complex was recognized to have an essential role in sensing gentle touch nearly 50 years ago. However, whether Merkel cells or afferent fibres themselves sense mechanical force is still debated, and the molecular mechanism of mechanotransduction is unknown. Synapse-like junctions are observed between Merkel cells and associated afferents, and yet it is unclear whether Merkel cells are inherently mechanosensitive or whether they can rapidly transmit such information to the neighbouring nerve. Here we show that Merkel cells produce touch-sensitive currents in vitro. Piezo2, a mechanically activated cation channel, is expressed in Merkel cells. We engineered mice deficient in Piezo2 in the skin, but not in sensory neurons, and show that Merkel-cell mechanosensitivity completely depends on Piezo2. In these mice, slowly adapting responses in vivo mediated by the Merkel cell-neurite complex show reduced static firing rates, and moreover, the mice display moderately decreased behavioural responses to gentle touch. Our results indicate that Piezo2 is the Merkel-cell mechanotransduction channel and provide the first line of evidence that Piezo channels have a physiological role in mechanosensation in mammals. Furthermore, our data present evidence for a two-receptor-site model, in which both Merkel cells and innervating afferents act together as mechanosensors. The two-receptor system could provide this mechanoreceptor complex with a tuning mechanism to achieve highly sophisticated responses to a given mechanical stimulus.


Assuntos
Canais Iônicos/metabolismo , Mecanotransdução Celular , Células de Merkel/metabolismo , Tato/fisiologia , Potenciais de Ação , Animais , Condutividade Elétrica , Feminino , Técnicas In Vitro , Canais Iônicos/deficiência , Canais Iônicos/genética , Masculino , Mecanotransdução Celular/genética , Camundongos , Camundongos Knockout , Neuritos/metabolismo , Neurônios Aferentes/metabolismo , Pele/citologia , Pele/inervação , Tato/genética
6.
Proc Natl Acad Sci U S A ; 108(48): 19413-8, 2011 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-22087007

RESUMO

The voltage-gated sodium channel Na(v)1.8 is known to function in the transmission of pain signals induced by cold, heat, and mechanical stimuli. Sequence variants of human Na(v)1.8 have been linked to altered cardiac conduction. We identified an allele of Scn10a encoding the α-subunit of Na(v)1.8 among mice homozygous for N-ethyl-N-nitrosourea-induced mutations. The allele creates a dominant neurobehavioral phenotype termed Possum, characterized by transient whole-body tonic immobility induced by pinching the skin at the back of the neck ("scruffing"). The Possum mutation enhanced Na(v)1.8 sodium currents and neuronal excitability and heightened sensitivity of mutants to cold stimuli. Striking electroencephalographic changes were observed concomitant with the scruffing-induced behavioral change. In addition, electrocardiography demonstrated that Possum mice exhibited marked sinus bradycardia and R-R variability upon scruffing, abrogated by infusion of atropine. However, atropine failed to prevent or mitigate the tonic immobility response. Hyperactive sodium conduction via Na(v)1.8 thus leads to a complex neurobehavioral phenotype, which resembles catatonia in schizophrenic humans and tonic immobility in other mammals upon application of a discrete stimulus; no other form of mechanosensory stimulus could induce the immobility phenotype. Our data confirm the involvement of Na(v)1.8 in transducing pain initiated by cold and additionally implicate Na(v)1.8 in previously unknown functions in the central nervous system and heart.


Assuntos
Resposta de Imobilidade Tônica/fisiologia , Mutação/genética , Fenótipo , Canais de Sódio/genética , Animais , Atropina/farmacologia , Bradicardia/genética , Eletrocardiografia , Eletroencefalografia , Resposta de Imobilidade Tônica/efeitos dos fármacos , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.8 , Canais de Sódio/fisiologia
7.
Nat Commun ; 2: 369, 2011 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-21712817

RESUMO

Nitric oxide (NO) is an unstable signalling molecule synthesized de novo mainly from L-arginine by NO synthase (NOS) enzymes. Nitrite reduction can also produce NO, predominantly within body fluids (for example, saliva, sweat and blood plasma) and under extreme hypoxic and acidic conditions. It remains unknown if intracellular canonical signalling pathways regulate nitrite-dependent NO production. Here we examine NO production in the skin, a hypoxic tissue enriched in nitrites wherein NO has important roles in wound healing and other biological processes. We show that activation of TRPV3, a heat-activated transient receptor potential ion channel expressed in keratinocytes, induces NO production via a nitrite-dependent pathway. TRPV3 and nitrite are involved in keratinocyte migration in vitro and in wound healing and thermosensory behaviours in vivo. Our study demonstrates that activation of an ion channel can induce NOS-independent NO production in keratinocytes.


Assuntos
Queratinócitos/metabolismo , Óxido Nítrico/biossíntese , Canais de Cátion TRPV/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Proliferação de Células , Células Cultivadas , Primers do DNA/genética , Eletroporação , Queratinócitos/fisiologia , Camundongos , Nitritos/metabolismo , Reação em Cadeia da Polimerase , Cicatrização/fisiologia
8.
J Neurosci ; 30(45): 15165-74, 2010 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-21068322

RESUMO

TRPA1 is a nonselective cation channel expressed by nociceptors. Although it is widely accepted that TRPA1 serves as a broad irritancy receptor for a variety of reactive chemicals, its role in cold sensation remains controversial. Here, we demonstrate that mild cooling markedly increases agonist-evoked rat TRPA1 currents. In the absence of an agonist, even noxious cold only increases current amplitude slightly. These results suggest that TRPA1 is a key mediator of cold hypersensitivity in pathological conditions in which reactive oxygen species and proinflammatory activators of the channel are present, but likely plays a comparatively minor role in acute cold sensation. Supporting this, cold hypersensitivity can be induced in wild-type but not Trpa1(-/-) mice by subcutaneous administration of a TRPA1 agonist. Furthermore, the selective TRPA1 antagonist HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide] reduces cold hypersensitivity in rodent models of inflammatory and neuropathic pain.


Assuntos
Temperatura Baixa , Hiperalgesia/metabolismo , Nociceptores/fisiologia , Sensação Térmica/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Eletrofisiologia , Gânglios Espinais/fisiologia , Hiperalgesia/fisiopatologia , Camundongos , Camundongos Knockout , Ratos , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/antagonistas & inibidores
9.
Science ; 330(6000): 55-60, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20813920

RESUMO

Mechanical stimuli drive many physiological processes, including touch and pain sensation, hearing, and blood pressure regulation. Mechanically activated (MA) cation channel activities have been recorded in many cells, but the responsible molecules have not been identified. We characterized a rapidly adapting MA current in a mouse neuroblastoma cell line. Expression profiling and RNA interference knockdown of candidate genes identified Piezo1 (Fam38A) to be required for MA currents in these cells. Piezo1 and related Piezo2 (Fam38B) are vertebrate multipass transmembrane proteins with homologs in invertebrates, plants, and protozoa. Overexpression of mouse Piezo1 or Piezo2 induced two kinetically distinct MA currents. Piezos are expressed in several tissues, and knockdown of Piezo2 in dorsal root ganglia neurons specifically reduced rapidly adapting MA currents. We propose that Piezos are components of MA cation channels.


Assuntos
Cátions/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Neurônios/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/química , Clonagem Molecular , Gânglios Espinais/citologia , Canais Iônicos/análise , Canais Iônicos/química , Canais Iônicos/genética , Potenciais da Membrana , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Pressão , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/genética , Transfecção
10.
Nat Neurosci ; 13(6): 708-14, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20414199

RESUMO

TRPV1 is the founding and best-studied member of the family of temperature-activated transient receptor potential ion channels (thermoTRPs). Voltage, chemicals and heat allosterically gate TRPV1. Molecular determinants of TRPV1 activation by capsaicin, allicin, acid, ammonia and voltage have been identified. However, the structures and mechanisms mediating TRPV1's pronounced temperature sensitivity remain unclear. Recent studies of the related channel TRPV3 identified residues in the pore region that are required for heat activation. We used both random and targeted mutagenesis screens of rat TRPV1 and identified point mutations in the outer pore region that specifically impair temperature activation. Single-channel analysis indicated that TRPV1 mutations disrupted heat sensitivity by ablating long channel openings, which are part of the temperature-gating pathway. We propose that sequential occupancy of short and long open states on activation provides a mechanism for enhancing temperature sensitivity. Our results suggest that the outer pore is important for the heat sensitivity of thermoTRPs.


Assuntos
Ativação do Canal Iônico , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Temperatura , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Cinética , Potenciais da Membrana , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Técnicas de Patch-Clamp , Mutação Puntual , Probabilidade , Estabilidade Proteica , Estrutura Terciária de Proteína , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Canais de Cátion TRPV/genética
11.
Neuron ; 64(4): 498-509, 2009 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-19945392

RESUMO

Transient receptor potential A1 (TRPA1) ion channel senses a variety of noxious stimuli and is involved in nociception. Many TRPA1 agonists covalently modify the channel, which can lead to desensitization. The fate of modified TRPA1 and the mechanism of preserving its response to subsequent stimuli are not understood. Moreover, inflammatory signals sensitize TRPA1 by involving protein kinase A (PKA) and phospholipase C (PLC) through unknown means. We show that TRPA1-mediated nocifensive behavior can be sensitized in vivo via PKA/PLC signaling and by activating TRPA1 with the ligand mustard oil (MO). Interestingly, both stimuli increased TRPA1 membrane levels in vitro. Tetanus toxin attenuated the response to the second of two pulses of MO in neurons, suggesting that vesicle fusion increases functional surface TRPA1. Capacitance recordings suggest that MO can induce exocytosis. We propose that TRPA1 translocation to the membrane might represent one of the mechanisms controlling TRPA1 functionality upon acute activation or inflammatory signals.


Assuntos
Membrana Celular/metabolismo , Nociceptores/fisiologia , Dor/metabolismo , Transdução de Sinais/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/genética , Membrana Celular/patologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Humanos , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Dor/genética , Dor/patologia , Transporte Proteico/genética , Transporte Proteico/fisiologia , Transdução de Sinais/genética , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/deficiência , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/fisiologia , Fosfolipases Tipo C/fisiologia
12.
PLoS One ; 4(10): e7596, 2009 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-19893614

RESUMO

Nitric oxide (NO) can induce acute pain in humans and plays an important role in pain sensitization caused by inflammation and injury in animal models. There is evidence that NO acts both in the central nervous system via a cyclic GMP pathway and in the periphery on sensory neurons through unknown mechanisms. It has recently been suggested that TRPV1 and TRPA1, two polymodal ion channels that sense noxious stimuli impinging on peripheral nociceptors, are activated by NO in heterologous systems. Here, we investigate the relevance of this activation. We demonstrate that NO donors directly activate TRPV1 and TRPA1 in isolated inside-out patch recordings. Cultured primary sensory neurons display both TRPV1- and TRPA1-dependent responses to NO donors. BH4, an essential co-factor for NO production, causes activation of a subset of DRG neurons as assayed by calcium imaging, and this activation is at least partly dependent on nitric oxide synthase activity. We show that BH4-induced calcium influx is ablated in DRG neurons from TRPA1/TRPV1 double knockout mice, suggesting that production of endogenous levels of NO can activate these ion channels. In behavioral assays, peripheral NO-induced nociception is compromised when TRPV1 and TRPA1 are both ablated. These results provide genetic evidence that the peripheral nociceptive action of NO is mediated by both TRPV1 and TRPA1.


Assuntos
Óxido Nítrico/metabolismo , Nociceptores/metabolismo , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Canais de Potencial de Receptor Transitório/fisiologia , Animais , Comportamento Animal , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Gânglios Espinais/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Dor , Canal de Cátion TRPA1
13.
Nat Chem Biol ; 5(3): 183-90, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19202543

RESUMO

Zinc is an essential biological trace element. It is required for the structure or function of over 300 proteins, and it is increasingly recognized for its role in cell signaling. However, high concentrations of zinc have cytotoxic effects, and overexposure to zinc can cause pain and inflammation through unknown mechanisms. Here we show that zinc excites nociceptive somatosensory neurons and causes nociception in mice through TRPA1, a cation channel previously shown to mediate the pungency of wasabi and cinnamon through cysteine modification. Zinc activates TRPA1 through a unique mechanism that requires zinc influx through TRPA1 channels and subsequent activation via specific intracellular cysteine and histidine residues. TRPA1 is highly sensitive to intracellular zinc, as low nanomolar concentrations activate TRPA1 and modulate its sensitivity. These findings identify TRPA1 as an important target for the sensory effects of zinc and support an emerging role for zinc as a signaling molecule that can modulate sensory transmission.


Assuntos
Canais de Potencial de Receptor Transitório/efeitos dos fármacos , Zinco/toxicidade , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Dor/induzido quimicamente , Conformação Proteica , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/fisiologia , Zinco/farmacocinética
14.
J Neurosci ; 29(1): 153-8, 2009 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-19129393

RESUMO

Maintaining physiological pH is required for survival, and exposure to alkaline chemicals such as ammonia (smelling salts) elicits severe pain and inflammation through unknown mechanisms. TRPV1, the capsaicin receptor, is an integrator of noxious stimuli including heat and extracellular acidic pH. Here, we report that ammonia activates TRPV1, TRPA1 (another polymodal nocisensor), and other unknown receptor(s) expressed in sensory neurons. Ammonia and intracellular alkalization activate TRPV1 through a mechanism that involves a cytoplasmic histidine residue, not used by other TRPV1 agonists such as heat, capsaicin or low pH. Our studies show that TRPV1 detects both acidic and basic deviations from homeostatic pH.


Assuntos
Ácidos/farmacologia , Cloreto de Amônio/farmacologia , Células Receptoras Sensoriais/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Animais , Cálcio/metabolismo , Capsaicina/farmacologia , Linhagem Celular Transformada , Gânglios Espinais/citologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Mutagênese Sítio-Dirigida/métodos , Técnicas de Patch-Clamp , Pirazinas/farmacologia , Piridinas/farmacologia , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/deficiência , Transfecção/métodos
15.
Proc Natl Acad Sci U S A ; 106(5): 1626-31, 2009 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-19164517

RESUMO

Temperature-activated transient receptor potential ion channels (thermoTRPs) are polymodal detectors of various stimuli including temperature, voltage, and chemicals. To date, it is not known how TRP channels integrate the action of such disparate stimuli. Identifying specific residues required for channel-activation by distinct stimuli is necessary for understanding overall TRP channel function. TRPV3 is activated by warm temperatures and various chemicals, and is modulated by voltage. One potent activator of TRPV3 is 2-aminoethyl diphenylborinate (2-APB), a synthetic chemical that modulates many TRP channels. In a high-throughput mutagenesis screen of approximately 14,000 mutated mouse TRPV3 clones, we found 2 residues (H426 and R696) specifically required for sensitivity of TRPV3 to 2-APB, but not to camphor or voltage. The cytoplasmic N-terminal mutation H426N in human, dog, and frog TRPV3 also effectively abolished 2-APB activation without affecting camphor responses. Interestingly, chicken TRPV3 is weakly sensitive to 2-APB, and the equivalent residue at 426 is an asparagine (N). Mutating this residue to histidine induced 2-APB sensitivity of chicken TRPV3 to levels comparable for other TRPV3 orthologs. The cytoplasmic C-terminal mutation R696K in the TRP box displayed 2-APB specific deficits only in the presence of extracellular calcium, suggesting involvement in gating. TRPV4, a related thermoTRP, is 2-APB insensitive and has variant sequences at both residues identified here. Remarkably, mutating these 2 residues in TRPV4 to TRPV3 sequences (N426H and W737R) was sufficient to induce TRPV3-like 2-APB sensitivity. Therefore, 2-APB activation of TRPV3 is separable from other activation mechanisms, and depends on 2 cytoplasmic residues.


Assuntos
Compostos de Boro/farmacologia , Canais de Cátion TRPV/efeitos dos fármacos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Linhagem Celular , Primers do DNA , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genética , Temperatura
16.
Nat Neurosci ; 11(9): 1007-13, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19160498

RESUMO

Ion channels can be activated (gated) by a variety of stimuli, including chemicals, voltage, mechanical force or temperature. Although molecular mechanisms of ion channel gating by chemical and voltage stimuli are understood in principal, the mechanisms of temperature activation remain unknown. The transient receptor potential channel TRPV3 is a nonselective cation channel that is activated by warm temperatures and sensory chemicals such as camphor. Here we screened approcimately 14,000 random mutant clones of mouse TRPV3 and identified five single point mutations that specifically abolish heat activation but do not perturb chemical activation or voltage modulation. Notably, all five mutations are located in the putative sixth transmembrane helix and the adjacent extracellular loop in the pore region of mouse TRPV3. Although distinct in sequence, we found that the corresponding loop of frog TRPV3 is also specifically required for heat activation. These findings demonstrate that the temperature sensitivity of TRPV3 is separable from all other known activation mechanisms and implicate a specific region in temperature sensing.


Assuntos
Temperatura Alta , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Canais de Cátion TRPV/química , Canais de Cátion TRPV/fisiologia , Animais , Biofísica , Compostos de Boro/farmacologia , Linhagem Celular Transformada , Clonagem Molecular , Estimulação Elétrica , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Modelos Moleculares , Mutação/fisiologia , Técnicas de Patch-Clamp , Canais de Cátion TRPV/genética , Transfecção/métodos , Xenopus/genética
17.
Mol Pain ; 3: 40, 2007 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-18086313

RESUMO

Mechanical hyperalgesia is a clinically-relevant form of pain sensitization that develops through largely unknown mechanisms. TRPA1, a Transient Receptor Potential ion channel, is a sensor of pungent chemicals that may play a role in acute noxious mechanosensation and cold thermosensation. We have developed a specific small molecule TRPA1 inhibitor (AP18) that can reduce cinnameldehyde-induced nociception in vivo. Interestingly, AP18 is capable of reversing CFA-induced mechanical hyperalgesia in mice. Although TRPA1-deficient mice develop normal CFA-induced hyperalgeisa, AP18 is ineffective in the knockout mice, consistent with an on-target mechanism. Therefore, TRPA1 plays a role in sensitization of nociception, and that compensation in TRPA1-deficient mice masks this requirement.


Assuntos
Hiperalgesia/metabolismo , Canais de Potencial de Receptor Transitório/fisiologia , Animais , Comportamento Animal/efeitos dos fármacos , Bradicinina/efeitos adversos , Células CHO , Sinalização do Cálcio/efeitos dos fármacos , Cricetinae , Cricetulus , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Humanos , Hiperalgesia/genética , Hiperalgesia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos , Medição da Dor/métodos , Ratos , Ratos Sprague-Dawley , Canal de Cátion TRPA1 , Transfecção/métodos , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/deficiência , Canais de Potencial de Receptor Transitório/genética , Xenopus
18.
J Neurosci ; 27(42): 11412-5, 2007 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-17942735

RESUMO

Tissue damage and its downstream consequences are experimentally assayed by formaldehyde application, which indiscriminately modifies proteins and is presumed to cause pain through broadly acting mechanisms. Here we show that formaldehyde activates the ion channel TRPA1 and that TRPA1-deficient mice exhibit dramatically reduced formaldehyde-induced pain responses. 4-Hydroxynonenal, a reactive chemical produced endogenously during oxidative stress, and other related aldehydes also activate TRPA1 in vitro. Furthermore, painful responses to iodoacetamide, a nonspecific cysteine-alkylating compound, are abolished in TRPA1-deficient mice. Therefore, although these reactive chemicals modify many proteins, the associated pain appears mainly dependent on a single ion channel.


Assuntos
Dor/induzido quimicamente , Dor/metabolismo , Canais de Potencial de Receptor Transitório/fisiologia , Aldeídos/toxicidade , Animais , Linhagem Celular , Formaldeído/toxicidade , Humanos , Iodoacetamida/toxicidade , Camundongos , Camundongos Knockout , Medição da Dor/métodos , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/deficiência , Canais de Potencial de Receptor Transitório/genética
19.
Neuron ; 54(3): 371-8, 2007 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-17481391

RESUMO

ThermoTRPs, a subset of the Transient Receptor Potential (TRP) family of cation channels, have been implicated in sensing temperature. TRPM8 and TRPA1 are both activated by cooling; however, it is unclear whether either ion channel is required for thermosensation in vivo. We show that mice lacking TRPM8 have severe behavioral deficits in response to cold stimuli. In thermotaxis assays of temperature gradient and two-temperature choice assays, TRPM8-deficient mice exhibit strikingly reduced avoidance of cold temperatures. TRPM8-deficient mice also lack behavioral response to cold-inducing icilin application and display an attenuated response to acetone, an unpleasant cold stimulus. However, TRPM8-deficient mice have normal nociceptive-like responses to subzero centigrade temperatures, suggesting the presence of at least one additional noxious cold receptor. Finally, we show that TRPM8 mediates the analgesic effect of moderate cooling after administration of formalin, a painful stimulus. Therefore, depending on context, TRPM8 contributes to sensing unpleasant cold stimuli or mediating the effects of cold analgesia.


Assuntos
Temperatura Baixa , Canais de Cátion TRPM/fisiologia , Sensação Térmica/fisiologia , Animais , Comportamento Animal/fisiologia , Cálcio/metabolismo , Comportamento de Escolha/efeitos dos fármacos , Comportamento de Escolha/fisiologia , Formaldeído/farmacologia , Camundongos , Camundongos Knockout , Medição da Dor/métodos , Pirimidinonas/farmacologia , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Limiar Sensorial/efeitos dos fármacos , Limiar Sensorial/fisiologia , Canais de Cátion TRPM/deficiência , Fatores de Tempo
20.
Nat Neurosci ; 9(4): 493-500, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16520735

RESUMO

Menthol is a cooling compound derived from mint leaves and is extensively used as a flavoring chemical. Menthol activates transient receptor potential melastatin 8 (TRPM8), an ion channel also activated by cold, voltage and phosphatidylinositol-4,5-bisphosphate (PIP2). Here we investigated the mechanism by which menthol activates mouse TRPM8. Using a new high-throughput approach, we screened a random mutant library consisting of approximately 14,000 individual TRPM8 mutants for clones that are affected in their response to menthol while retaining channel function. We identified determinants of menthol sensitivity in two regions: putative transmembrane segment 2 (S2) and the C-terminal TRP domain. Analysis of these mutants indicated that activation by menthol involves a gating mechanism distinct and separable from gating by cold, voltage or PIP2. Notably, TRP domain mutations mainly attenuated menthol efficacy, suggesting that this domain influences events downstream of initial binding. In contrast, S2 mutations strongly shifted the concentration dependence of menthol activation, raising the possibility that S2 influences menthol binding.


Assuntos
Temperatura Baixa , Mentol/metabolismo , Canais de Cátion TRPM/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Biblioteca Gênica , Mentol/química , Camundongos , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Pirimidinonas/metabolismo , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genética
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