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1.
J Physiol ; 597(17): 4661-4675, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31290157

RESUMO

KEY POINTS: HCN ion channels conducting the Ih current control the frequency of firing in peripheral sensory neurons signalling pain. Previous studies have demonstrated a major role for the HCN2 subunit in chronic pain but the potential involvement of HCN3 in pain has not been investigated. HCN3 was found to be widely expressed in all classes of sensory neurons (small, medium, large) where it contributes to Ih . HCN3 deletion increased the firing rate of medium but not small, sensory neurons. Pain sensitivity both acutely and following neuropathic injury was largely unaffected by HCN3 deletion, with the exception of a small decrease of mechanical hyperalgesia in response to a pinprick. We conclude that HCN3 plays little role in either acute or chronic pain sensation. ABSTRACT: HCN ion channels govern the firing rate of action potentials in the pacemaker region of the heart and in pain-sensitive (nociceptive) nerve fibres. Intracellular cAMP promotes activation of the HCN4 and HCN2 isoforms, whereas HCN1 and HCN3 are relatively insensitive to cAMP. HCN2 modulates action potential firing rate in nociceptive neurons and plays a critical role in all modes of inflammatory and neuropathic pain, although the role of HCN3 in nociceptive excitability and pain is less studied. Using antibody staining, we found that HCN3 is expressed in all classes of somatosensory neurons. In small nociceptive neurons, genetic deletion of HCN2 abolished the voltage shift of the Ih current carried by HCN isoforms following cAMP elevation, whereas the voltage shift was retained following deletion of HCN3, consistent with the sensitivity of HCN2 but not HCN3 to cAMP. Deletion of HCN3 had little effect on the evoked firing frequency in small neurons but enhanced the firing of medium-sized neurons, showing that HCN3 makes a significant contribution to the input resistance only in medium-sized neurons. Genetic deletion of HCN3 had no effect on acute thresholds to heat or mechanical stimuli in vivo and did not affect inflammatory pain measured with the formalin test. Nerve-injured HCN3 knockout mice exhibited similar levels of mechanical allodynia and thermal hyperalgesia to wild-type mice but reduced mechanical hyperalgesia in response to a pinprick. These results show that HCN3 makes some contribution to excitability, particularly in medium-sized neurons, although it has no major influence on acute or neuropathic pain processing.

2.
Pain ; 160(11): 2554-2565, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31188268

RESUMO

Preclinical studies suggest that type 2 hyperpolarization-activated cyclic nucleotide gated ion channels (HCN2) are necessary for neuropathic pain. This trial assessed the influence of ivabradine, a nonselective HCN channel blocker, on capsaicin-induced hyperalgesia and pain in healthy human subjects. An enriched population comprising subjects who developed >20 cm of punctate hyperalgesia from topical capsaicin (0.5% cream applied onto 9 cm area) was identified. These subjects then received ivabradine (15 mg) or placebo 1 hour before capsaicin application in randomly allocated order in a crossover study. The forearm site for capsaicin alternated with each application of the cream. The interval of time from screening to the first and to the second treatment visits was at least 3 and 5 weeks, respectively, to minimize carryover effects. Fifty-five participants were screened, of which 25 completed at least 1 treatment visit. Intention-to-treat hierarchical analysis revealed no significant effects of the drug on primary trial outcome, defined as a difference in effects of placebo and ivabradine on the area of punctate hyperalgesia (ivabradine - placebo: mean = 3.22 cm, 95% confidence interval: = -4.04 to 10.48, P = 0.37). However, ivabradine caused a slowing of heart rate (difference of 10.10 beats per minute [95% confidence interval -6.48 to -13.73; P-value <0.0001]). We conclude that ivabradine lacks analgesic effects in the capsaicin pain model at a dose that caused appreciable slowing of heart rate and, hence, is unlikely to prove a useful analgesic in humans. More selective drugs are required to establish a role of HCN2 for pain in humans.

4.
Pflugers Arch ; 470(5): 787-798, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29552700

RESUMO

The abilities to detect warmth and heat are critical for the survival of all animals, both in order to be able to identify suitable thermal environments for the many different activities essential for life and to avoid damage caused by extremes of temperature. Several ion channels belonging to the TRP family are activated by non-noxious warmth or by heat and are therefore plausible candidates for thermal detectors, but identifying those that actually regulate warmth and heat detection in intact animals has proven problematic. TRPM2 has recently emerged as a likely candidate for the detector of non-noxious warmth, as it is expressed in sensory neurons, and mice show deficits in the detection of warmth when TRPM2 is genetically deleted. TRPM2 is a chanzyme, containing a thermally activated TRP ion channel domain attached to a C-terminal motif, derived from a mitochondrial ADP ribose pyrophosphatase, that confers on the channel sensitivity to ADP ribose and reactive oxygen species such as hydrogen peroxide. Several open questions remain. Male mammals prefer cooler environments than female, but the molecular basis of this sex difference is unknown. TRPM2 plays a role in regulating body temperature, but are other warmth-detecting mechanisms also involved? TRPM2 is expressed in autonomic neurons, but does it confer a sensory function in addition to the well-known motor functions of autonomic neurons? TRPM2 is thought to play important roles in the immune system, in pain and in insulin secretion, but the mechanisms are unclear. TRPM2 has to date received less attention than many other members of the TRP family but is rapidly assuming importance both in normal physiology and as a key target in disease pathology.

5.
Sci Transl Med ; 9(409): eaam6072, 2017 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-28954930

RESUMO

Diabetic patients frequently suffer from continuous pain that is poorly treated by currently available analgesics. We used mouse models of type 1 and type 2 diabetes to investigate a possible role for the hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) ion channels as drivers of diabetic pain. Blocking or genetically deleting HCN2 channels in small nociceptive neurons suppressed diabetes-associated mechanical allodynia and prevented neuronal activation of second-order neurons in the spinal cord in mice. In addition, we found that intracellular cyclic adenosine monophosphate (cAMP), a positive HCN2 modulator, is increased in somatosensory neurons in an animal model of painful diabetes. We propose that the increased intracellular cAMP drives diabetes-associated pain by facilitating HCN2 activation and consequently promoting repetitive firing in primary nociceptive nerve fibers. Our results suggest that HCN2 may be an analgesic target in the treatment of painful diabetic neuropathy.


Assuntos
Neuropatias Diabéticas/complicações , Neuropatias Diabéticas/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Dor/complicações , Dor/metabolismo , Canais de Potássio/metabolismo , Analgésicos , Animais , Benzazepinas/farmacologia , Benzazepinas/uso terapêutico , AMP Cíclico/metabolismo , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/patologia , Modelos Animais de Doenças , Deleção de Genes , Hiperalgesia/complicações , Hiperalgesia/tratamento farmacológico , Ivabradina , Nociceptividade , Dor/tratamento farmacológico , Dor/patologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células Receptoras Sensoriais/metabolismo , Pele/inervação , Corno Dorsal da Medula Espinal/metabolismo , Estreptozocina
6.
PLoS One ; 12(1): e0170097, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28076424

RESUMO

The TRPA1 ion channel is expressed in nociceptive (pain-sensitive) somatosensory neurons and is activated by a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here, we investigate the enhancement of TRPA1 function caused by inflammatory mediators, which is thought to be important in lung conditions such as asthma and COPD. Protein kinase A is an important kinase acting downstream of inflammatory mediators to cause sensitization of TRPA1. By using site-directed mutagenesis, patch-clamp electrophysiology and calcium imaging we identify four amino acid residues, S86, S317, S428, and S972, as the principal targets of PKA-mediated phosphorylation and sensitization of TRPA1.


Assuntos
Canais de Cálcio/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Canais de Receptores Transientes de Potencial/fisiologia , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Monoterpenos/farmacologia , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Canal de Cátion TRPA1 , Transfecção , Canais de Receptores Transientes de Potencial/genética , Canais de Receptores Transientes de Potencial/metabolismo
7.
Biochem J ; 473(18): 2717-36, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27621481

RESUMO

Nociception - the ability to detect painful stimuli - is an invaluable sense that warns against present or imminent damage. In patients with chronic pain, however, this warning signal persists in the absence of any genuine threat and affects all aspects of everyday life. Neuropathic pain, a form of chronic pain caused by damage to sensory nerves themselves, is dishearteningly refractory to drugs that may work in other types of pain and is a major unmet medical need begging for novel analgesics. Hyperpolarisation-activated cyclic nucleotide (HCN)-modulated ion channels are best known for their fundamental pacemaker role in the heart; here, we review data demonstrating that the HCN2 isoform acts in an analogous way as a 'pacemaker for pain', in that its activity in nociceptive neurons is critical for the maintenance of electrical activity and for the sensation of chronic pain in pathological pain states. Pharmacological block or genetic deletion of HCN2 in sensory neurons provides robust pain relief in a variety of animal models of inflammatory and neuropathic pain, without any effect on normal sensation of acute pain. We discuss the implications of these findings for our understanding of neuropathic pain pathogenesis, and we outline possible future opportunities for the development of efficacious and safe pharmacotherapies in a range of chronic pain syndromes.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Neuralgia/metabolismo , Humanos
8.
Nature ; 536(7617): 460-3, 2016 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-27533035

RESUMO

Thermally activated ion channels are known to detect the entire thermal range from extreme heat (TRPV2), painful heat (TRPV1, TRPM3 and ANO1), non-painful warmth (TRPV3 and TRPV4) and non-painful coolness (TRPM8) through to painful cold (TRPA1). Genetic deletion of each of these ion channels, however, has only modest effects on thermal behaviour in mice, with the exception of TRPM8, the deletion of which has marked effects on the perception of moderate coolness in the range 10-25 °C. The molecular mechanism responsible for detecting non-painful warmth, in particular, is unresolved. Here we used calcium imaging to identify a population of thermally sensitive somatosensory neurons which do not express any of the known thermally activated TRP channels. We then used a combination of calcium imaging, electrophysiology and RNA sequencing to show that the ion channel generating heat sensitivity in these neurons is TRPM2. Autonomic neurons, usually thought of as exclusively motor, also express TRPM2 and respond directly to heat. Mice in which TRPM2 had been genetically deleted showed a striking deficit in their sensation of non-noxious warm temperatures, consistent with the idea that TRPM2 initiates a 'warm' signal which drives cool-seeking behaviour.


Assuntos
Temperatura Alta , Canais de Cátion TRPM/metabolismo , Animais , Aprendizagem da Esquiva , Cálcio/análise , Cálcio/metabolismo , Sinalização do Cálcio , Eletrofisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Análise de Sequência de RNA , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/genética
9.
J Physiol ; 594(22): 6643-6660, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27307078

RESUMO

KEY POINTS: The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in nociceptive neurons and its activation causes ongoing pain and inflammation; TRPA1 is thought to play an important role in inflammation in the airways. TRPA1 is sensitised by repeated stimulation with chemical agonists in a calcium-free environment and this sensitisation is very long lasting following agonist removal. We show that agonist-induced sensitisation is independent of the agonist's binding site and is also independent of ion channel trafficking or of other typical signalling pathways. We find that sensitisation is intrinsic to the TRPA1 protein and is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials. Agonist-induced sensitisation may provide an explanation for sensitisation following long-term exposure to harmful irritants and pollutants, particularly in the airways. ABSTRACT: The TRPA1 ion channel is expressed in nociceptive (pain-sensitive) neurons and responds to a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here we show that in the absence of extracellular calcium the current passing through TRPA1 gradually increases (sensitises) during prolonged application of agonists. Activation by an agonist is essential, because activation of TRPA1 by membrane depolarisation did not cause sensitisation. Sensitisation is independent of the site of action of the agonist, because covalent and non-covalent agonists were equally effective, and is long lasting following agonist removal. Mutating N-terminal cysteines, the target of covalent agonists, did not affect sensitisation by the non-covalent agonist carvacrol, which activates by binding to a different site. Sensitisation is unaffected by agents blocking ion channel trafficking or by block of signalling pathways involving ATP, protein kinase A or the formation of lipid rafts, and does not require ion flux through the channel. Examination of the voltage dependence of TRPA1 activation shows that sensitisation is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials, and is therefore intrinsic to the TRPA1 channel. Sensitisation may play a role in exacerbating the pain caused by prolonged activation of TRPA1.


Assuntos
Monoterpenos/farmacologia , Canais de Receptores Transientes de Potencial/agonistas , Canais de Receptores Transientes de Potencial/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Feminino , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transdução de Sinais/efeitos dos fármacos
10.
Pain ; 155(9): 1708-19, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24861581

RESUMO

Previous studies have shown that hyperpolarisation-activated cyclic nucleotide-gated (HCN)-2 ion channels regulate the firing frequency of nociceptive sensory neurons and thus play a central role in both inflammatory and neuropathic pain conditions. Here we use ivabradine, a clinically approved anti-anginal agent that blocks all HCN channel isoforms approximately equally, to investigate the effect on inflammatory and neuropathic pain of HCN ion channel block. We show that ivabradine does not have major off-target effects on a sample group of Na, Ca, and K ion channels, and that it is peripherally restricted because it is a substrate for the P-glycoprotein (PgP) multidrug transporter that is expressed in the blood-brain barrier. Its effects are therefore likely to be due to an action on HCN ion channels in peripheral sensory neurons. Using patch clamp electrophysiology, we found that ivabradine was a use-dependent blocker of native HCN channels expressed in small sensory neurons. Ivabradine suppressed the action potential firing that is induced in nociceptive neurons by elevation of intracellular cAMP. In the formalin model of inflammatory pain, ivabradine reduced pain behaviour only in the second (inflammatory) phase. In nerve injury and chemotherapy models of neuropathic pain, we observed rapid and effective analgesia as effective as that with gabapentin. We conclude that both inflammatory and neuropathic pain are rapidly inhibited by blocking HCN-dependent repetitive firing in peripheral nociceptive neurons.


Assuntos
Benzazepinas/uso terapêutico , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Inflamação/tratamento farmacológico , Neuralgia/tratamento farmacológico , Animais , Benzazepinas/farmacologia , Células Cultivadas , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Inflamação/metabolismo , Ivabradina , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Neuralgia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp
11.
Pharmacol Ther ; 143(3): 316-22, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24727631

RESUMO

Cellular responsiveness to external stimuli can be altered by extracellular mediators which activate membrane receptors, in turn signalling to the intracellular space via calcium, cyclic nucleotides, membrane lipids or enzyme activity. These signalling events trigger a cascade leading to an effector which can be a channel, an enzyme or a transcription factor. The effectiveness of these intracellular events is enhanced when they are maintained in close proximity by anchoring proteins, which assemble complexes of signalling molecules such as kinases together with their targets, and in this way enhance both the speed and the precision of intracellular signalling. The A kinase anchoring protein (AKAP) family are adaptor proteins originally named for their ability to associate Protein Kinase A and its targets, but several other enzymes bound by AKAPs have now been found and a wide variety of target structures has been described. This review provides an overview of anchoring proteins involved in pain signalling. The key anchoring proteins and their ion channel targets in primary sensory neurons responding to painful stimuli (nociceptors) are discussed.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Dor/metabolismo , Animais , Humanos , Transdução de Sinais
12.
Pflugers Arch ; 466(12): 2229-41, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24643480

RESUMO

Transient receptor potential cation channel, subfamily V, member 1 (TRPV1) plays a key role in sensing environmental hazards and in enhanced pain sensation following inflammation. A considerable proportion of TRPV1-expressing cells also express transient receptor potential cation channel, subfamily A, member 1 (TRPA1). There is evidence for a TRPV1-TRPA1 interaction that is predominantly calcium-dependent, and it has been suggested that the two proteins might form a heteromeric channel. Here, we constructed subunit concatemers to search for direct evidence for such an interaction. We found that a TRPV1::TRPV1 concatemer and TRPV1 formed channels with similar properties. A TRPV1::TRPA1 concatemer was responsive to TRPV1 agonists capsaicin, acidic pH and ethanol, but not to TRPA1 agonists. Isolated TRPV1 and TRPV1::TRPA1 imaged by atomic force microscopy (AFM) both had molecular volumes consistent with the formation of tetrameric channels. Antibodies decorated epitope tags on TRPV1 with a four-fold symmetry, as expected for a homotetramer. In contrast, pairs of antibodies decorated tags on TRPV1::TRPA1 predominantly at 180°, indicating the formation of a channel consisting of two TRPV1::TRPA1 concatemers arranged face to face. TRPV1::TRPA1 was sensitized by PKC activation and could be inhibited by a TRPV1 antagonist. TRPV1::TRPA1 was activated by heat and displayed a threshold and temperature coefficient similar to TRPV1. However, the channel formed by TRPV1::TRPA1 has only two binding sites for capsaicin and shows less total current and a smaller capsaicin-induced shift in voltage-dependent gating than TRPV1::TRPV1 or TRPV1. We conclude that the presence of TRPA1 exerts a functional inhibition on TRPV1.


Assuntos
Canais de Cálcio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica , Canais de Cátion TRPV/metabolismo , Canais de Receptores Transientes de Potencial/metabolismo , Sítios de Ligação , Canais de Cálcio/química , Capsaicina/farmacologia , Células HEK293 , Humanos , Ativação do Canal Iônico , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/química , Ligação Proteica , Canal de Cátion TRPA1 , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/química , Canais de Receptores Transientes de Potencial/agonistas , Canais de Receptores Transientes de Potencial/química
13.
J Neurosci ; 33(21): 9184-9193, 2013 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-23699529

RESUMO

Inflammation causes hyperalgesia, an enhanced sensitivity to noxious stimuli. Transient receptor potential vanilloid 1 (TRPV1), a thermo-TRP ion channel activated by painful levels of heat, is an important contributor because hyperalgesia is reduced when TRPV1 is either genetically deleted or pharmacologically blocked. Inflammatory mediators such as prostaglandin-E2 or bradykinin cause hyperalgesia by activating cellular kinases that phosphorylate TRPV1, a process that has recently been shown to rely on a scaffolding protein, AKAP79, to target the kinases to TRPV1. Here we use Förster resonance energy transfer, immunoprecipitation, and TRPV1 membrane trafficking experiments to identify a key region on AKAP79, between amino acids 326-336, which is responsible for its interaction with TRPV1. A peptide identical to this domain inhibited sensitization of TRPV1 in vitro, and when covalently linked to a TAT peptide to promote uptake across the cell membrane the peptide inhibited in vivo inflammatory hyperalgesia in mice. Critically, it did so without affecting pain thresholds in the absence of inflammation. These results suggest that antagonizing the TRPV1-AKAP79 interaction will be a useful strategy for inhibiting inflammatory hyperalgesia.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Hiperalgesia/metabolismo , Limiar da Dor/fisiologia , Canais de Cátion TRPV/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Cálcio/metabolismo , Capsaicina/farmacologia , Carragenina/toxicidade , Linhagem Celular Transformada , Cercopithecus aethiops , Feminino , Gânglios Espinais/citologia , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Imunoprecipitação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Neurônios , Limiar da Dor/efeitos dos fármacos , Técnicas de Patch-Clamp , Peptídeos/uso terapêutico , Canais de Cátion TRPV/genética , Transfecção
14.
J Neurosci ; 33(17): 7407-14, 2013 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-23616546

RESUMO

Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel that plays a key role in enhanced pain sensation after inflammation, but directly blocking TRPV1 causes hyperthermia and decreased sensitivity to painful levels of heat in animals and humans. Here we explore an alternative analgesic strategy in which the modulation of TRPV1 is inhibited by antagonizing the interaction between TRPV1 and A kinase anchoring protein 79 (AKAP79), a scaffolding protein essential for positioning serine-threonine kinases adjacent to target phosphorylation sites. We first defined key residues in the domain in TRPV1 that interacts with AKAP79, and we then used this information to construct short peptides capable of preventing TRPV1-AKAP79 interaction. An effective peptide, when coupled to a TAT sequence conferring cell permeability, was found to be analgesic in three mouse models of inflammatory hyperalgesia. These results demonstrate the potential value of interfering with the interaction between TRPV1 and AKAP79 as a novel analgesic strategy.


Assuntos
Hiperalgesia/metabolismo , Hiperalgesia/prevenção & controle , Mediadores da Inflamação/fisiologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/fisiologia , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Feminino , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Hiperalgesia/genética , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Ligação Proteica , Canais de Cátion TRPV/genética
15.
Nat Cell Biol ; 14(8): 851-8, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22750945

RESUMO

Activation of the TRPM8 ion channel in sensory nerve endings produces a sensation of pleasant coolness. Here we show that inflammatory mediators such as bradykinin and histamine inhibit TRPM8 in intact sensory nerves, but do not do so through conventional signalling pathways. The G-protein subunit Gα(q) instead binds to TRPM8 and when activated by a Gq-coupled receptor directly inhibits ion channel activity. Deletion of Gα(q) largely abolished inhibition of TRPM8, and inhibition was rescued by a Gα(q) chimaera whose ability to activate downstream signalling pathways was completely ablated. Activated Gα(q) protein, but not Gßγ, potently inhibits TRPM8 in excised patches. We conclude that Gα(q) pre-forms a complex with TRPM8 and inhibits activation of TRPM8, following activation of G-protein-coupled receptors, by a direct action. This signalling mechanism may underlie the abnormal cold sensation caused by inflammation.


Assuntos
Temperatura Baixa , Neurônios/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Animais , Células Cultivadas , Cristalografia por Raios X , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neurônios/citologia , Ligação Proteica , Transdução de Sinais
16.
Proc Natl Acad Sci U S A ; 109(30): 12022-7, 2012 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-22778440

RESUMO

Over the past 50 y, behavioral experiments have produced a large body of evidence for the existence of a magnetic sense in a wide range of animals. However, the underlying sensory physiology remains poorly understood due to the elusiveness of the magnetosensory structures. Here we present an effective method for isolating and characterizing potential magnetite-based magnetoreceptor cells. In essence, a rotating magnetic field is employed to visually identify, within a dissociated tissue preparation, cells that contain magnetic material by their rotational behavior. As a tissue of choice, we selected trout olfactory epithelium that has been previously suggested to host candidate magnetoreceptor cells. We were able to reproducibly detect magnetic cells and to determine their magnetic dipole moment. The obtained values (4 to 100 fAm(2)) greatly exceed previous estimates (0.5 fAm(2)). The magnetism of the cells is due to a µm-sized intracellular structure of iron-rich crystals, most likely single-domain magnetite. In confocal reflectance imaging, these produce bright reflective spots close to the cell membrane. The magnetic inclusions are found to be firmly coupled to the cell membrane, enabling a direct transduction of mechanical stress produced by magnetic torque acting on the cellular dipole in situ. Our results show that the magnetically identified cells clearly meet the physical requirements for a magnetoreceptor capable of rapidly detecting small changes in the external magnetic field. This would also explain interference of ac powerline magnetic fields with magnetoreception, as reported in cattle.


Assuntos
Campos Eletromagnéticos , Óxido Ferroso-Férrico/metabolismo , Magnetismo , Mucosa Olfatória/metabolismo , Células Receptoras Sensoriais/fisiologia , Truta , Migração Animal/fisiologia , Animais , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Microscopia Eletrônica de Varredura , Modelos Biológicos , Orientação/fisiologia , Células Receptoras Sensoriais/ultraestrutura , Especificidade da Espécie
17.
Trends Pharmacol Sci ; 33(8): 456-63, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22613784

RESUMO

Acute nociceptive pain is caused by the direct action of a noxious stimulus on pain-sensitive nerve endings, whereas inflammatory pain (both acute and chronic) arises from the actions of a wide range of inflammatory mediators released following tissue injury. Neuropathic pain, which is triggered by nerve damage, is often considered to be very different in its origins, and is particularly difficult to treat effectively. Here we review recent evidence showing that members of the hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channel family - better known for their role in the pacemaker potential of the heart - play important roles in both inflammatory and neuropathic pain. Deletion of the HCN2 isoform from nociceptive neurons abolishes heat-evoked inflammatory pain and all aspects of neuropathic pain, but acute pain sensation is unaffected. This work shows that inflammatory and neuropathic pain have much in common, and suggests that selective blockers of HCN2 may have value as analgesics in the treatment of pain.


Assuntos
Dor Aguda/metabolismo , Canais Iônicos/metabolismo , Neuralgia/metabolismo , Dor Nociceptiva/metabolismo , Potenciais de Ação , Dor Aguda/etiologia , Animais , AMP Cíclico/metabolismo , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Inflamação/metabolismo , Neuralgia/etiologia , Dor Nociceptiva/etiologia , Canais de Potássio , Prostaglandinas E/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Células Receptoras Sensoriais/metabolismo
18.
Br J Pharmacol ; 165(4): 787-801, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21797839

RESUMO

The thermo-transient receptor potentials (TRPs), a recently discovered family of ion channels activated by temperature, are expressed in primary sensory nerve terminals where they provide information about thermal changes in the environment. Six thermo-TRPs have been characterised to date: TRP vanilloid (TRPV) 1 and 2 are activated by painful levels of heat, TRPV3 and 4 respond to non-painful warmth, TRP melastatin 8 is activated by non-painful cool temperatures, while TRP ankyrin (TRPA) 1 is activated by painful cold. The thermal thresholds of many thermo-TRPs are known to be modulated by extracellular mediators, released by tissue damage or inflammation, such as bradykinin, PG and growth factors. There have been intensive efforts recently to develop antagonists of thermo-TRP channels, particularly of the noxious thermal sensors TRPV1 and TRPA1. Blockers of these channels are likely to have therapeutic uses as novel analgesics, but may also cause unacceptable side effects. Controlling the modulation of thermo-TRPs by inflammatory mediators may be a useful alternative strategy in developing novel analgesics.


Assuntos
Canais de Receptores Transientes de Potencial/fisiologia , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Dor/fisiopatologia , Receptores Acoplados a Proteínas-G/fisiologia , Temperatura Ambiente , Canais de Receptores Transientes de Potencial/agonistas , Canais de Receptores Transientes de Potencial/antagonistas & inibidores
19.
Science ; 333(6048): 1462-6, 2011 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-21903816

RESUMO

The rate of action potential firing in nociceptors is a major determinant of the intensity of pain. Possible modulators of action potential firing include the HCN ion channels, which generate an inward current, I(h), after hyperpolarization of the membrane. We found that genetic deletion of HCN2 removed the cyclic adenosine monophosphate (cAMP)-sensitive component of I(h) and abolished action potential firing caused by an elevation of cAMP in nociceptors. Mice in which HCN2 was specifically deleted in nociceptors expressing Na(V)1.8 had normal pain thresholds, but inflammation did not cause hyperalgesia to heat stimuli. After a nerve lesion, these mice showed no neuropathic pain in response to thermal or mechanical stimuli. Neuropathic pain is therefore initiated by HCN2-driven action potential firing in Na(V)1.8-expressing nociceptors.


Assuntos
Inflamação/fisiopatologia , Canais Iônicos/metabolismo , Neuralgia/fisiopatologia , Nociceptores/fisiologia , Dor/fisiopatologia , Potenciais de Ação , Animais , Temperatura Baixa , AMP Cíclico/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Temperatura Alta , Hiperalgesia/fisiopatologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/genética , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.8 , Limiar da Dor , Técnicas de Patch-Clamp , Canais de Potássio , Canais de Sódio/metabolismo
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