RESUMEN
The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole-genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]), significantly increased brood size and ovulation rate, as well as alleviating the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Expression of GEX-3 in the soma is required to rescue the brood size defects in pezo-1(R2405P) animals. Actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the PIEZO coordinates with the cytoskeleton regulator to maintain the F-actin network and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.
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Actinas , Artrogriposis , Oftalmoplejía , Enfermedades de la Retina , Animales , Femenino , Humanos , Actinas/genética , Artrogriposis/genética , Caenorhabditis elegans/genética , Canales Iónicos , Mutación/genética , PolimerizacionRESUMEN
The sense of touch is crucial for cognitive, emotional, and social development and relies on mechanically activated (MA) ion channels that transduce force into an electrical signal. Despite advances in the molecular characterization of these channels, the physiological factors that control their activity are poorly understood. Here, we used behavioral assays, electrophysiological recordings, and various mouse strains (males and females analyzed separately) to investigate the role of the calmodulin-like Ca2+ sensor, caldendrin, as a key regulator of MA channels and their roles in touch sensation. In mice lacking caldendrin (Cabp1 KO), heightened responses to tactile stimuli correlate with enlarged MA currents with lower mechanical thresholds in dorsal root ganglion neurons (DRGNs). The expression pattern of caldendrin in the DRG parallels that of the major MA channel required for touch sensation, PIEZO2. In transfected cells, caldendrin interacts with and inhibits the activity of PIEZO2 in a manner that requires an alternatively spliced sequence in the N-terminal domain of caldendrin. Moreover, targeted genetic deletion of caldendrin in Piezo2-expressing DRGNs phenocopies the tactile hypersensitivity of complete Cabp1 KO mice. We conclude that caldendrin is an endogenous repressor of PIEZO2 channels and their contributions to touch sensation in DRGNs.
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Canales Iónicos , Tacto , Animales , Femenino , Masculino , Ratones , Canales Iónicos/genética , Mecanotransducción Celular/fisiología , Neuronas/metabolismo , Tacto/fisiologíaRESUMEN
The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]) , significantly restored the small brood size and low ovulation rate, as well as alleviated the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Auxin-inducible degradation of GEX-3 revealed that only somatic-specific degradation of GEX-3 restored the reduced brood size in the pezo-1(R2405P) mutants. Additionally, actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the cytoskeleton plays an important role in regulating PIEZO channel activity and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.
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Angelman syndrome (AS) is a neurogenetic disorder characterized by intellectual disability and atypical behaviors. AS results from loss of expression of the E3 ubiquitin-protein ligase UBE3A from the maternal allele in neurons. Individuals with AS display impaired coordination, poor balance, and gait ataxia. PIEZO2 is a mechanosensitive ion channel essential for coordination and balance. Here, we report that PIEZO2 activity is reduced in Ube3a deficient male and female mouse sensory neurons, a human Merkel cell carcinoma cell line and female human iPSC-derived sensory neurons with UBE3A knock-down, and de-identified stem cell-derived neurons from individuals with AS. We find that loss of UBE3A decreases actin filaments and reduces PIEZO2 expression and function. A linoleic acid (LA)-enriched diet increases PIEZO2 activity, mechano-excitability, and improves gait in male AS mice. Finally, LA supplementation increases PIEZO2 function in stem cell-derived neurons from individuals with AS. We propose a mechanism whereby loss of UBE3A expression reduces PIEZO2 function and identified a fatty acid that enhances channel activity and ameliorates AS-associated mechano-sensory deficits.
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Síndrome de Angelman , Canales Iónicos , Ácido Linoleico , Animales , Femenino , Humanos , Masculino , Ratones , Alelos , Síndrome de Angelman/tratamiento farmacológico , Síndrome de Angelman/genética , Modelos Animales de Enfermedad , Discapacidad Intelectual , Canales Iónicos/genética , Ácido Linoleico/farmacologíaRESUMEN
This Viewpoint, which accompanies a Special Issue focusing on membrane mechanosensors, discusses unifying and unique features of both established and emerging mechanosensitive (MS) membrane proteins, their distribution across protein families and phyla, and current and future challenges in the study of these important proteins and their partners. MS membrane proteins are essential for tissue development, cellular motion, osmotic homeostasis, and sensing external and self-generated mechanical cues like those responsible for touch and proprioception. Though researchers' attention and this Viewpoint focus on a few famous ion channels that are considered the usual suspects as MS mechanosensors, we also discuss some of the more unusual suspects, such as G-protein coupled receptors. As the field continues to grow, so too will the list of proteins suspected to function as mechanosensors and the diversity of known MS membrane proteins.
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Canales Iónicos , Proteínas de la Membrana , Canales Iónicos/metabolismo , Proteínas de la Membrana/metabolismo , Mecanotransducción Celular/fisiologíaRESUMEN
Distal arthrogryposis (DA) is a collection of rare disorders that are characterized by congenital joint contractures. Most DA mutations are in muscle- and joint-related genes, and the anatomical defects originate cell-autonomously within the musculoskeletal system. However, gain-of-function mutations in PIEZO2, a principal mechanosensor in somatosensation, cause DA subtype 5 (DA5) through unknown mechanisms. We show that expression of a gain-of-function PIEZO2 mutation in proprioceptive sensory neurons that mainly innervate muscle spindles and tendons is sufficient to induce DA5-like phenotypes in mice. Overactive PIEZO2 causes anatomical defects through increased activity within the peripheral nervous system during postnatal development. Furthermore, botulinum toxin (Botox) and a dietary fatty acid that modulates PIEZO2 activity reduce DA5-like deficits. This reveals a role for somatosensory neurons: Excessive mechanosensation within these neurons disrupts musculoskeletal development.
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Artrogriposis , Contractura , Canales Iónicos , Mecanotransducción Celular , Células Receptoras Sensoriales , Animales , Ratones , Artrogriposis/genética , Artrogriposis/fisiopatología , Contractura/genética , Contractura/fisiopatología , Mecanotransducción Celular/genética , Mutación , Células Receptoras Sensoriales/fisiología , Canales Iónicos/genéticaRESUMEN
TRPV4 channel activation in endothelial cells leads to vasodilation, while impairment of TRPV4 activity is implicated in vascular dysfunction. Strategies that increase TRPV4 activity could enhance vasodilation and ameliorate vascular disorders. Here, we show that supplementation with eicosapentaenoic acid (EPA), an ω-3 polyunsaturated fatty acid known to have beneficial cardiovascular effects, increases TRPV4 activity in human endothelial cells of various vascular beds. Mice carrying the C. elegans FAT-1 enzyme, which converts ω-6 to ω-3 polyunsaturated fatty acids, display higher EPA content and increased TRPV4-mediated vasodilation in mesenteric arteries. Likewise, mice fed an EPA-enriched diet exhibit enhanced and prolonged TRPV4-dependent vasodilation in an endothelial cell-specific manner. We also show that EPA supplementation reduces TRPV4 desensitization, which contributes to the prolonged vasodilation. Neutralization of positive charges in the TRPV4 N terminus impairs the effect of EPA on channel desensitization. These findings highlight the beneficial effects of manipulating fatty acid content to enhance TRPV4-mediated vasodilation.
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Ácidos Grasos Omega-3 , Vasodilatación , Animales , Caenorhabditis elegans , Dieta , Células Endoteliales , Ácidos Grasos Omega-3/farmacología , Humanos , Ratones , Canales Catiónicos TRPV/genéticaRESUMEN
Intestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6-/- mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.
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Endotoxemia , Etanol , Histidina , Mucosa Intestinal , Canales Catiónicos TRPV , Acetaldehído/toxicidad , Animales , Células CACO-2 , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Etanol/toxicidad , Histidina/farmacología , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Ratones , Canales Catiónicos TRPV/efectos de los fármacos , Canales Catiónicos TRPV/metabolismoRESUMEN
Purpose: The aim of this study is to evaluate the oncological care during the first state of national emergency due to the COVID-19 pandemic in several public cancer hospitals in Peru. Materials and Methods: A multicentric cross-sectional descriptive study was conducted by interviewing adult cancer patients diagnosed and treated between January 2019 and February 2020 from 18 hospitals. This study was carried out in September 2020, the last month of the first state of national emergency. Demographic and clinical characteristics were evaluated, including COVID-19 status and cancer treatment features. Results: A total of 1472 patients were included; the median age was 55 years (range 19-97). Most patients (85.8%, n = 1263) had solid neoplasia, 13.5% (n = 198) hematologic neoplasia, and 0.7% (n = 11) others. SARS-CoV-2 infection was confirmed in 8.6% (n = 126), 1.2% (n = 18) were probable, 1.6% (n = 24) suspected, and 88.6% (n = 1304) negative cases. Overall, 51.6% of patients (n = 759) had cancer treatment delays, 42.5% (n = 626) changed treatment delivery (endovenous to oral systemic therapy), and 12.6% (n = 185) of cases cancer therapy was discontinued. In total, 10.3% (n = 117) of patients whose disease was controlled or in remission, experienced progression of disease during the state of emergency. A total of 6.7% (n = 98) of patients died, of whom 73.5% (n = 72) died from disease progression; 18.4% (n = 18) from SARS-CoV-2 infection and 8.1% (n = 8) from undetermined causes. Patients with hematological malignancies [hazard ratio (HR): 5.11 (95% confidence interval (CI): 1.99-13.07)] and no response to therapy before the onset of the pandemic [5.01 (1.44-17.42)] had an increased risk of death among COVID-19 infected individuals, whereas advanced clinical stage [5.09 (2.37-10.95)] and discontinuation of treatment [3.66 (1.97-6.78)] were risk factors among non-COVID-19 patients. Conclusion: Our study suggests that the COVID-19 pandemic has an adverse impact on the outcomes of Peruvian cancer patients. In our cohort, cancer mortality was higher than COVID-19 disease mortality.
RESUMEN
The seventh session of the Oncological Pathology Conference (JoPaO) entitled 'Pathological Anatomy in the context of the National Cancer Law: An overview of the Latin American experience', was held virtually on July 15, 22 and 23. Peru was the headquarters for this event, where 17 national and international professors of high academic standing participated. They interacted in a multidisciplinary context through talks with national panellists and the general public. The recent promulgation of the 'National Cancer Law' fosters the development of discussion forums to analyse the national realities and uphold continuous learning about experiences in other Latin American countries with successful cancer programmes, in which pathology holds a principal role. The topics addressed during this JoPaO included the exchange of Latin American cancer management experiences, an emphasis on investments in and the development of strategic plans to improve care, the use of new technologies, laboratory quality control, and the need to advance scientific research.
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PIEZO channels are force sensors essential for physiological processes, including baroreception and proprioception. The Caenorhabditis elegans genome encodes an orthologue gene of the Piezo family, pezo-1, which is expressed in several tissues, including the pharynx. This myogenic pump is an essential component of the C. elegans alimentary canal, whose contraction and relaxation are modulated by mechanical stimulation elicited by food content. Whether pezo-1 encodes a mechanosensitive ion channel and contributes to pharyngeal function remains unknown. Here, we leverage genome editing, genetics, microfluidics, and electropharyngeogram recording to establish that pezo-1 is expressed in the pharynx, including in a proprioceptive-like neuron, and regulates pharyngeal function. Knockout (KO) and gain-of-function (GOF) mutants reveal that pezo-1 is involved in fine-tuning pharyngeal pumping frequency, as well as sensing osmolarity and food mechanical properties. Using pressure-clamp experiments in primary C. elegans embryo cultures, we determine that pezo-1 KO cells do not display mechanosensitive currents, whereas cells expressing wild-type or GOF PEZO-1 exhibit mechanosensitivity. Moreover, infecting the Spodoptera frugiperda cell line with a baculovirus containing the G-isoform of pezo-1 (among the longest isoforms) demonstrates that pezo-1 encodes a mechanosensitive channel. Our findings reveal that pezo-1 is a mechanosensitive ion channel that regulates food sensation in worms.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Canales Iónicos/genética , Neuronas/metabolismo , SensaciónRESUMEN
Membrane remodeling by inflammatory mediators influences the function of sensory ion channels. The capsaicin- and heat-activated transient receptor potential vanilloid 1 (TRPV1) channel contributes to neurogenic inflammation and pain hypersensitivity, in part because of its potentiation downstream of phospholipase C-coupled receptors that regulate phosphoinositide lipid content. Here, we determined the effect of phosphoinositide lipids on TRPV1 function by combining genetic dissection, diet supplementation, and behavioral, biochemical, and functional analyses in Caenorhabditis elegans As capsaicin elicits heat and pain sensations in mammals, transgenic TRPV1 worms exhibit an aversive response to capsaicin. TRPV1 worms with low levels of phosphoinositide lipids display an enhanced response to capsaicin, whereas phosphoinositide lipid supplementation reduces TRPV1-mediated responses. A worm carrying a TRPV1 construct lacking the distal C-terminal domain features an enhanced response to capsaicin, independent of the phosphoinositide lipid content. Our results demonstrate that TRPV1 activity is enhanced when the phosphoinositide lipid content is reduced, and the C-terminal domain is key to determining agonist response in vivo.
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Caenorhabditis elegans/fisiología , Metabolismo de los Lípidos , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/deficiencia , Canales Catiónicos TRPV/fisiología , Animales , Conducta Animal , Proteínas de Caenorhabditis elegans/biosíntesis , Señalización del Calcio/efectos de los fármacos , Capsaicina/farmacología , Dieta , Suplementos Dietéticos , Células HEK293 , Humanos , Neuronas/metabolismo , Fosfatidilinositoles/farmacología , Canales Catiónicos TRPV/genéticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
PIEZO2 is the essential transduction channel for touch discrimination, vibration, and proprioception. Mice and humans lacking Piezo2 experience severe mechanosensory and proprioceptive deficits and fail to develop tactile allodynia. Bradykinin, a proalgesic agent released during inflammation, potentiates PIEZO2 activity. Molecules that decrease PIEZO2 function could reduce heightened touch responses during inflammation. Here, we find that the dietary fatty acid margaric acid (MA) decreases PIEZO2 function in a dose-dependent manner. Chimera analyses demonstrate that the PIEZO2 beam is a key region tuning MA-mediated channel inhibition. MA reduces neuronal action potential firing elicited by mechanical stimuli in mice and rat neurons and counteracts PIEZO2 sensitization by bradykinin. Finally, we demonstrate that this saturated fatty acid decreases PIEZO2 currents in touch neurons derived from human induced pluripotent stem cells. Our findings report on a natural product that inhibits PIEZO2 function and counteracts neuronal mechanical sensitization and reveal a key region for channel inhibition.
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Ácidos Grasos/administración & dosificación , Canales Iónicos/antagonistas & inhibidores , Mecanotransducción Celular/efectos de los fármacos , Neuronas/efectos de los fármacos , Propiocepción/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Algoritmos , Animales , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/fisiología , Canales Iónicos/genética , Canales Iónicos/metabolismo , Mecanotransducción Celular/genética , Mecanotransducción Celular/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Neuronas/fisiología , Propiocepción/genética , Propiocepción/fisiología , Ratas , Tacto/efectos de los fármacos , Tacto/fisiologíaRESUMEN
Caenorhabditis elegans is a powerful animal model in which transgenesis, behavior, and physiology can be merged to study in vivo the effect of natural and synthetic agonists in sensory ion channels. Worms have polymodal sensory neurons (like the ASH pair) that couple ion channel activation with a robust and easily scorable aversive-like behavior. We expressed the transient receptor potential vanilloid 1 (TRPV1) channel from rat (r) in worms' ASH neurons and determined its sensitivity to the tarantula double-knot toxin (DkTx) and the active component of chili peppers (capsaicin). This chapter describes protocols for generating and maintaining transgenic rTRPV1 worms to determine dose-dependent behavior. The goal is to provide an efficient tool to characterize the function of sensory channels (wild type and mutants) in vivo.
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Caenorhabditis elegans/metabolismo , Animales , Capsaicina/farmacología , Genotipo , Canales Iónicos/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Venenos de Araña/farmacología , Canales Catiónicos TRPV/metabolismoRESUMEN
TRP channels of the transient receptor potential ion channel superfamily are involved in a wide variety of mechanosensory processes, including touch sensation, pain, blood pressure regulation, bone loading and detection of cerebrospinal fluid flow. However, in many instances it is unclear whether TRP channels are the primary transducers of mechanical force in these processes. In this study, we tested stretch activation of eleven TRP channels from six mammalian subfamilies. We found that these TRP channels were insensitive to short membrane stretches in cellular systems. Furthermore, we purified TRPC6 and demonstrated its insensitivity to stretch in liposomes, an artificial bilayer system free from cellular components. Additionally, we demonstrated that, when expressed in C. elegans neurons, mouse TRPC6 restores the mechanoresponse of a touch insensitive mutant but requires diacylglycerol for activation. These results strongly suggest that the mammalian members of the TRP ion channel family are insensitive to tension induced by cell membrane stretching and, thus, are more likely to be activated by cytoplasmic tethers or downstream components and to act as amplifiers of cellular mechanosensory signaling cascades.
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Canal Catiónico TRPC6/química , Animales , Células CHO , Caenorhabditis elegans/metabolismo , Cricetulus , Electrofisiología , Células HEK293 , Células HeLa , Humanos , Mecanotransducción Celular/fisiología , Neuronas/metabolismo , Proteolípidos/químicaRESUMEN
Mechanosensitive ion channels rely on membrane composition to transduce physical stimuli into electrical signals. The Piezo1 channel mediates mechanoelectrical transduction and regulates crucial physiological processes, including vascular architecture and remodeling, cell migration, and erythrocyte volume. The identity of the membrane components that modulate Piezo1 function remain largely unknown. Using lipid profiling analyses, we here identify dietary fatty acids that tune Piezo1 mechanical response. We find that margaric acid, a saturated fatty acid present in dairy products and fish, inhibits Piezo1 activation and polyunsaturated fatty acids (PUFAs), present in fish oils, modulate channel inactivation. Force measurements reveal that margaric acid increases membrane bending stiffness, whereas PUFAs decrease it. We use fatty acid supplementation to abrogate the phenotype of gain-of-function Piezo1 mutations causing human dehydrated hereditary stomatocytosis. Beyond Piezo1, our findings demonstrate that cell-intrinsic lipid profile and changes in the fatty acid metabolism can dictate the cell's response to mechanical cues.
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Anemia Hemolítica Congénita/dietoterapia , Grasas de la Dieta/metabolismo , Hidropesía Fetal/dietoterapia , Activación del Canal Iónico/fisiología , Canales Iónicos/metabolismo , Anemia Hemolítica Congénita/genética , Animales , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Grasas de la Dieta/administración & dosificación , Ácidos Grasos/metabolismo , Ácidos Grasos Insaturados/administración & dosificación , Ácidos Grasos Insaturados/metabolismo , Mutación con Ganancia de Función , Células HEK293 , Humanos , Hidropesía Fetal/genética , Canales Iónicos/genética , Metabolismo de los Lípidos/fisiología , Ratones , Microscopía de Fuerza Atómica , Técnicas de Placa-ClampRESUMEN
Many neurotoxins inflict pain by targeting receptors expressed on nociceptors, such as the polymodal cationic channel TRPV1. The tarantula double-knot toxin (DkTx) is a peptide with an atypical bivalent structure, providing it with the unique capability to lock TRPV1 in its open state and evoke an irreversible channel activation. Here, we describe a distinct gating mechanism of DkTx-evoked TRPV1 activation. Interestingly, DkTx evokes significantly smaller TRPV1 macroscopic currents than capsaicin, with a significantly lower unitary conductance. Accordingly, while capsaicin evokes aversive behaviors in TRPV1-transgenic Caenorhabditis elegans, DkTx fails to evoke such response at physiological concentrations. To determine the structural feature(s) responsible for this phenomenon, we engineered and evaluated a series of mutated toxins and TRPV1 channels. We found that elongating the DkTx linker, which connects its two knots, increases channel conductance compared with currents elicited by the native toxin. Importantly, deletion of the TRPV1 pore turret, a stretch of amino acids protruding out of the channel's outer pore region, is sufficient to produce both full conductance and aversive behaviors in response to DkTx. Interestingly, this deletion decreases the capsaicin-evoked channel activation. Taken together with structure modeling analysis, our results demonstrate that the TRPV1 pore turret restricts DkTx-mediated pore opening, probably through steric hindrance, limiting the current size and mitigating the evoked downstream physiological response. Overall, our findings reveal that DkTx and capsaicin elicit distinct TRPV1 gating mechanisms and subsequent pain responses. Our results also indicate that the TRPV1 pore turret regulates the mechanisms of channel gating and permeation.
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Capsaicina/toxicidad , Neurotoxinas/toxicidad , Canales Catiónicos TRPV/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neurotoxinas/química , Neurotoxinas/genética , Técnicas de Placa-Clamp , Venenos de Araña/toxicidad , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/genéticaRESUMEN
Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.
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Análisis Espectral/métodos , Canales Catiónicos TRPV/genética , Células HEK293 , Humanos , Canales Catiónicos TRPV/metabolismoRESUMEN
Membrane lipid composition and remodeling influence the function of ion channels. Polyunsaturated fatty acids (PUFAs) and their derivatives modulate ion channel function; whether this effect occurs directly by binding to the protein or indirectly through alteration of membranes' mechanical properties has been difficult to distinguish. There are a large number of studies addressing the effect of fatty acids; recent structural and functional analyses have identified binding sites and provided further evidence for the role of the plasma membrane in ion channel function. Here, we review cation channels that do not share a common topology or lipid-binding signature sequence, but for which there are recent compelling data that support both direct and indirect modulation by PUFAs or their derivatives.