RESUMEN
Endothelial cells (ECs) line the wall of blood vessels and regulate arterial contractility to tune regional organ blood flow and systemic pressure. Chloride (Cl-) is the most abundant anion in ECs and the Cl- sensitive With-No-Lysine (WNK) kinase is expressed in this cell type. Whether intracellular Cl- signaling and WNK kinase regulate EC function to alter arterial contractility is unclear. Here, we tested the hypothesis that intracellular Cl- signaling in ECs regulates arterial contractility and examined the signaling mechanisms involved, including the participation of WNK kinase. Our data obtained using two-photon microscopy and cell-specific inducible knockout mice indicated that acetylcholine, a prototypical vasodilator, stimulated a rapid reduction in intracellular Cl- concentration ([Cl-]i) due to the activation of TMEM16A, a Cl- channel, in ECs of resistance-size arteries. TMEM16A channel-mediated Cl- signaling activated WNK kinase, which phosphorylated its substrate proteins SPAK and OSR1 in ECs. OSR1 potentiated transient receptor potential vanilloid 4 (TRPV4) currents in a kinase-dependent manner and required a conserved binding motif located in the channel C terminus. Intracellular Ca2+ signaling was measured in four dimensions in ECs using a high-speed lightsheet microscope. WNK kinase-dependent activation of TRPV4 channels increased local intracellular Ca2+ signaling in ECs and produced vasodilation. In summary, we show that TMEM16A channel activation reduces [Cl-]i, which activates WNK kinase in ECs. WNK kinase phosphorylates OSR1 which then stimulates TRPV4 channels to produce vasodilation. Thus, TMEM16A channels regulate intracellular Cl- signaling and WNK kinase activity in ECs to control arterial contractility.
Asunto(s)
Cloruros , Proteínas Serina-Treonina Quinasas , Ratones , Animales , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Cloruros/metabolismo , Células Endoteliales/metabolismo , Canales Catiónicos TRPV/metabolismo , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
Canal Catiónico TRPC6/química , Animales , Células CHO , Caenorhabditis elegans/metabolismo , Cricetulus , Electrofisiología , Células HEK293 , Células HeLa , Humanos , Mecanotransducción Celular/fisiología , Neuronas/metabolismo , Proteolípidos/químicaRESUMEN
The kidney maintains the internal milieu by regulating the retention and excretion of proteins, ions, and small molecules. The glomerular podocyte forms the slit diaphragm of the ultrafiltration filter, whose damage leads to progressive kidney failure and focal segmental glomerulosclerosis (FSGS). The canonical transient receptor potential 6 (TRPC6) ion channel is expressed in the podocyte, and mutations in its cytoplasmic domain cause FSGS in humans. In vitro evaluation of disease-causing mutations in TRPC6 has revealed that these genetic alterations result in abnormal ion channel gating. However, the mechanism whereby the cytoplasmic domain modulates TRPC6 function is largely unknown. Here, we report a cryo-EM structure of the cytoplasmic domain of murine TRPC6 at 3.8 Å resolution. The cytoplasmic fold of TRPC6 is characterized by an inverted dome-like chamber pierced by four radial horizontal helices that converge into a vertical coiled-coil at the central axis. Unlike other TRP channels, TRPC6 displays a unique domain swap that occurs at the junction of the horizontal helices and coiled-coil. Multiple FSGS mutations converge at the buried interface between the vertical coiled-coil and the ankyrin repeats, which form the dome, suggesting these regions are critical for allosteric gating modulation. This functionally critical interface is a potential target for drug design. Importantly, dysfunction in other family members leads to learning deficits (TRPC1/4/5) and ataxia (TRPC3). Our data provide a structural framework for the mechanistic investigation of the TRPC family.
Asunto(s)
Microscopía por Crioelectrón , Citoplasma/metabolismo , Canal Catiónico TRPC6/química , Canal Catiónico TRPC6/metabolismo , Animales , Células HEK293 , Humanos , Ratones , Mutación , Dominios Proteicos , Canal Catiónico TRPC6/genéticaRESUMEN
The transient receptor potential ion channels support Ca2+ permeation in many organs, including the heart, brain, and kidney. Genetic mutations in transient receptor potential cation channel subfamily C member 3 (TRPC3) are associated with neurodegenerative diseases, memory loss, and hypertension. To better understand the conformational changes that regulate TRPC3 function, we solved the cryo-EM structures for the full-length human TRPC3 and its cytoplasmic domain (CPD) in the apo state at 5.8- and 4.0-Å resolution, respectively. These structures revealed that the TRPC3 transmembrane domain resembles those of other TRP channels and that the CPD is a stable module involved in channel assembly and gating. We observed the presence of a C-terminal domain swap at the center of the CPD where horizontal helices (HHs) transition into a coiled-coil bundle. Comparison of TRPC3 structures revealed that the HHs can reside in two distinct positions. Electrophysiological analyses disclosed that shortening the length of the C-terminal loop connecting the HH with the TRP helices increases TRPC3 activity and that elongating the length of the loop has the opposite effect. Our findings indicate that the C-terminal loop affects channel gating by altering the allosteric coupling between the cytoplasmic and transmembrane domains. We propose that molecules that target the HH may represent a promising strategy for controlling TRPC3-associated neurological disorders and hypertension.
Asunto(s)
Activación del Canal Iónico , Canales Catiónicos TRPC/química , Regulación Alostérica , Repetición de Anquirina , Células HEK293 , Humanos , Mutación , Conformación Proteica en Hélice alfa , Dominios Proteicos , Canales Catiónicos TRPC/genéticaRESUMEN
Vampire bats (Desmodus rotundus) are obligate blood feeders that have evolved specialized systems to suit their sanguinary lifestyle. Chief among such adaptations is the ability to detect infrared radiation as a means of locating hotspots on warm-blooded prey. Among vertebrates, only vampire bats, boas, pythons and pit vipers are capable of detecting infrared radiation. In each case, infrared signals are detected by trigeminal nerve fibres that innervate specialized pit organs on the animal's face. Thus, vampire bats and snakes have taken thermosensation to the extreme by developing specialized systems for detecting infrared radiation. As such, these creatures provide a window into the molecular and genetic mechanisms underlying evolutionary tuning of thermoreceptors in a species-specific or cell-type-specific manner. Previously, we have shown that snakes co-opt a non-heat-sensitive channel, vertebrate TRPA1 (transient receptor potential cation channel A1), to produce an infrared detector. Here we show that vampire bats tune a channel that is already heat-sensitive, TRPV1, by lowering its thermal activation threshold to about 30 °C. This is achieved through alternative splicing of TRPV1 transcripts to produce a channel with a truncated carboxy-terminal cytoplasmic domain. These splicing events occur exclusively in trigeminal ganglia, and not in dorsal root ganglia, thereby maintaining a role for TRPV1 as a detector of noxious heat in somatic afferents. This reflects a unique organization of the bat Trpv1 gene that we show to be characteristic of Laurasiatheria mammals (cows, dogs and moles), supporting a close phylogenetic relationship with bats. These findings reveal a novel molecular mechanism for physiological tuning of thermosensory nerve fibres.
Asunto(s)
Empalme Alternativo/genética , Quirópteros/genética , Quirópteros/fisiología , Rayos Infrarrojos , Sensación/fisiología , Canales Catiónicos TRPV/genética , Ganglio del Trigémino/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Quirópteros/anatomía & histología , Quirópteros/clasificación , Cara/anatomía & histología , Cara/inervación , Conducta Alimentaria/fisiología , Células HEK293 , Calor , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Filogenia , Conducta Predatoria/fisiología , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/metabolismoRESUMEN
The coupled interplay between activation and inactivation gating is a functional hallmark of K(+) channels. This coupling has been experimentally demonstrated through ion interaction effects and cysteine accessibility, and is associated with a well defined boundary of energetically coupled residues. The structure of the K(+) channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation-inactivation gating. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K(+) channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K(+) channels.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Activación del Canal Iónico , Canales de Potasio/química , Canales de Potasio/metabolismo , Streptomyces lividans/química , Regulación Alostérica , Proteínas Bacterianas/genética , Cisteína/genética , Cisteína/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Fenilalanina/metabolismo , Canales de Potasio/genética , Conformación Proteica , Canales de Potasio de la Superfamilia Shaker/química , Canales de Potasio de la Superfamilia Shaker/genética , Canales de Potasio de la Superfamilia Shaker/metabolismo , Relación Estructura-ActividadRESUMEN
Snakes possess a unique sensory system for detecting infrared radiation, enabling them to generate a 'thermal image' of predators or prey. Infrared signals are initially received by the pit organ, a highly specialized facial structure that is innervated by nerve fibres of the somatosensory system. How this organ detects and transduces infrared signals into nerve impulses is not known. Here we use an unbiased transcriptional profiling approach to identify TRPA1 channels as infrared receptors on sensory nerve fibres that innervate the pit organ. TRPA1 orthologues from pit-bearing snakes (vipers, pythons and boas) are the most heat-sensitive vertebrate ion channels thus far identified, consistent with their role as primary transducers of infrared stimuli. Thus, snakes detect infrared signals through a mechanism involving radiant heating of the pit organ, rather than photochemical transduction. These findings illustrate the broad evolutionary tuning of transient receptor potential (TRP) channels as thermosensors in the vertebrate nervous system.
Asunto(s)
Crotalus/fisiología , Calor , Rayos Infrarrojos , Fototransducción/fisiología , Fototransducción/efectos de la radiación , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Boidae/genética , Boidae/metabolismo , Pollos , Clonación Molecular , Crotalus/anatomía & histología , Crotalus/genética , Crotalus/metabolismo , Datos de Secuencia Molecular , Conducta Predatoria/fisiología , Conducta Predatoria/efectos de la radiación , Ratas , Células Receptoras Sensoriales/metabolismo , Canales de Potencial de Receptor Transitorio/genética , Ganglio del Trigémino/citología , Ganglio del Trigémino/metabolismoRESUMEN
Transient receptor potential (TRP) channels are polymodal signal detectors that respond to a wide array of physical and chemical stimuli, making them important components of sensory systems in both vertebrate and invertebrate organisms. Mammalian TRPA1 channels are activated by chemically reactive irritants, whereas snake and Drosophila TRPA1 orthologs are preferentially activated by heat. By comparing human and rattlesnake TRPA1 channels, we have identified two portable heat-sensitive modules within the ankyrin repeat-rich aminoterminal cytoplasmic domain of the snake ortholog. Chimeric channel studies further demonstrate that sensitivity to chemical stimuli and modulation by intracellular calcium also localize to the N-terminal ankyrin repeat-rich domain, identifying this region as an integrator of diverse physiological signals that regulate sensory neuron excitability. These findings provide a framework for understanding how restricted changes in TRPA1 sequence account for evolution of physiologically diverse channels, also identifying portable modules that specify thermosensitivity.
Asunto(s)
Proteínas de Drosophila/química , Canales Catiónicos TRPC/química , Canales de Potencial de Receptor Transitorio/química , Animales , Crotalus , Citoplasma/metabolismo , Dimerización , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Electrofisiología/métodos , Calor , Humanos , Canales Iónicos , Oocitos/metabolismo , Mutación Puntual , Estructura Terciaria de Proteína , ARN Complementario/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Canal Catiónico TRPA1 , Canales Catiónicos TRPC/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Xenopus , Xenopus laevis/metabolismo , Pez CebraRESUMEN
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.
Asunto(s)
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
Emerging evidence suggests that K(+) channel inactivation involves coupling between residues in adjacent regions of the channel. Human ether-a-go-go-related gene-1 (hERG1) K(+) channels undergo a fast inactivation gating process that is crucial for maintaining electrical stability in the heart. The molecular mechanisms that drive inactivation in hERG1 channels are unknown. Using alanine scanning mutagenesis, we show that a pore helix residue (Thr-618) that points toward the S5 segment is critical for normal inactivation gating. Amino acid substitutions at position 618 modulate the free energy of inactivation gating, causing enhanced or reduced inactivation. Mutation of an S5 residue that is predicted to be adjacent to Thr-618 (W568L) abolishes inactivation and alters ion selectivity. The introduction of the Thr-618-equivalent residue in Kv1.5 enhances inactivation. Molecular dynamic simulations of the Kv1.2 tetramer reveal van der Waals coupling between hERG1 618- and 568-equivalent residues and a significant increase in interaction energies when threonine is introduced at the 618-equivalent position. We propose that coupling between the S5 segment and pore helix may participate in the inactivation process in hERG1 channels.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/metabolismo , Activación del Canal Iónico/fisiología , Proteínas Musculares/metabolismo , Sustitución de Aminoácidos , Animales , Canales de Potasio Éter-A-Go-Go/genética , Humanos , Modelos Moleculares , Proteínas Musculares/genética , Mutagénesis , Mutación Missense , Miocardio/metabolismo , Estructura Secundaria de Proteína , Xenopus laevisRESUMEN
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.
Asunto(s)
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.
Asunto(s)
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
In the prokaryotic potassium channel KcsA activation gating at the inner bundle gate is followed by C-type inactivation at the selectivity filter. Entry into the C-type inactivated state has been directly linked to the strength of the H-bond interaction between residues Glu-71 and Asp-80 behind the filter, and is allosterically triggered by the rearrangement of the inner bundle gate. Here, we show that H-bond pairing between residues Trp-67 and Asp-80, conserved in most K⺠channels, constitutes another critical interaction that determines the rate and extent of KcsA C-type inactivation. Disruption of the equivalent interaction in Shaker (Trp-434-Asp-447) and Kv1.2 (Trp-366-Asp-379) leads also to modulation of the inactivation process, suggesting that these residues also play an analogous role in the inactivation gating of Kv channels. The present results show that in KcsA C-type inactivation gating is governed by a multipoint hydrogen-bond network formed by the triad Trp-67-Glu71-Asp-80. This triad exerts a critical role in the dynamics and conformational stability of the selectivity filter and might serve as a general modulator of selectivity filter gating in other members of the K⺠channel family.
Asunto(s)
Proteínas Bacterianas/metabolismo , Activación del Canal Iónico , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Ácido Aspártico/metabolismo , Proteínas Bacterianas/química , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Canales de Potasio/química , Unión Proteica , Ratas , Triptófano/metabolismo , XenopusRESUMEN
The prokaryotic K(+) channel KcsA, although lacking a 'standard' voltage-sensing domain, shows voltage-dependent gating that leads to an increase in steady-state open probability of almost two orders of magnitude between +150 and -150 mV. Here we show that voltage-dependent gating in KcsA is associated with the movement of approximately 0.7 equivalent electronic charges. This charge movement produces an increase in the rate of entry into a long-lived inactivated state and seems to be independent of the proton-activation mechanism. Charge neutralization at position 71 renders the channel essentially voltage-independent by preventing entry into the inactivated state. A mechanism for voltage-dependent gating at the selectivity filter is proposed that is based on the reorientation of the carboxylic moiety of Glu71 and its influence in the conformational dynamics of the selectivity filter.
Asunto(s)
Proteínas Bacterianas/metabolismo , Canales de Potasio/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Concentración de Iones de Hidrógeno , Activación del Canal Iónico , Potenciales de la Membrana , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Canales de Potasio/química , Canales de Potasio/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces lividans/genética , Streptomyces lividans/metabolismoRESUMEN
We show that in the potassium channel KcsA, proton-dependent activation is followed by an inactivation process similar to C-type inactivation, and this process is suppressed by an E71A mutation in the pore helix. EPR spectroscopy demonstrates that the inner gate opens maximally at low pH regardless of the magnitude of the single-channel-open probability, implying that stationary gating originates mostly from rearrangements at the selectivity filter. Two E71A crystal structures obtained at 2.5 A reveal large structural excursions of the selectivity filter during ion conduction and provide a glimpse of the range of conformations available to this region of the channel during gating. These data establish a mechanistic basis for the role of the selectivity filter during channel activation and inactivation.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Activación del Canal Iónico , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Canales de Potasio/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , Streptomyces lividans/genética , Streptomyces lividans/metabolismo , TermodinámicaRESUMEN
The overactivation of transient receptor potential canonical 3 (TRPC3) is associated with neurodegenerative diseases and hypertension. Pyrazole 3 (Pyr3) is reported as the most selective TRPC3 inhibitor, but it has two inherent structural limitations: (1) the labile ester moiety leads to its rapid hydrolysis to the inactive Pyr8 in vivo, and (2) the alkylating trichloroacrylic amide moiety is known to be toxic. To circumvent these limitations, we designed a series of conformationally restricted Pyr3 analogues and reported that compound 20 maintains high potency and selectivity for human TRPC3 over its closely related TRP channels. It has significantly improved metabolic stability compared with Pyr3 and has a good safety profile. Preliminary evaluation of 20 demonstrated its ability to rescue Aß-induced neuron damage with similar potency to that of Pyr3 in vitro. Collectively, these results suggest that 20 represents a promising scaffold to potentially ameliorate the symptoms associated with TRPC3-mediated neurological and cardiovascular disorders.
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.
Asunto(s)
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
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
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.