RESUMEN
The dispanins are a family of 15 transmembrane proteins that have diverse and often unclear physiological functions. Many dispanins, including synapse differentiation induced gene 1 (SynDIG1), proline-rich transmembrane protein 1 (PRRT1)/SynDIG4, and PRRT2, are expressed in the central nervous system (CNS), where they are involved in the development of synapses, regulation of neurotransmitter release, and interactions with ion channels, including AMPA receptors (AMPARs). Others, including transmembrane protein 233 (TMEM233) and trafficking regulator of GLUT4-1 (TRARG1), are expressed in the peripheral nervous system (PNS); however, the function of these dispanins is less clear. Recently, a family of neurotoxins isolated from the giant Australian stinging tree was shown to target TMEM233 to modulate the function of voltage-gated sodium (NaV) channels, suggesting that the dispanins are inherently druggable. Here, we review current knowledge about the structure and function of the dispanins, in particular TMEM233 and its two most closely related homologs PRRT2 and TRARG1, which may be drug targets involved in neurological disease.
Asunto(s)
Proteínas de la Membrana , Enfermedades del Sistema Nervioso , Humanos , Animales , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades del Sistema Nervioso/fisiopatología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiologíaRESUMEN
Erythromelalgia (EM), is a familial pain syndrome characterized by episodic 'burning' pain, warmth, and erythema. EM is caused by monoallelic variants in SCN9A, which encodes the voltage-gated sodium channel (NaV) NaV1.7. Over 25 different SCN9A mutations attributed to EM have been described to date, all identified in the SCN9A transcript utilizing exon 6N. Here we report a novel SCN9A missense variant identified in seven related individuals with stereotypic episodes of bilateral lower limb pain presenting in childhood. The variant, XM_011511617.3:c.659G>C;p.(Arg220Pro), resides in the exon 6A of SCN9A, an exon previously shown to be selectively incorporated by developmentally regulated alternative splicing. The mutation is located in the voltage-sensing S4 segment of domain I, which is important for regulating channel activation. Functional analysis showed the p.Arg220Pro mutation altered voltage-dependent activation and delayed channel inactivation, consistent with a NaV1.7 gain-of-function molecular phenotype. These results demonstrate that alternatively spliced isoforms of SCN9A should be included in all genomic testing of EM.
Asunto(s)
Eritromelalgia , Humanos , Eritromelalgia/genética , Mutación Missense/genética , Canal de Sodio Activado por Voltaje NAV1.7/genética , Dolor/genética , Mutación , Exones/genéticaRESUMEN
Bivalency is a prevalent natural mechanism to enhance receptor avidity. Various two-domain disulfide-rich peptides exhibiting bivalent action have been identified from animal venoms. A unique characteristic of these peptides is that they induce a pharmacological response different from that provoked by any of the constituent domains. The enhanced potency and avidity of such peptides is therefore a consequence of their domain fusion by a peptide linker. The role of the linker itself, beyond conjugation, remains unclear. Here, we investigate how the linker affects the bivalency of the capsaicin receptor (TRPV1) agonist DkTx. We recombinantly produced isotope labelled DkTx using a protein splicing approach, to solve the high-resolution solution structure of DkTx, revealing residual linker order stabilised by linker-domain interactions leading to biased domain orientations. The significance of this was studied using a combination of mutagenesis, spin relaxation studies and electrophysiology measurements. Our results reveal that disrupting the pre-organisation of the domains of DkTx is accompanied by reductions in potency and onset of avidity. Our findings support a model of pre-configured two-domain binding, in favour of the previously suggested sequential binding model. This highlights the significance of ordered elements in linker design and the natural evolution of these in bivalent toxins.
Asunto(s)
Toxinas Biológicas , Animales , Péptidos , Fenómenos ElectrofisiológicosRESUMEN
Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain, and other neurotoxic symptoms in vertebrates. Here, we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.
Asunto(s)
Hormigas , Mordeduras y Picaduras , Dolor , Péptidos , Toxinas Biológicas , Bloqueadores del Canal de Sodio Activado por Voltaje , Canales de Sodio Activados por Voltaje , Animales , Hormigas/patogenicidad , Hormigas/fisiología , Mordeduras y Picaduras/complicaciones , Dolor/inducido químicamente , Dolor/complicaciones , Péptidos/química , Péptidos/farmacología , Péptidos/toxicidad , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Toxinas Biológicas/química , Toxinas Biológicas/farmacología , Toxinas Biológicas/toxicidad , Vertebrados , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/toxicidad , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
Aristotelia chilensis or "maqui" is a tree native to Chile used in the folk medicine of the Mapuche people as an anti-inflammatory agent for the treatment of digestive ailments, fever, and skin lesions. Maqui fruits are black berries which are considered a "superfruit" with notable potential health benefits, promoted to be an antioxidant, cardioprotective, and anti-inflammatory. Maqui leaves contain non-iridoid monoterpene indole alkaloids which have previously been shown to act on nicotinic acetylcholine receptors, potassium channels, and calcium channels. Here, we isolated a new alkaloid from maqui leaves, now called makomakinol, together with the known alkaloids aristoteline, hobartine, and 3-formylindole. Moreover, the polyphenols quercetine, ethyl caffeate, and the terpenes, dihydro-ß-ionone and terpin hydrate, were also obtained. In light of the reported analgesic and anti-nociceptive properties of A. chilensis, in particular a crude mixture of alkaloids containing aristoteline and hobartinol (PMID 21585384), we therefore evaluated the activity of aristoteline and hobartine on NaV1.8, a key NaV isoform involved in nociception, using automated whole-cell patch-clamp electrophysiology. Aristoteline and hobartine both inhibited Nav1.8 with an IC50 of 68 ± 3 µM and 54 ± 1 µM, respectively. Hobartine caused a hyperpolarizing shift of the voltage-dependence of the activation, whereas aristoteline did not change the voltage-dependence of the activation or inactivation. The inhibitory activity of these alkaloids on NaV channels may contribute to the reported analgesic properties of Aristotelia chilensis used by the Mapuche people.
Asunto(s)
Alcaloides , Elaeocarpaceae , Humanos , Alcaloides/farmacología , Alcaloides Indólicos , Extractos Vegetales/farmacología , Analgésicos/farmacología , AntiinflamatoriosRESUMEN
Voltage-gated sodium (NaV) channels are transmembrane proteins that play a critical role in electrical signaling in the nervous system and other excitable tissues. µ-Conotoxins are peptide toxins from the venoms of marine cone snails (genus Conus) that block NaV channels with nanomolar potency. Most species of the subgenera Textilia and Afonsoconus are difficult to acquire; therefore, their venoms have yet to be comprehensively interrogated for µ-conotoxins. The goal of this study was to find new µ-conotoxins from species of the subgenera Textilia and Afonsoconus and investigate their selectivity at human NaV channels. Using RNA-seq of the venom gland of Conus (Textilia) bullatus, we identified 12 µ-conotoxin (or µ-conotoxin-like) sequences. Based on these sequences we designed primers which we used to identify additional µ-conotoxin sequences from DNA extracted from historical specimens of species from Textilia and Afonsoconus. We synthesized six of these µ-conotoxins and tested their activity on human NaV1.1-NaV1.8. Five of the six synthetic peptides were potent blockers of human NaV channels. Of these, two peptides (BuIIIB and BuIIIE) were potent blockers of hNaV1.3. Three of the peptides (BuIIIB, BuIIIE and AdIIIA) had submicromolar activity at hNaV1.7. This study serves as an example of the identification of new peptide toxins from historical DNA and provides new insights into structure-activity relationships of µ-conotoxins with activity at hNaV1.3 and hNaV1.7.
Asunto(s)
Conotoxinas , Caracol Conus , Toxinas Biológicas , Humanos , Animales , Conotoxinas/farmacología , Proteínas de la Membrana , Canales de Sodio/genéticaRESUMEN
Numerous spider venom-derived gating modifier toxins exhibit conformational heterogeneity during purification by reversed-phase high-performance liquid chromatography (RP-HPLC). This conformational exchange is especially peculiar for peptides containing an inhibitor cystine knot motif, which confers excellent structural stability under conditions that are not conducive to disulfide shuffling. This phenomenon is often attributed to proline cis/trans isomerization but has also been observed in peptides that do not contain a proline residue. Pn3a is one such peptide forming two chromatographically distinguishable peaks that readily interconvert following the purification of either conformer. The nature of this exchange was previously uncharacterized due to the fast rate of conversion in solution, making isolation of the conformers impossible. In the present study, an N-terminal modification of Pn3a enabled the isolation of the individual conformers, allowing activity assays to be conducted on the individual conformers using electrophysiology. The conformers were analyzed separately by nuclear magnetic resonance spectroscopy (NMR) to study their structural differences. RP-HPLC and NMR were used to study the mechanism of exchange. The later-eluting conformer was the active conformer with a rigid structure that corresponds to the published structure of Pn3a, while NMR analysis revealed the earlier-eluting conformer to be inactive and disordered. The exchange was found to be pH-dependent, arising in acidic solutions, possibly due to reversible disruption and formation of intramolecular salt bridges. This study reveals the nature of non-proline conformational exchange observed in Pn3a and possibly other disulfide-rich peptides, highlighting that the structure and activity of some disulfide-stabilized peptides can be dramatically susceptible to disruption.
RESUMEN
Disulfide-rich peptide toxins have long been studied for their ability to inhibit voltage-gated sodium channel subtype NaV1.7, a validated target for the treatment of pain. In this study, we sought to combine the pore blocking activity of conotoxins with the gating modifier activity of spider toxins to design new bivalent inhibitors of NaV1.7 with improved potency and selectivity. To do this, we created an array of heterodimeric toxins designed to target human NaV1.7 by ligating a conotoxin to a spider toxin and assessed the potency and selectivity of the resulting bivalent toxins. A series of spider-derived gating modifier toxins (GpTx-1, ProTx-II, gHwTx-IV, JzTx-V, CcoTx-1, and Pn3a) and two pore-blocker µ-conotoxins, SxIIIC and KIIIA, were used for this study. We employed either enzymatic ligation with sortase A for C- to N-terminal ligation or click chemistry for N- to N-terminal ligation. The bivalent peptide resulting from ligation of ProTx-II and SxIIIC (Pro[LPATG6]Sx) was shown to be the best combination as native ProTx-II potency at hNaV1.7 was conserved following ligation. At hNaV1.4, a synergistic effect between the pore blocker and gating modifier toxin moieties was observed, resulting in altered sodium channel subtype selectivity compared to the parent peptides. Further studies including mutant bivalent peptides and mutant hNaV1.7 channels suggested that gating modifier toxins have a greater contribution to the potency of the bivalent peptides than pore blockers. This study delineated potential benefits and drawbacks of designing pharmacological hybrid peptides targeting hNaV1.7.
Asunto(s)
Péptidos , Humanos , Péptidos/farmacologíaRESUMEN
Stings of certain ant species (Hymenoptera: Formicidae) can cause intense, long-lasting nociception. Here we show that the major contributors to these symptoms are venom peptides that modulate the activity of voltage-gated sodium (NaV) channels, reducing their voltage threshold for activation and inhibiting channel inactivation. These peptide toxins are likely vertebrate-selective, consistent with a primarily defensive function. They emerged early in the Formicidae lineage and may have been a pivotal factor in the expansion of ants.
Asunto(s)
Venenos de Hormiga , Hormigas , Toxinas Biológicas , Animales , Dolor , Canales de Sodio , VertebradosRESUMEN
Voltage-gated sodium (NaV) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons.
Asunto(s)
Toxinas Biológicas , Urtica dioica , Australia , Dolor , Péptidos , Canal de Sodio Activado por Voltaje NAV1.7/metabolismoRESUMEN
Venom-derived peptides targeting ion channels involved in pain are regarded as a promising alternative to current, and often ineffective, chronic pain treatments. Many peptide toxins are known to specifically and potently block established therapeutic targets, among which the voltage-gated sodium and calcium channels are major contributors. Here, we report on the discovery and characterization of a novel spider toxin isolated from the crude venom of Pterinochilus murinus that shows inhibitory activity at both hNaV 1.7 and hCaV 3.2 channels, two therapeutic targets implicated in pain pathways. Bioassay-guided HPLC fractionation revealed a 36-amino acid peptide with three disulfide bridges named µ/ω-theraphotoxin-Pmu1a (Pmu1a). Following isolation and characterization, the toxin was chemically synthesized and its biological activity was further assessed using electrophysiology, revealing Pmu1a to be a toxin that potently blocks both hNaV 1.7 and hCaV 3. Nuclear magnetic resonance structure determination of Pmu1a shows an inhibitor cystine knot fold that is the characteristic of many spider peptides. Combined, these data show the potential of Pmu1a as a basis for the design of compounds with dual activity at the therapeutically relevant hCaV 3.2 and hNaV 1.7 voltage-gated channels.
Asunto(s)
Venenos de Araña , Arañas , Animales , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Venenos de Araña/farmacología , Venenos de Araña/química , Venenos de Araña/metabolismo , Dolor , Péptidos/farmacología , Espectroscopía de Resonancia Magnética , Arañas/metabolismoRESUMEN
Most species of bee are capable of delivering a defensive sting which is often painful. A solitary lifestyle is the ancestral state of bees and most extant species are solitary, but information on bee venoms comes predominantly from studies on eusocial species. In this study we investigated the venom composition of the Australian great carpenter bee, Xylocopa aruana Ritsema, 1876. We show that the venom is relatively simple, composed mainly of one small amphipathic peptide (XYTX1-Xa1a), with lesser amounts of an apamin homologue (XYTX2-Xa2a) and a venom phospholipase-A2 (PLA2). XYTX1-Xa1a is homologous to, and shares a similar mode-of-action to melittin and the bombilitins, the major components of the venoms of the eusocial Apis mellifera (Western honeybee) and Bombus spp. (bumblebee), respectively. XYTX1-Xa1a and melittin directly activate mammalian sensory neurons and cause spontaneous pain behaviours in vivo, effects which are potentiated in the presence of venom PLA2. The apamin-like peptide XYTX2-Xa2a was a relatively weak blocker of small conductance calcium-activated potassium (KCa) channels and, like A. mellifera apamin and mast cell-degranulating peptide, did not contribute to pain behaviours in mice. While the composition and mode-of-action of the venom of X. aruana are similar to that of A. mellifera, the greater potency, on mammalian sensory neurons, of the major pain-causing component in A. mellifera venom may represent an adaptation to the distinct defensive pressures on eusocial Apidae.
Asunto(s)
Venenos de Abeja , Toxinas Biológicas , Abejas , Ratones , Animales , Meliteno , Apamina , Australia , Venenos de Abeja/química , Fosfolipasas A2 , Péptidos , Dolor/inducido químicamente , MamíferosRESUMEN
µ-Conotoxins are small, potent, peptide voltage-gated sodium (NaV) channel inhibitors characterised by a conserved cysteine framework. Despite promising in vivo studies indicating analgesic potential of these compounds, selectivity towards the therapeutically relevant subtype NaV1.7 has so far been limited. We recently identified a novel µ-conotoxin, SxIIIC, which potently inhibits human NaV1.7 (hNaV1.7). SxIIIC has high sequence homology with other µ-conotoxins, including SmIIIA and KIIIA, yet shows different NaV channel selectivity for mammalian subtypes. Here, we evaluated and compared the inhibitory potency of µ-conotoxins SxIIIC, SmIIIA and KIIIA at hNaV channels by whole-cell patch-clamp electrophysiology and discovered that these three closely related µ-conotoxins display unique selectivity profiles with significant variations in inhibitory potency at hNaV1.7. Analysis of other µ-conotoxins at hNaV1.7 shows that only a limited number are capable of inhibition at this subtype and that differences between the number of residues in loop 3 appear to influence the ability of µ-conotoxins to inhibit hNaV1.7. Through mutagenesis studies, we confirmed that charged residues in this region also affect the selectivity for hNaV1.4. Comparison of µ-conotoxin NMR solution structures identified differences that may contribute to the variance in hNaV1.7 inhibition and validated the role of the loop 1 extension in SxIIIC for improving potency at hNaV1.7, when compared to KIIIA. This work could assist in designing µ-conotoxin derivatives specific for hNaV1.7.
Asunto(s)
Conotoxinas , Bloqueadores del Canal de Sodio Activado por Voltaje , Analgésicos/química , Analgésicos/farmacología , Animales , Conotoxinas/química , Conotoxinas/farmacología , Cisteína , Humanos , Canal de Sodio Activado por Voltaje NAV1.4 , Canal de Sodio Activado por Voltaje NAV1.7 , Péptidos , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacologíaRESUMEN
The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while ß/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (NaV) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that ß/δ-Uf2a is a potent modulator of human NaV1.5 (EC50: 55 nM), NaV1.6 (EC50: 0.86 nM), and NaV1.7 (EC50: 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides.
Asunto(s)
Neurotoxinas , Péptidos , Toxinas Biológicas , Urticaceae , Humanos , Neurotoxinas/química , Dolor , Técnicas de Placa-Clamp , Péptidos/química , Péptidos/toxicidad , Toxinas Biológicas/química , Urticaceae/química , Canales de Sodio Activados por Voltaje/efectos de los fármacosRESUMEN
QX-314 is a quaternary permanently charged lidocaine derivative that inhibits voltage-gated sodium channels (NaV). As it is membrane impermeable, it is generally considered that QX-314 applied externally is inactive, unless it can gain access to the internal local anesthetic binding site via another entry pathway. Here, we characterized the electrophysiological effects of QX-314 on NaV1.7 heterologously expressed in HEK293 cells, and found that at high concentrations, external QX-314 inhibited NaV1.7 current (IC50 2.0 ± 0.3 mM) and shifted the voltage-dependence to more depolarized potentials (ΔV50 +10.6 mV). Unlike lidocaine, the activity of external QX-314 was not state- or use-dependent. The effect of externally applied QX-314 on NaV1.7 channel biophysics differed to that of internally applied QX-314, suggesting QX-314 has an additional externally accessible site of action. In line with this hypothesis, disruption of the local anesthetic binding site in a [F1748A]NaV1.7 mutant reduced the potency of lidocaine by 40-fold, but had no effect on the potency or activity of externally applied QX-314. Therefore, we conclude, using an expression system where QX-314 was unable to cross the membrane, that externally applied QX-314 is able to inhibit NaV1.7 peak current at low millimolar concentrations.
Asunto(s)
Anestésicos Locales , Lidocaína , Anestésicos Locales/farmacología , Células HEK293 , Humanos , Lidocaína/análogos & derivados , Lidocaína/farmacología , Sodio/metabolismo , Bloqueadores de los Canales de Sodio/farmacologíaRESUMEN
µ-Conotoxins are components of cone snail venom, well-known for their analgesic activity through potent inhibition of voltage-gated sodium channel (NaV) subtypes, including NaV1.7. These small, disulfide-rich peptides are typically stabilized by three disulfide bonds arranged in a 'native' CysI-CysIV, CysII-CysV, CysIII-CysVI pattern of disulfide connectivity. However, µ-conotoxin KIIIA, the smallest and most studied µ-conotoxin with inhibitory activity at NaV1.7, forms two distinct disulfide bond isomers during thermodynamic oxidative folding, including Isomer 1 (CysI-CysV, CysII-CysIV, CysIII-CysVI) and Isomer 2 (CysI-CysVI, CysII-CysIV, CysIII-CysV), but not the native µ-conotoxin arrangement. To date, there has been no study on the structure and activity of KIIIA comprising the native µ-conotoxin disulfide bond arrangement. Here, we evaluated the synthesis, potency, sodium channel subtype selectivity, and 3D structure of the three isomers of KIIIA. Using a regioselective disulfide bond-forming strategy, we synthetically produced the three µ-conotoxin KIIIA isomers displaying distinct bioactivity and NaV subtype selectivity across human NaV channel subtypes 1.2, 1.4, and 1.7. We show that Isomer 1 inhibits NaV subtypes with a rank order of potency of NaV1.4 > 1.2 > 1.7 and Isomer 2 in the order of NaV1.4≈1.2 > 1.7, while the native isomer inhibited NaV1.4 > 1.7≈1.2. The three KIIIA isomers were further evaluated by NMR solution structure analysis and molecular docking with hNaV1.2. Our study highlights the importance of investigating alternate disulfide isomers, as disulfide connectivity affects not only the overall structure of the peptides but also the potency and subtype selectivity of µ-conotoxins targeting therapeutically relevant NaV subtypes.
Asunto(s)
Conotoxinas , Bloqueadores del Canal de Sodio Activado por Voltaje , Canales de Sodio Activados por Voltaje , Conotoxinas/química , Conotoxinas/farmacología , Disulfuros/química , Disulfuros/farmacología , Humanos , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Canales de Sodio Activados por Voltaje/química , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
Advances in peptide and protein therapeutics increased the need for rapid and cost-effective polypeptide prototyping. While in vitro translation systems are well suited for fast and multiplexed polypeptide prototyping, they suffer from misfolding, aggregation and disulfide-bond scrambling of the translated products. Here we propose that efficient folding of in vitro produced disulfide-rich peptides and proteins can be achieved if performed in an aggregation-free and thermodynamically controlled folding environment. To this end, we modify an E. coli-based in vitro translation system to allow co-translational capture of translated products by affinity matrix. This process reduces protein aggregation and enables productive oxidative folding and recycling of misfolded states under thermodynamic control. In this study we show that the developed approach is likely to be generally applicable for prototyping of a wide variety of disulfide-constrained peptides, macrocyclic peptides with non-native bonds and antibody fragments in amounts sufficient for interaction analysis and biological activity assessment.
Asunto(s)
Sistema Libre de Células/efectos de los fármacos , Medicamentos Genéricos/química , Medicamentos Genéricos/farmacología , Péptidos/química , Péptidos/farmacología , Animales , Anticuerpos , Análisis Costo-Beneficio , Interpretación Estadística de Datos , Disulfuros , Drosophila melanogaster , Escherichia coli , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Leishmania , Péptidos/genética , Agregado de Proteínas , Dominios Proteicos , ARN Ribosómico 16S , Biología Sintética , TermodinámicaRESUMEN
Drimys winteri J.R.Forst. & G.Forst, a South American evergreen shrub that is used by the Mapuche people for treatment of several painful conditions, contains polygodial, a lipophilic drimane-type sesquiterpene dialdehyde with known activity at transient receptor potential channel family members including TRPA1 and TRPV1. We sought to assess the activity of polygodial at NaV1.7 and NaV1.8, two key isoforms of the voltage-gated sodium channel family involved in nociception. Polygodial was isolated from D. winteri by thin-layer chromatography and analysed structurally by 1 D and 2 D nuclear magnetic resonance (NMR) spectroscopy. Activity at heterologously expressed NaV1.7 and NaV1.8 was assessed using automated whole-cell patch-clamp electrophysiology. Here, we show that polygodial inhibits members of the voltage-gated sodium channel family, specifically NaV1.7 and NaV1.8, without changing the voltage-dependence of activation or inactivation. Activity of polygodial at voltage-gated sodium channels may contribute to the previously reported antinociceptive properties.
Asunto(s)
Drimys , Sesquiterpenos , Canales de Sodio Activados por Voltaje , Humanos , Sesquiterpenos/farmacologíaRESUMEN
In this work, we investigated the in vitro neurotoxicity of Calliophis intestinalis venom using chick biventer cervicis neuromuscular preparations and electrophysiological analysis of voltage-gated sodium (NaV) channels expressed in HEK293 cells. We found that the indirect twitches of the neuromuscular preparations decreased over time when exposed to venom. However, the responses of these preparations to the agonists acetylcholine, carbachol, and potassium chloride were not changed after incubation with the venom. Our electrophysiological experiments show that C. intestinalis venom acts as a NaV channel antagonist-the first known from a vertebrate venom-by decreasing the peak current of NaV1.4 channels without changing the kinetics of activation or inactivation. Our proteomic results accord with earlier analyses and find that the venom contains three-finger toxins, cysteine-rich secretory proteins, kunitz peptides, phospholipase A2s, snake venom metalloproteases, and vespryns. Some of the three-finger toxins are similar to the δ-elapitoxins from the venom of the closely related Calliophis bivirgatus. However, δ-elapitoxins act as NaV channel agonists in C. bivirgatus whereas C. intestinalis venom contains NaV channel antagonists. The toxins and mechanisms responsible for the neuromuscular symptoms remain unclear as does the identity of the NaV channel antagonists. These aspects of this unusual venom require further study.