RESUMO
Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.
Assuntos
Nucléolo Celular/metabolismo , Gânglios Espinais/metabolismo , Cinesinas/metabolismo , Neurônios/metabolismo , Fosfoproteínas/química , Proteínas de Ligação a RNA/química , Nervo Isquiático/metabolismo , Sequência de Aminoácidos , Animais , Transporte Axonal/genética , Linhagem Celular Tumoral , Nucléolo Celular/ultraestrutura , Gânglios Espinais/citologia , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Cinesinas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Neurônios/citologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Cultura Primária de Células , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Nervo Isquiático/citologia , NucleolinaRESUMO
The interaction of insect-selective scorpion depressant ß-toxins (LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNav1-1a, was investigated using site-directed mutagenesis, electrophysiological analyses, and structural modeling. Focusing on the pharmacologically defined binding site-4 of scorpion ß-toxins at the voltage-sensing domain II (VSD-II), we found that charge neutralization of D802 in VSD-II greatly enhanced the channel sensitivity to Lqh-dprIT3. This was consistent with the high sensitivity of the splice variant BgNav2-1, bearing G802, to Lqh-dprIT3, and low sensitivity of BgNav2-1 mutant, G802D, to the toxin. Further mutational and electrophysiological analyses revealed that the sensitivity of the WT = D802E < D802G < D802A < D802K channel mutants to Lqh-dprIT3 correlated with the depolarizing shifts of activation in toxin-free channels. However, the sensitivity of single mutants involving IIS4 basic residues (K4E = WT << R1E < R2E < R3E) or double mutants (D802K = K4E/D802K = R3E/D802K > R2E/D802K > R1E/D802K > WT) did not correlate with the activation shifts. Using the cryo-EM structure of the Periplaneta americana channel, NavPaS, as a template and the crystal structure of LqhIT2, we constructed structural models of LqhIT2 and Lqh-dprIT3-c in complex with BgNav1-1a. These models along with the mutational analysis suggest that depressant toxins approach the salt-bridge between R1 and D802 at VSD-II to form contacts with linkers IIS1-S2, IIS3-S4, IIIP5-P1 and IIIP2-S6. Elimination of this salt-bridge enables deeper penetration of the toxin into a VSD-II gorge to form new contacts with the channel, leading to increased channel sensitivity to Lqh-dprIT3.
Assuntos
Neópteros/metabolismo , Venenos de Escorpião/metabolismo , Escorpiões/metabolismo , Canais de Sódio/metabolismo , Animais , Sítios de Ligação/genética , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Modelos Moleculares , Mutação , Neópteros/genética , Oócitos/metabolismo , Oócitos/fisiologia , Técnicas de Patch-Clamp/métodos , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Venenos de Escorpião/química , Venenos de Escorpião/genética , Escorpiões/genética , Canais de Sódio/química , Canais de Sódio/genética , XenopusRESUMO
Voltage-dependent potassium channels (Kvs) gate in response to changes in electrical membrane potential by coupling a voltage-sensing module with a K+-selective pore. Animal toxins targeting Kvs are classified as pore blockers, which physically plug the ion conduction pathway, or as gating modifiers, which disrupt voltage sensor movements. A third group of toxins blocks K+ conduction by an unknown mechanism via binding to the channel turrets. Here, we show that Conkunitzin-S1 (Cs1), a peptide toxin isolated from cone snail venom, binds at the turrets of Kv1.2 and targets a network of hydrogen bonds that govern water access to the peripheral cavities that surround the central pore. The resulting ectopic water flow triggers an asymmetric collapse of the pore by a process resembling that of inherent slow inactivation. Pore modulation by animal toxins exposes the peripheral cavity of K+ channels as a novel pharmacological target and provides a rational framework for drug design.
Assuntos
Membrana Celular/efeitos dos fármacos , Proteínas de Drosophila/antagonistas & inibidores , Ativação do Canal Iônico/efeitos dos fármacos , Canal de Potássio Kv1.2/antagonistas & inibidores , Venenos de Moluscos/toxicidade , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Animais , Membrana Celular/metabolismo , Cristalografia por Raios X , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Desenho de Fármacos , Feminino , Ligação de Hidrogênio/efeitos dos fármacos , Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.2/isolamento & purificação , Canal de Potássio Kv1.2/metabolismo , Dose Letal Mediana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Venenos de Moluscos/química , Mutação , Oócitos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/isolamento & purificação , Superfamília Shaker de Canais de Potássio/metabolismo , Água/química , Água/metabolismo , Xenopus laevisRESUMO
Animal venoms are considered as a promising source of new drugs. Sea anemones release polypeptides that affect electrical activity of neurons of their prey. Voltage dependent sodium (Nav) channels are the common targets of Av1, Av2, and Av3 toxins from Anemonia viridis and CgNa from Condylactis gigantea. The toxins bind to the extracellular side of a channel and slow its fast inactivation, but molecular details of the binding modes are not known. Electrophysiological measurements on Periplaneta americana neuronal preparation revealed differences in potency of these toxins to increase nerve activity. Av1 and CgNa exhibit the strongest effects, while Av2 the weakest effect. Extensive molecular docking using a modern SMINA computer method revealed only partial overlap among the sets of toxins' and channel's amino acid residues responsible for the selectivity and binding modes. Docking positions support earlier supposition that the higher neuronal activity observed in electrophysiology should be attributed to hampering the fast inactivation gate by interactions of an anemone toxin with the voltage driven S4 helix from domain IV of cockroach Nav channel (NavPaS). Our modelling provides new data linking activity of toxins with their mode of binding in site 3 of NavPaS channel.
Assuntos
Peptídeos/química , Canais de Sódio/química , Canais de Sódio/metabolismo , Peçonhas/química , Sequência de Aminoácidos , Aminoácidos/química , Animais , Sítios de Ligação , Baratas , Fenômenos Eletrofisiológicos , Conformação Molecular , Simulação de Acoplamento Molecular , Neurônios/efeitos dos fármacos , Anêmonas-do-MarRESUMO
Av3 is a peptide neurotoxin from the sea anemone Anemonia viridis that shows specificity for arthropod voltage-gated sodium channels (Navs). Interestingly, Av3 competes with a scorpion α-toxin on binding to insect Navs and similarly inhibits the inactivation process, and thus has been classified as 'receptor site-3 toxin', although the two peptides are structurally unrelated. This raises questions as to commonalities and differences in the way both toxins interact with Navs. Recently, site-3 was partly resolved for scorpion α-toxins highlighting S1-S2 and S3-S4 external linkers at the DIV voltage-sensor module and the juxtaposed external linkers at the DI pore module. To uncover channel determinants involved in Av3 specificity for arthropods, the toxin was examined on channel chimaeras constructed with the external linkers of the mammalian brain Nav1.2a, which is insensitive to Av3, in the background of the Drosophila DmNav1. This approach highlighted the role of linker DI/SS2-S6, adjacent to the channel pore, in determining Av3 specificity. Point mutagenesis at DI/SS2-S6 accompanied by functional assays highlighted Trp404 and His405 as a putative point of Av3 interaction with DmNav1. His405 conservation in arthropod Navs compared with tyrosine in vertebrate Navs may represent an ancient substitution that explains the contemporary selectivity of Av3. Trp404 and His405 localization near the membrane surface and the hydrophobic bioactive surface of Av3 suggest that the toxin possibly binds at a cleft by DI/S6. A partial overlap in receptor site-3 of both toxins nearby DI/S6 may explain their binding competition capabilities.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/química , Drosophila/metabolismo , Toxinas Marinhas/química , Anêmonas-do-Mar/metabolismo , Bloqueadores dos Canais de Sódio/química , Canais de Sódio/química , Canais de Sódio/metabolismo , Animais , Sítios de Ligação , Drosophila/efeitos dos fármacos , Drosophila/genética , Proteínas de Drosophila/genética , Toxinas Marinhas/metabolismo , Toxinas Marinhas/toxicidade , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidade , Anêmonas-do-Mar/química , Bloqueadores dos Canais de Sódio/metabolismo , Bloqueadores dos Canais de Sódio/toxicidade , Canais de Sódio/genética , Xenopus laevisRESUMO
The α-scorpions toxins bind to the resting state of Na(+) channels and inhibit fast inactivation by interaction with a receptor site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ~73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the receptor site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na(+) channels and reveals its mode of interaction with a gating modifier toxin.
Assuntos
Venenos de Escorpião/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismo , Aminoácidos/metabolismo , Ativação do Canal Iônico , Cinética , Modelos Moleculares , Mutação/genética , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Activation of voltage-gated sodium (Na(v)) channels initiates and propagates action potentials in electrically excitable cells. ß-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of Na(V) channels. CssIV stabilizes the voltage sensor in domain II in its activated state via a voltage-sensor trapping mechanism. Amino acid residues required for the action of CssIV have been identified in the S1-S2 and S3-S4 extracellular loops of domain II. The extracellular loops of domain III are also involved in toxin action, but individual amino acid residues have not been identified. We used site-directed mutagenesis and voltage clamp recording to investigate amino acid residues of domain III that are involved in CssIV action. In the IIISS2-S6 loop, five substitutions at four positions altered voltage-sensor trapping by CssIV(E15A). Three substitutions (E1438A, D1445A, and D1445Y) markedly decreased voltage-sensor trapping, whereas the other two substitutions (N1436G and L1439A) increased voltage-sensor trapping. These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with CssIV. N1436G enhanced voltage-sensor trapping via increased binding affinity to the resting state, whereas L1439A increased voltage-sensor trapping efficacy. Based on these results, a three-dimensional model of the toxin-channel interaction was developed using the Rosetta modeling method. These data provide additional molecular insight into the voltage-sensor trapping mechanism of toxin action and define a three-point interaction site for ß-scorpion toxins on Na(V) channels. Binding of α- and ß-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on Na(V) channels acts synergistically to modify channel gating and paralyze prey.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Venenos de Escorpião/farmacologia , Substituição de Aminoácidos , Animais , Linhagem Celular , Ativação do Canal Iônico/genética , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RatosRESUMO
Voltage-gated sodium (Na(v)) channels are the molecular targets of ß-scorpion toxins, which shift the voltage dependence of activation to more negative membrane potentials by a voltage sensor-trapping mechanism. Molecular determinants of ß-scorpion toxin (CssIV) binding and action on rat brain sodium channels are located in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular linkers of the voltage-sensing module in domain II. In IIS1-S2, mutations of two amino acid residues (Glu(779) and Pro(782)) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly(845), define a hot spot of high-impact residues. Two of these substitutions (A841N and L846A) reduced voltage sensor trapping. The other three substitutions (N842R, V843A, and E844N) increased voltage sensor trapping. These bidirectional effects suggest that the IIS3-S4 loop plays a primary role in determining both toxin affinity and efficacy. A high resolution molecular model constructed with the Rosetta-Membrane modeling system reveals interactions of amino acid residues in sodium channels that are crucial for toxin action with residues in CssIV that are required for its effects. In this model, the wedge-shaped CssIV inserts between the IIS1-S2 and IIS3-S4 loops of the voltage sensor, placing key amino acid residues in position to interact with binding partners in these extracellular loops. These results provide new molecular insights into the voltage sensor-trapping model of toxin action and further define the molecular requirements for the development of antagonists that can prevent or reverse toxicity of scorpion toxins.
Assuntos
Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Venenos de Escorpião/química , Venenos de Escorpião/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismo , Animais , Ativação do Canal Iônico , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.2 , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Relação Estrutura-AtividadeRESUMO
Neurotoxin receptor site-3 at voltage-gated Na(+) channels is recognized by various peptide toxin inhibitors of channel inactivation. Despite extensive studies of the effects of these toxins, their mode of interaction with the channel remained to be described at the molecular level. To identify channel constituents that interact with the toxins, we exploited the opposing preferences of LqhαIT and Lqh2 scorpion α-toxins for insect and mammalian brain Na(+) channels. Construction of the DIV/S1-S2, DIV/S3-S4, DI/S5-SS1, and DI/SS2-S6 external loops of the rat brain rNa(v)1.2a channel (highly sensitive to Lqh2) in the background of the Drosophila DmNa(v)1 channel (highly sensitive to LqhαIT), and examination of toxin activity on the channel chimera expressed in Xenopus oocytes revealed a substantial decrease in LqhαIT effect, whereas Lqh2 was as effective as at rNa(v)1.2a. Further substitutions of individual loops and specific residues followed by examination of gain or loss in Lqh2 and LqhαIT activities highlighted the importance of DI/S5-S6 (pore module) and the C-terminal region of DIV/S3 (gating module) of rNa(v)1.2a for Lqh2 action and selectivity. In contrast, a single substitution of Glu-1613 to Asp at DIV/S3-S4 converted rNa(v)1.2a to high sensitivity toward LqhαIT. Comparison of depolarization-driven dissociation of Lqh2 and mutant derivatives off their binding site at rNa(v)1.2a mutant channels has suggested that the toxin core domain interacts with the gating module of DIV. These results constitute the first step in better understanding of the way scorpion α-toxins interact with voltage-gated Na(+)-channels at the molecular level.
Assuntos
Venenos de Escorpião/metabolismo , Escorpiões/metabolismo , Canais de Sódio/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , DNA Complementar/metabolismo , Drosophila , Conformação Molecular , Dados de Sequência Molecular , Mutagênese , Mutação , Neurotoxinas/metabolismo , Ratos , Anêmonas-do-Mar , Homologia de Sequência de Aminoácidos , XenopusRESUMO
Scorpion ß-toxins bind to the extracellular regions of the voltage-sensing module of domain II and to the pore module of domain III in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain II in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant ß-toxin, Lqh-dprIT(3), from Leiurus quinquestriatus hebraeus with insect sodium channels from Blattella germanica (BgNa(v)). Like other scorpion ß-toxins, Lqh-dprIT(3) shifts the voltage dependence of activation of BgNa(v) channels expressed in Xenopus oocytes to more negative membrane potentials but only after strong depolarizing prepulses. Notably, among 10 BgNa(v) splice variants tested for their sensitivity to the toxin, only BgNa(v)1-1 was hypersensitive due to an L1285P substitution in IIIS1 resulting from a U-to-C RNA-editing event. Furthermore, charge reversal of a negatively charged residue (E1290K) at the extracellular end of IIIS1 and the two innermost positively charged residues (R4E and R5E) in IIIS4 also increased the channel sensitivity to Lqh-dprIT(3). Besides enhancement of toxin sensitivity, the R4E substitution caused an additional 20-mV negative shift in the voltage dependence of activation of toxin-modified channels, inducing a unique toxin-modified state. Our findings provide the first direct evidence for the involvement of the domain III voltage-sensing module in the action of scorpion ß-toxins. This hypersensitivity most likely reflects an increase in IIS4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation.
Assuntos
Blattellidae/metabolismo , Proteínas de Insetos/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Venenos de Escorpião/farmacologia , Canais de Sódio/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Processamento Alternativo/fisiologia , Substituição de Aminoácidos , Animais , Blattellidae/química , Blattellidae/genética , Expressão Gênica , Proteínas de Insetos/química , Proteínas de Insetos/genética , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Venenos de Escorpião/química , Escorpiões/química , Canais de Sódio/química , Canais de Sódio/genética , XenopusRESUMO
Jellyfish, hydras, corals and sea anemones (phylum Cnidaria) are known for their venomous stinging cells, nematocytes, used for prey and defence. Here we show, however, that the potent Type I neurotoxin of the sea anemone Nematostella vectensis, Nv1, is confined to ectodermal gland cells rather than nematocytes. We demonstrate massive Nv1 secretion upon encounter with a crustacean prey. Concomitant discharge of nematocysts probably pierces the prey, expediting toxin penetration. Toxin efficiency in sea water is further demonstrated by the rapid paralysis of fish or crustacean larvae upon application of recombinant Nv1 into their medium. Analysis of other anemone species reveals that in Anthopleura elegantissima, Type I neurotoxins also appear in gland cells, whereas in the common species Anemonia viridis, Type I toxins are localized to both nematocytes and ectodermal gland cells. The nematocyte-based and gland cell-based envenomation mechanisms may reflect substantial differences in the ecology and feeding habits of sea anemone species. Overall, the immunolocalization of neurotoxins to gland cells changes the common view in the literature that sea anemone neurotoxins are produced and delivered only by stinging nematocytes, and raises the possibility that this toxin-secretion mechanism is an ancestral evolutionary state of the venom delivery machinery in sea anemones.
Assuntos
Venenos de Cnidários/metabolismo , Neurotoxinas/metabolismo , Anêmonas-do-Mar/fisiologia , Animais , Artemia , Evolução Biológica , Venenos de Cnidários/genética , Venenos de Cnidários/toxicidade , Imuno-Histoquímica , Neurotoxinas/genética , Neurotoxinas/toxicidade , Comportamento Predatório , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Anêmonas-do-Mar/anatomia & histologia , Anêmonas-do-Mar/genética , Peixe-ZebraRESUMO
Scorpion ß-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin-based therapeutics to prevent lethality from scorpion envenomation.
Assuntos
Substituição de Aminoácidos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Biológicos , Venenos de Escorpião/genética , Venenos de Escorpião/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Animais , Sítios de Ligação , Mordeduras e Picadas/terapia , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Camundongos , Mutação , Ratos , Ratos Wistar , Venenos de Escorpião/antagonistas & inibidores , Venenos de Escorpião/uso terapêutico , EscorpiõesRESUMO
Alpha-neurotoxins target voltage-gated sodium channels (Na(v)s) and constitute an important component in the venom of Buthidae scorpions. These toxins are short polypeptides highly conserved in sequence and three-dimensional structure, and yet they differ greatly in activity and preference for insect and various mammalian Na(v)s. Despite extensive studies of the structure-function relationship of these toxins, only little is known about their evolution and phylogeny. Using a broad data set based on published sequences and rigorous cloning, we reconstructed a reliable phylogenetic tree of scorpion alpha-toxins and estimated the evolutionary forces involved in the diversification of their genes using maximum likelihood-based methods. Although the toxins are largely conserved, four positions were found to evolve under positive selection, of which two (10 and 18; numbered according to LqhalphaIT and Lqh2 from the Israeli yellow scorpion Leiurus quinquestriatus hebraeus) have been previously shown to affect toxin activity. The putative role of the other two positions (39 and 41) was analyzed by mutagenesis of Lqh2 and LqhalphaIT. Whereas substitution P41K in Lqh2 did not alter its activity, substitution K41P in LqhalphaIT significantly decreased the activity at insect and mammalian Na(v)s. Surprisingly, not only that substitution A39L in both toxins increased their activity by 10-fold but also LqhalphaIT(A39L) was active at the mammalian brain channel rNa(v)1.2a, which otherwise is hardly affected by LqhalphaIT, and Lqh2(A39L) was active at the insect channel, DmNa(v)1, which is almost insensitive to Lqh2. Thus, position 39 is involved not only in activity but also in toxin selectivity. Overall, this study describes evolutionary forces involved in the diversification of scorpion alpha-toxins, highlights the key role of positions under positive selection for selectivity and potency, and raises new questions as to the toxin-channel face of interaction.
Assuntos
Aminoácidos/genética , Evolução Molecular , Venenos de Escorpião/genética , Venenos de Escorpião/farmacologia , Seleção Genética , Sequência de Aminoácidos , Substituição de Aminoácidos/efeitos dos fármacos , Substituição de Aminoácidos/genética , Animais , Sequência de Bases , Insetos , Ativação do Canal Iônico/efeitos dos fármacos , Funções Verossimilhança , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/farmacologia , Filogenia , Ratos , Venenos de Escorpião/química , Venenos de Escorpião/metabolismo , Escorpiões/classificação , Escorpiões/genética , Canais de Sódio/metabolismoRESUMO
Many venomous organisms carry in their arsenal short polypeptides that block K+ channels in a highly selective manner. These toxins may compete with the permeating ions directly via a "plug" mechanism or indirectly via a "pore-collapse" mechanism. An alternative "lid" mechanism was proposed but remained poorly defined. Here we study the Drosophila Shaker channel block by Conkunitzin-S1 and Conkunitzin-C3, two highly similar toxins derived from cone venom. Despite their similarity, the two peptides exhibited differences in their binding poses and biophysical assays, implying discrete action modes. We show that while Conkunitzin-S1 binds tightly to the channel turret and acts via a "pore-collapse" mechanism, Conkunitzin-C3 does not contact this region. Instead, Conk-C3 uses a non-conserved Arg to divert the permeant ions and trap them in off-axis cryptic sites above the SF, a mechanism we term a "molecular-lid". Our study provides an atomic description of the "lid" K+ blocking mode and offers valuable insights for the design of therapeutics based on venom peptides.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Peptídeos/farmacologia , Canais de Potássio/metabolismo , Potássio/metabolismo , Venenos de Escorpião/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Biofísica/métodos , Xenopus laevis/metabolismoRESUMO
Scorpion α-toxins bind at the pharmacologically-defined site-3 on the sodium channel and inhibit channel inactivation by preventing the outward movement of the voltage sensor in domain IV (IVS4), whereas scorpion ß-toxins bind at site-4 on the sodium channel and enhance channel activation by trapping the voltage sensor of domain II (IIS4) in its outward position. However, limited information is available on the role of the voltage-sensing modules (VSM, comprising S1-S4) of domains I and III in toxin actions. We have previously shown that charge reversing substitutions of the innermost positively-charged residues in IIIS4 (R4E, R5E) increase the activity of an insect-selective site-4 scorpion toxin, Lqh-dprIT3-c, on BgNav1-1a, a cockroach sodium channel. Here we show that substitutions R4E and R5E in IIIS4 also increase the activity of two site-3 toxins, LqhαIT from Leiurusquinquestriatus hebraeus and insect-selective Av3 from Anemonia viridis. Furthermore, charge reversal of either of two conserved negatively-charged residues, D1K and E2K, in IIIS2 also increase the action of the site-3 and site-4 toxins. Homology modeling suggests that S2-D1 and S2-E2 interact with S4-R4 and S4-R5 in the VSM of domain III (III-VSM), respectively, in the activated state of the channel. However, charge swapping between S2-D1 and S4-R4 had no compensatory effects on gating or toxin actions, suggesting that charged residue interactions are complex. Collectively, our results highlight the involvement of III-VSM in the actions of both site 3 and site 4 toxins, suggesting that charge reversing substitutions in III-VSM allosterically facilitate IIS4 or IVS4 voltage sensor trapping by these toxins.
Assuntos
Venenos de Cnidários/farmacologia , Drosophila melanogaster/genética , Proteínas de Insetos/genética , Venenos de Escorpião/farmacologia , Canais de Sódio/genética , Animais , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Proteínas de Insetos/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Canais de Sódio/metabolismoRESUMO
The scorpion alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus) is active at various mammalian voltage-gated sodium channels (Na(v)s) and is inactive at insect Na(v)s. To resolve the molecular basis of this preference we used the following strategy: 1) Lqh2 was expressed in recombinant form and key residues important for activity at the rat brain channel rNa(v)1.2a were identified by mutagenesis. These residues form a bipartite functional surface made of a conserved "core domain" (residues of the loops connecting the secondary structure elements of the molecule core), and a variable "NC domain" (five-residue turn and the C-tail) as was reported for other scorpion alpha-toxins. 2) The functional role of the two domains was validated by their stepwise construction on the similar scaffold of the anti-insect toxin LqhalphaIT. Analysis of the activity of the intermediate constructs highlighted the critical role of Phe(15) of the core domain in toxin potency at rNa(v)1.2a, and has suggested that the shape of the NC-domain is important for toxin efficacy. 3) Based on these findings and by comparison with other scorpion alpha-toxins we were able to eliminate the activity of Lqh2 at rNa(v)1.4 (skeletal muscle), hNa(v)1.5 (cardiac), and rNa(v)1.6 channels, with no hindrance of its activity at Na(v)1.1-1.3. These results suggest that by employing a similar approach the design of further target-selective sodium channel modifiers is imminent.
Assuntos
Ativação do Canal Iônico , Venenos de Escorpião/química , Venenos de Escorpião/metabolismo , Canais de Sódio/metabolismo , Animais , Humanos , Modelos Moleculares , Mutagênese , Proteínas Mutantes/metabolismo , Estrutura Secundária de Proteína , Ratos , Ratos Wistar , Propriedades de Superfície , XenopusRESUMO
How is neuropathic pain regulated in peripheral sensory neurons? Importins are key regulators of nucleocytoplasmic transport. In this study, we found that importin α3 (also known as karyopherin subunit alpha 4) can control pain responsiveness in peripheral sensory neurons in mice. Importin α3 knockout or sensory neuron-specific knockdown in mice reduced responsiveness to diverse noxious stimuli and increased tolerance to neuropathic pain. Importin α3-bound c-Fos and importin α3-deficient neurons were impaired in c-Fos nuclear import. Knockdown or dominant-negative inhibition of c-Fos or c-Jun in sensory neurons reduced neuropathic pain. In silico screens identified drugs that mimic importin α3 deficiency. These drugs attenuated neuropathic pain and reduced c-Fos nuclear localization. Thus, perturbing c-Fos nuclear import by importin α3 in peripheral neurons can promote analgesia.
Assuntos
Dor Crônica/fisiopatologia , Neuralgia/fisiopatologia , Células Receptoras Sensoriais/fisiologia , alfa Carioferinas/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Benzofenonas/farmacologia , Dor Crônica/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Isoxazóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuralgia/genética , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição AP-1/metabolismo , alfa Carioferinas/genéticaRESUMO
Sea anemones are sessile predators that use a variety of toxins to paralyze prey and foe. Among these toxins, Types I, II and III are short peptides that affect voltage-gated sodium channels. Anemonia viridis is the only sea anemone species that produces both Types I and III neurotoxin. Although the two toxin types are unrelated in sequence and three-dimensional structure, cloning and comparative analysis of their loci revealed a highly similar sequence at the 5' region, which encodes a signal peptide. This similarity was likely generated by gene fusion and could be advantageous in transcript stability and intracellular trafficking and secretion. In addition, these analyses identified the processed pseudogenes of the two gene families in the genome of A. viridis, probably resulting from retrotransposition events. As presence of processed pseudogenes in the genome requires transcription in germ-line cells, we analyzed oocyte-rich ovaries and found that indeed they contain Types I and III transcripts. This result raises questions regarding the role of toxin transcripts in these tissues. Overall, the retrotransposition and gene fusion events suggest that the genes of both Types I and III neurotoxins evolved in a similar fashion and share a partial common ancestry.
Assuntos
Evolução Molecular , Fusão Gênica , Neurotoxinas/genética , Retroelementos/genética , Anêmonas-do-Mar/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , DNA Intergênico/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Neurotoxinas/química , Neurotoxinas/metabolismo , Filogenia , Pseudogenes/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Scorpion depressant beta-toxins show high preference for insect voltage-gated sodium channels (Na(v)s) and modulate their activation. Although their pharmacological and physiological effects were described, their three-dimensional structure and bioactive surface have never been determined. We utilized an efficient system for expression of the depressant toxin LqhIT2 (from Leiurus quinquestriatushebraeus), mutagenized its entire exterior, and determined its X-ray structure at 1.2 A resolution. The toxin molecule is composed of a conserved cysteine-stabilized alpha/beta-core (core-globule), and perpendicular to it an entity constituted from the N and C-terminal regions (NC-globule). The surface topology and overall hydrophobicity of the groove between the core and NC-globules (N-groove) is important for toxin activity and plays a role in selectivity to insect Na(v)s. The N-groove is flanked by Glu24 and Tyr28, which belong to the "pharmacophore" of scorpion beta-toxins, and by the side-chains of Trp53 and Asn58 that are important for receptor site recognition. Substitution of Ala13 by Trp in the N-groove uncoupled activity from binding, suggesting that this region of the molecule is also involved in "voltage-sensor trapping", the mode of action that typifies scorpion beta-toxins. The involvement of the N-groove in recognition of the receptor site, which seems to require a defined topology, as well as in sensor trapping, which involves interaction with a moving channel region, is puzzling. On the basis of the mutagenesis studies we hypothesize that following binding to the receptor site, the toxin undergoes a conformational change at the N-groove region that facilitates the trapping of the voltage-sensor in its activated position.
Assuntos
Venenos de Escorpião/química , Escorpiões/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Av3 is a short peptide toxin from the sea anemone Anemonia viridis shown to be active on crustaceans and inactive on mammals. It inhibits inactivation of Na(v)s (voltage-gated Na+ channels) like the structurally dissimilar scorpion alpha-toxins and type I sea anemone toxins that bind to receptor site-3. To examine the potency and mode of interaction of Av3 with insect Na(v)s, we established a system for its expression, mutagenized it throughout, and analysed it in toxicity, binding and electrophysiological assays. The recombinant Av3 was found to be highly toxic to blowfly larvae (ED50=2.65+/-0.46 pmol/100 mg), to compete well with the site-3 toxin LqhalphaIT (from the scorpion Leiurus quinquestriatus) on binding to cockroach neuronal membranes (K(i)=21.4+/-7.1 nM), and to inhibit the inactivation of Drosophila melanogaster channel, DmNa(v)1, but not that of mammalian Na(v)s expressed in Xenopus oocytes. Moreover, like other site-3 toxins, the activity of Av3 was synergically enhanced by ligands of receptor site-4 (e.g. scorpion beta-toxins). The bioactive surface of Av3 was found to consist mainly of aromatic residues and did not resemble any of the bioactive surfaces of other site-3 toxins. These analyses have portrayed a toxin that might interact with receptor site-3 in a different fashion compared with other ligands of this site. This assumption was corroborated by a D1701R mutation in DmNa(v)1, which has been shown to abolish the activity of all other site-3 ligands, except Av3. All in all, the present study provides further evidence for the heterogeneity of receptor site-3, and raises Av3 as a unique model for design of selective anti-insect compounds.