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1.
J Med Chem ; 63(21): 12773-12785, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33078946

RESUMO

Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of NaV channel ligands. We conjugated µ-conotoxin KIIIA, which occludes the pore of the NaV channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40-120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4-24-fold higher potency compared to those of the monovalent peptides for the human NaV1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of NaV channels.


Assuntos
Ligantes , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Potenciais de Ação/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Conotoxinas/química , Conotoxinas/metabolismo , Reação de Cicloadição , Humanos , Concentração Inibidora 50 , Cinética , Simulação de Acoplamento Molecular , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.7/química , Técnicas de Patch-Clamp , Polietilenos/química , Venenos de Aranha/síntese química , Venenos de Aranha/química , Venenos de Aranha/metabolismo , Aranhas/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/síntese química , Bloqueadores do Canal de Sódio Disparado por Voltagem/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia
2.
Int J Mol Sci ; 21(7)2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32276507

RESUMO

Myotonia congenita (MC) is a rare disorder characterized by stiffness and weakness of the limb and trunk muscles. Mutations in the SCN4A gene encoding the alpha-subunit of the voltage-gated sodium channel Nav1.4 have been reported to be responsible for sodium channel myotonia (SCM). The Nav1.4 channel is expressed in skeletal muscles, and its related channelopathies affect skeletal muscle excitability, which can manifest as SCM, paramyotonia and periodic paralysis. In this study, the missense mutation p.V445M was identified in two individual families with MC. To determine the functional consequences of having a mutated Nav1.4 channel, whole-cell patch-clamp recording of transfected Chinese hamster ovary cells was performed. Evaluation of the transient Na+ current found that a hyperpolarizing shift occurs at both the activation and inactivation curves with an increase of the window currents in the mutant channels. The Nav1.4 channel's co-expression with the Navß4 peptide can generate resurgent Na+ currents at repolarization following a depolarization. The magnitude of the resurgent currents is higher in the mutant than in the wild-type (WT) channel. Although the decay kinetics are comparable between the mutant and WT channels, the time to the peak of resurgent Na+ currents in the mutant channel is significantly protracted compared with that in the WT channel. These findings suggest that the p.V445M mutation in the Nav1.4 channel results in an increase of both sustained and resurgent Na+ currents, which may contribute to hyperexcitability with repetitive firing and is likely to facilitate recurrent myotonia in SCM patients.


Assuntos
Mutação de Sentido Incorreto , Miotonia Congênita/genética , Miotonia Congênita/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.4/fisiologia , Sequência de Aminoácidos , Animais , Grupo com Ancestrais do Continente Asiático , Células CHO , Canalopatias/genética , Canalopatias/metabolismo , Canalopatias/fisiopatologia , Cricetulus , Feminino , Humanos , Masculino , Miotonia Congênita/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Técnicas de Patch-Clamp , Linhagem
3.
Biochim Biophys Acta Biomembr ; 1862(2): 183129, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31738900

RESUMO

Slow inactivation in voltage-gated Na+ channels (Navs) plays an important physiological role in excitable tissues (muscle, heart, nerves) and mutations that disrupt Nav slow inactivation can result in pathophysiologies (myotonia, arrhythmias, epilepsy). While the molecular mechanisms responsible for slow inactivation remain elusive, previous studies have suggested a role for the pore-lining D1-S6 helix. The goals of this research were to determine if (1) cysteine substitutions in D1-S6 affect gating kinetics and (2) methanethiosulfonate ethylammonium (MTSEA) accessibility changes in different kinetic states. Site-directed mutagenesis in the human skeletal muscle isoform hNav1.4 was used to substitute cysteine for eleven amino acids in D1-S6 from L433 to L443. Mutants were expressed in HEK cells and recorded from with whole-cell patch clamp. All mutations affected one or more baseline kinetics of the sodium channel, including activation, fast inactivation, and slow inactivation. Substitution of cysteine (for nonpolar residues) adjacent to polar residues destabilized slow inactivation in G434C, F436C, I439C, and L441C. Cysteine substitution without adjacent polar residues enhanced slow inactivation in L438C and N440C, and disrupted possible H-bonds involving Y437:D4 S4-S5 and N440:D4-S6. MTSEA exposure in closed, fast-inactivated, or slow-inactivated states in most mutants had little-to-no effect. In I439C, MTSEA application in closed, fast-inactivated, and slow-inactivated states produced irreversible reduction in current, suggesting I439C accessibility to MTSEA in all three kinetic states. D1-S6 is important for Nav gating kinetics, stability of slow-inactivated state, structural contacts, and state-dependent positioning. However, prominent reconfiguration of D1-S6 may not occur in slow inactivation.


Assuntos
Substituição de Aminoácidos , Cisteína/genética , Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.4/química , Cisteína/química , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/química , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Ligação Proteica , Domínios Proteicos
4.
Mar Drugs ; 17(7)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269696

RESUMO

Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer's activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble.


Assuntos
Conotoxinas/farmacocinética , Canal de Sódio Disparado por Voltagem NAV1.4/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Conotoxinas/química , Dissulfetos/química , Isomerismo , Simulação de Acoplamento Molecular , Conformação Proteica , Eletricidade Estática , Relação Estrutura-Atividade , Bloqueadores do Canal de Sódio Disparado por Voltagem/química
5.
J Biomol NMR ; 73(10-11): 531-544, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31281943

RESUMO

Assignment of backbone resonances is a necessary initial step in every protein NMR investigation. Standard assignment procedure is based on the set of 3D triple-resonance (1H-13C-15N) spectra and requires at least several days of experimental measurements. This limits its application to the proteins with low stability. To speed up the assignment procedure, combinatorial selective labeling (CSL) can be used. In this case, sequence-specific information is extracted from 2D spectra measured for several selectively 13C,15N-labeled samples, produced in accordance with a special CSL scheme. Here we review previous applications of the CSL approach and present novel deterministic 'CombLabel' algorithm, which generates CSL schemes minimizing the number of labeled samples and their price and maximizing assignment information that can be obtained for a given protein sequence. Theoretical calculations revealed that CombLabel software outperformed previously proposed stochastic algorithms. Current implementation of CombLabel robustly calculates CSL schemes containing up to six samples, which is sufficient for moderately sized (up to 200 residues) proteins. As a proof of concept, we calculated CSL scheme for the first voltage-sensing domain of human Nav1.4 channel, a 134 residue four helical transmembrane protein having extremely low stability in micellar solution (half-life ~ 24 h at 45 °C). Application of CSL doubled the extent of backbone resonance assignment, initially obtained by conventional approach. The obtained assignment coverage (~ 50%) is sufficient for ligand screening and mapping of binding interfaces.


Assuntos
Sequência de Aminoácidos , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular/métodos , Humanos , Canal de Sódio Disparado por Voltagem NAV1.4/química , Estudo de Prova de Conceito , Ligação Proteica , Domínios Proteicos , Software , Coloração e Rotulagem , Fatores de Tempo
6.
Nat Commun ; 10(1): 1514, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944319

RESUMO

Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2+-dependent inactivation; no such inactivation is observed in the cardiac Na+ channel (NaV1.5). Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM's N-lobe binding outside the NaV C-terminal while CaM's C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Sítios de Ligação , Cálcio/química , Calmodulina/química , Calmodulina/genética , Humanos , Modelos Moleculares , Músculo Esquelético/metabolismo , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas
7.
Biochem Biophys Res Commun ; 506(4): 826-832, 2018 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389137

RESUMO

Voltage-gated sodium channels play important roles in human physiology. However, their complexity hinders the understanding of their physiology and pathology at atomic level. We took advantage of the structural reports of similar channels obtained by cryo-EM (EeNav1.4, and NavPaS), and constructed models of human Nav1.4 channels at closed and open states. The open-state model is very similar to the recently published cryo-EM structure of hNav1.4. The comparison of both models shows shifts of the voltage sensors (VS) of DIII and DIV. The activated position of VS-DII in the closed model was demonstrated by Ts1 docking, thereby confirming the requirement that VS-DI, VS-DII and VS-DIII must be activated for the channel to open. The interactions observed with VS-DIII suggest a stepwise, yet fast, transition from resting to activated state. These models provide structural insights on the closed-open transition of the channel.


Assuntos
Ativação do Canal Iônico , Modelos Biológicos , Músculo Esquelético/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Humanos , Simulação de Acoplamento Molecular
8.
Science ; 362(6412)2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30190309

RESUMO

Voltage-gated sodium (Nav) channels, which are responsible for action potential generation, are implicated in many human diseases. Despite decades of rigorous characterization, the lack of a structure of any human Nav channel has hampered mechanistic understanding. Here, we report the cryo-electron microscopy structure of the human Nav1.4-ß1 complex at 3.2-Å resolution. Accurate model building was made for the pore domain, the voltage-sensing domains, and the ß1 subunit, providing insight into the molecular basis for Na+ permeation and kinetic asymmetry of the four repeats. Structural analysis of reported functional residues and disease mutations corroborates an allosteric blocking mechanism for fast inactivation of Nav channels. The structure provides a path toward mechanistic investigation of Nav channels and drug discovery for Nav channelopathies.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.4/química , Subunidade beta-4 do Canal de Sódio Disparado por Voltagem/química , Regulação Alostérica , Sequência de Aminoácidos , Canalopatias/genética , Canalopatias/metabolismo , Microscopia Crioeletrônica , Descoberta de Drogas , Células HEK293 , Humanos , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/ultraestrutura , Domínios Proteicos , Subunidade beta-4 do Canal de Sódio Disparado por Voltagem/genética , Subunidade beta-4 do Canal de Sódio Disparado por Voltagem/ultraestrutura
9.
Biomol NMR Assign ; 12(2): 283-289, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29728980

RESUMO

Human voltage-gated sodium (NaV) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane ß-subunits. NaV pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all NaV isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of NaV1.2, NaV1.5, and NaV1.6 showed that CaM binding was mediated by the C-domain of CaM (CaMC), while the N-domain (CaMN) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other NaV isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of NaV1.1, NaV1.4, and NaV1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaMC; resonances of CaMN were identical to free CaM. Thus, CaMC residues contribute to the interface with the IQ motif, while CaMN is available to interact elsewhere on the channel.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Canais de Sódio Disparados por Voltagem/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1/química , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/química , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canais de Sódio Disparados por Voltagem/química
10.
Nature ; 557(7706): 590-594, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29769724

RESUMO

Potassium-sensitive hypokalaemic and normokalaemic periodic paralysis are inherited skeletal muscle diseases characterized by episodes of flaccid muscle weakness1,2. They are caused by single mutations in positively charged residues ('gating charges') in the S4 transmembrane segment of the voltage sensor of the voltage-gated sodium channel Nav1.4 or the calcium channel Cav1.11,2. Mutations of the outermost gating charges (R1 and R2) cause hypokalaemic periodic paralysis1,2 by creating a pathogenic gating pore in the voltage sensor through which cations leak in the resting state3,4. Mutations of the third gating charge (R3) cause normokalaemic periodic paralysis 5 owing to cation leak in both activated and inactivated states 6 . Here we present high-resolution structures of the model bacterial sodium channel NavAb with the analogous gating-charge mutations7,8, which have similar functional effects as in the human channels. The R2G and R3G mutations have no effect on the backbone structures of the voltage sensor, but they create an aqueous cavity near the hydrophobic constriction site that controls gating charge movement through the voltage sensor. The R3G mutation extends the extracellular aqueous cleft through the entire length of the activated voltage sensor, creating an aqueous path through the membrane. Conversely, molecular modelling shows that the R2G mutation creates a continuous aqueous path through the membrane only in the resting state. Crystal structures of NavAb(R2G) in complex with guanidinium define a potential drug target site. Molecular dynamics simulations illustrate the mechanism of Na+ permeation through the mutant gating pore in concert with conformational fluctuations of the gating charge R4. Our results reveal pathogenic mechanisms of periodic paralysis at the atomic level and suggest designs of drugs that may prevent ionic leak and provide symptomatic relief from hypokalaemic and normokalaemic periodic paralysis.


Assuntos
Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Paralisias Periódicas Familiares/metabolismo , Sítios de Ligação , Condutividade Elétrica , Guanidina/metabolismo , Humanos , Paralisia Periódica Hipopotassêmica/genética , Paralisia Periódica Hipopotassêmica/metabolismo , Ativação do Canal Iônico/genética , Simulação de Dinâmica Molecular , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Paralisias Periódicas Familiares/genética , Sódio/metabolismo , Termodinâmica
11.
Proc Natl Acad Sci U S A ; 115(17): 4495-4500, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29636418

RESUMO

Gating pore currents through the voltage-sensing domains (VSDs) of the skeletal muscle voltage-gated sodium channel NaV1.4 underlie hypokalemic periodic paralysis (HypoPP) type 2. Gating modifier toxins target ion channels by modifying the function of the VSDs. We tested the hypothesis that these toxins could function as blockers of the pathogenic gating pore currents. We report that a crab spider toxin Hm-3 from Heriaeus melloteei can inhibit gating pore currents due to mutations affecting the second arginine residue in the S4 helix of VSD-I that we have found in patients with HypoPP and describe here. NMR studies show that Hm-3 partitions into micelles through a hydrophobic cluster formed by aromatic residues and reveal complex formation with VSD-I through electrostatic and hydrophobic interactions with the S3b helix and the S3-S4 extracellular loop. Our data identify VSD-I as a specific binding site for neurotoxins on sodium channels. Gating modifier toxins may constitute useful hits for the treatment of HypoPP.


Assuntos
Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Neurotoxinas/toxicidade , Paralisia Periódica Hiperpotassêmica/metabolismo , Estrutura Secundária de Proteína , Venenos de Aranha/toxicidade , Substituição de Aminoácidos , Animais , Feminino , Células HEK293 , Humanos , Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Paralisia Periódica Hiperpotassêmica/genética , Paralisia Periódica Hiperpotassêmica/patologia , Xenopus laevis
12.
Curr Med Chem ; 25(42): 5822-5834, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29589531

RESUMO

Tocainide is an antiarrhythmic agent belonging to class IB that was primarily used for suppression of symptomatic ventricular arrhythmias. Tocainide was also reported to relieve pain such as tic douloureux, trigemina neuralgia in humans and tinnitus. Significant antinociception, as assayed on the hot-plate test, was observed after intraperitoneal injection of tocainide, too. By the mid-1980s tocainide was emerging as a more consistently effective treatment for myotonic disorders. Numerous reports of serious adverse reactions led to the use of tocainide being discontinued, even though research on tocainide and its analogues, endowed with a better pharmacological profile, is still in progress for their potential usefulness in the treatment of myotonias. This review is focused on the description of the different synthetic routes to racemic and optically active tocainide developed in the last decades, as well as analytical studies regarding enantioseparation methods. Finally, some analogues of tocainide reported in the literature, most of which with pharmacological studies, have been mentioned.


Assuntos
Antiarrítmicos/síntese química , Tocainide/análogos & derivados , Antiarrítmicos/farmacocinética , Antiarrítmicos/uso terapêutico , Arritmias Cardíacas/tratamento farmacológico , Meia-Vida , Humanos , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Relação Quantitativa Estrutura-Atividade , Tocainide/farmacocinética , Tocainide/uso terapêutico , Bloqueadores do Canal de Sódio Disparado por Voltagem/síntese química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico
13.
Sci Rep ; 8(1): 2041, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29391559

RESUMO

Mutations in NaV1.4, the skeletal muscle voltage-gated Na+ channel, underlie several skeletal muscle channelopathies. We report here the functional characterization of two substitutions targeting the R1451 residue and resulting in 3 distinct clinical phenotypes. The R1451L is a novel pathogenic substitution found in two unrelated individuals. The first individual was diagnosed with non-dystrophic myotonia, whereas the second suffered from an unusual phenotype combining hyperkalemic and hypokalemic episodes of periodic paralysis (PP). The R1451C substitution was found in one individual with a single attack of hypoPP induced by glucocorticoids. To elucidate the biophysical mechanism underlying the phenotypes, we used the patch-clamp technique to study tsA201 cells expressing WT or R1451C/L channels. Our results showed that both substitutions shifted the inactivation to hyperpolarized potentials, slowed the kinetics of inactivation, slowed the recovery from slow inactivation and reduced the current density. Cooling further enhanced these abnormalities. Homology modeling revealed a disruption of hydrogen bonds in the voltage sensor domain caused by R1451C/L. We concluded that the altered biophysical properties of R1451C/L well account for the PMC-hyperPP cluster and that additional factors likely play a critical role in the inter-individual differences of clinical expression resulting from R1451C/L.


Assuntos
Mutação de Sentido Incorreto , Transtornos Miotônicos/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética
14.
J Biol Chem ; 292(44): 18270-18280, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-28924048

RESUMO

Scorpion toxins can kill other animals by inducing paralysis and arrhythmia, which limits the potential applications of these agents in the clinical management of diseases. Antitumor-analgesic peptide (AGAP), purified from Buthus martensii Karsch, has been proved to possess analgesic and antitumor activities. Trp38, a conserved aromatic residue of AGAP, might play an important role in mediating AGAP activities according to the sequence and homology-modeling analyses. Therefore, an AGAP mutant, W38G, was generated, and effects of both AGAP and the mutant W38G were examined by whole-cell patch clamp techniques on the sodium channels hNav1.4 and hNav1.5, which were closely associated with the biotoxicity of skeletal and cardiac muscles, respectively. The data showed that both W38G and AGAP inhibited the peak currents of hNav1.4 and hNav1.5; however, W38G induced a much weaker inhibition of both channels than AGAP. Accordingly, W38G exhibited much less toxic effect on both skeletal and cardiac muscles than AGAP in vivo The analgesic activity of W38G and AGAP were verified in vivo as well, and W38G retained analgesic activity similar to AGAP. Inhibition to both Nav1.7 and Nav1.8 was involved in the analgesic mechanism of AGAP and W38G. These findings indicated that Trp38 was a key amino acid involved in the biotoxicity of AGAP, and the AGAP mutant W38G might be a safer alternative for clinical application because it retains the analgesic efficacy with less toxicity to skeletal and cardiac muscles.


Assuntos
Analgésicos não Entorpecentes/efeitos adversos , Antineoplásicos/efeitos adversos , Proteínas de Artrópodes/efeitos adversos , Mutação , Peptídeos/efeitos adversos , Venenos de Escorpião/efeitos adversos , Bloqueadores do Canal de Sódio Disparado por Voltagem/efeitos adversos , Substituição de Aminoácidos , Analgésicos não Entorpecentes/administração & dosagem , Analgésicos não Entorpecentes/farmacologia , Analgésicos não Entorpecentes/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/farmacologia , Proteínas de Artrópodes/uso terapêutico , Células CHO , Cricetulus , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Peptídeos/genética , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Distribuição Aleatória , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Venenos de Escorpião/genética , Venenos de Escorpião/farmacologia , Venenos de Escorpião/uso terapêutico , Escorpiões , Testes de Toxicidade Aguda , Testes de Toxicidade Subaguda , Bloqueadores do Canal de Sódio Disparado por Voltagem/administração & dosagem , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico
15.
Peptides ; 91: 13-19, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28300672

RESUMO

Non-Buthidae venomous scorpions are huge natural sources of toxin peptides; however, only a few studies have been done to understand their toxin peptides. Herein, we describe three new potential immunomodulating toxin peptides, Ctri18, Ctry68 and Ctry2908, from two non-Buthidae scorpions, Chaerilus tricostatus and Chaerilus tryznai. Sequence alignment analyses showed that Ctri18, Ctry68 and Ctry2908 are three new members of the scorpion toxin α-KTx15 subfamily. Electrophysiological experiments showed that Ctri18, Ctry68 and Ctry2908 blocked the Kv1.3 channel at micromole to nanomole levels, but had weak effects on potassium channel KCNQ1 and sodium channel Nav1.4, which indicated that Ctri18, Ctry68 and Ctry2908 might have specific inhibiting effects on the Kv1.3 channel. ELISA experiments showed that Ctri18, Ctry68 and Ctry2908 inhibited IL-2 cytokine secretions of activated T lymphocyte in human PBMCs. Excitingly, consistent with the good Kv1.3 channel inhibitory activity, Ctry2908 inhibited cytokine IL-2 secretion in nanomole level, which indicated that Ctry2908 might be a new lead drug template toward Kv1.3 channels. Together, these studies discovered three new toxin peptides, Ctri18, Ctry68 and Ctry2908, with Kv1.3 channel and IL-2 cytokine-inhibiting activities from two scorpions, C. tricostatus and C. tryznai, and highlighted that non-Buthidae venomous scorpions are new natural toxin peptide sources.


Assuntos
Interleucina-2/antagonistas & inibidores , Canal de Potássio Kv1.3/antagonistas & inibidores , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Escorpiões/química , Adulto , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , Relação Dose-Resposta a Droga , Feminino , Humanos , Canal de Potássio KCNQ1/antagonistas & inibidores , Masculino , Modelos Moleculares , Canal de Sódio Disparado por Voltagem NAV1.4/química , Peptídeos/química , Peptídeos/genética , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/isolamento & purificação , Bloqueadores dos Canais de Potássio/farmacologia , Venenos de Escorpião/genética , Venenos de Escorpião/isolamento & purificação , Escorpiões/genética , Linfócitos T/química
16.
J Gen Physiol ; 149(4): 465-481, 2017 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-28258204

RESUMO

Local anesthetics, antiarrhythmics, and anticonvulsants include both charged and electroneutral compounds that block voltage-gated sodium channels. Prior studies have revealed a common drug-binding region within the pore, but details about the binding sites and mechanism of block remain unclear. Here, we use the x-ray structure of a prokaryotic sodium channel, NavMs, to model a eukaryotic channel and dock representative ligands. These include lidocaine, QX-314, cocaine, quinidine, lamotrigine, carbamazepine (CMZ), phenytoin, lacosamide, sipatrigine, and bisphenol A. Preliminary calculations demonstrated that a sodium ion near the selectivity filter attracts electroneutral CMZ but repels cationic lidocaine. Therefore, we further docked electroneutral and cationic drugs with and without a sodium ion, respectively. In our models, all the drugs interact with a phenylalanine in helix IVS6. Electroneutral drugs trap a sodium ion in the proximity of the selectivity filter, and this same site attracts the charged group of cationic ligands. At this position, even small drugs can block the permeation pathway by an electrostatic or steric mechanism. Our study proposes a common pharmacophore for these diverse drugs. It includes a cationic moiety and an aromatic moiety, which are usually linked by four bonds.


Assuntos
Anestésicos Locais/farmacologia , Antiarrítmicos/farmacologia , Anticonvulsivantes/farmacologia , Simulação de Acoplamento Molecular , Canal de Sódio Disparado por Voltagem NAV1.4/química , Bloqueadores dos Canais de Sódio/farmacologia , Acetamidas/farmacologia , Animais , Compostos Benzidrílicos/farmacologia , Sítios de Ligação , Carbamazepina/farmacologia , Cocaína/farmacologia , Humanos , Lacosamida , Lamotrigina , Lidocaína/análogos & derivados , Lidocaína/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Fenóis/farmacologia , Fenitoína/farmacologia , Piperazinas/farmacologia , Ligação Proteica , Pirimidinas/farmacologia , Quinidina/farmacologia , Triazinas/farmacologia
17.
Eur J Pharmacol ; 796: 215-223, 2017 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-28057491

RESUMO

Mefloquine constitutes a multitarget antimalaric that inhibits cation currents. However, the effect and the binding site of this compound on Na+ channels is unknown. To address the mechanism of action of mefloquine, we employed two-electrode voltage clamp recordings on Xenopus laevis oocytes, site-directed mutagenesis of the rat Na+ channel, and a combined in silico approach using Molecular Dynamics and docking protocols. We found that mefloquine: i) inhibited Nav1.4 currents (IC50 =60µM), ii) significantly delayed fast inactivation but did not affect recovery from inactivation, iii) markedly the shifted steady-state inactivation curve to more hyperpolarized potentials. The presence of the ß1 subunit significantly reduced mefloquine potency, but the drug induced a significant frequency-independent rundown upon repetitive depolarisations. Computational and experimental results indicate that mefloquine overlaps the local anaesthetic binding site by docking at a hydrophobic cavity between domains DIII and DIV that communicates the local anaesthetic binding site with the selectivity filter. This is supported by the fact that mefloquine potency significantly decreased on mutant Nav1.4 channel F1579A and significantly increased on K1237S channels. In silico this compound docked above F1579 forming stable π-π interactions with this residue. We provide structure-activity insights into how cationic amphiphilic compounds may exert inhibitory effects by docking between the local anaesthetic binding site and the selectivity filter of a mammalian Na+ channel. Our proposed synergistic cycle of experimental and computational studies may be useful for elucidating binding sites of other drugs, thereby saving in vitro and in silico resources.


Assuntos
Anestésicos Locais/metabolismo , Anestésicos Locais/farmacologia , Mefloquina/metabolismo , Mefloquina/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Sítios de Ligação , Relação Dose-Resposta a Droga , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Lidocaína/metabolismo , Lidocaína/farmacologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Conformação Proteica , Ratos
18.
Biochim Biophys Acta Biomembr ; 1859(3): 493-506, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28065835

RESUMO

Voltage-gated Na+ channels are essential for the functioning of cardiovascular, muscular, and nervous systems. The α-subunit of eukaryotic Na+ channel consists of ~2000 amino acid residues and encloses 24 transmembrane (TM) helices, which form five membrane domains: four voltage-sensing (VSD) and one pore domain. The structural complexity significantly impedes recombinant production and structural studies of full-sized Na+ channels. Modular organization of voltage-gated channels gives an idea for studying of the isolated second VSD of human skeletal muscle Nav1.4 channel (VSD-II). Several variants of VSD-II (~150a.a., four TM helices) with different N- and C-termini were produced by cell-free expression. Screening of membrane mimetics revealed low stability of VSD-II samples in media containing phospholipids (bicelles, nanodiscs) associated with the aggregation of electrically neutral domain molecules. The almost complete resonance assignment of 13C,15N-labeled VSD-II was obtained in LPPG micelles. The secondary structure of VSD-II showed similarity with the structures of bacterial Na+ channels. The fragment of S4 TM helix between the first and second conserved Arg residues probably adopts 310-helical conformation. Water accessibility of S3 helix, observed by the Mn2+ titration, pointed to the formation of water-filled crevices in the micelle embedded VSD-II. 15N relaxation data revealed characteristic pattern of µs-ms time scale motions in the VSD-II regions sharing expected interhelical contacts. VSD-II demonstrated enhanced mobility at ps-ns time scale as compared to isolated VSDs of K+ channels. These results validate structural studies of isolated VSDs of Na+ channels and show possible pitfalls in application of this 'divide and conquer' approach.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.4/química , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Sistema Livre de Células , Glicolipídeos/química , Humanos , Fosfatos de Inositol/química , Manganês/química , Micelas , Músculo Esquelético/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Estrutura Secundária de Proteína , Alinhamento de Sequência
19.
Eur Biophys J ; 46(5): 485-494, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28012039

RESUMO

The mechanism of inactivation of mammalian voltage-gated Na+ channels involves transient interactions between intracellular domains resulting in direct pore occlusion by the IFM motif and concomitant extracellular interactions with the ß1 subunit. Navß1 subunits constitute single-pass transmembrane proteins that form protein-protein associations with pore-forming α subunits to allosterically modulate the Na+ influx into the cell during the action potential of every excitable cell in vertebrates. Here, we explored the role of the intracellular IFM motif of rNav1.4 (skeletal muscle isoform of the rat Na+ channel) on the α-ß1 functional interaction and showed for the first time that the modulation of ß1 is independent of the IFM motif. We found that: (1) Nav1.4 channels that lack the IFM inactivation particle can undergo a "C-type-like inactivation" albeit in an ultraslow gating mode; (2) ß1 can significantly accelerate the inactivation of Nav1.4 channels in the absence of the IFM motif. Previously, we identified two residues (T109 and N110) on the ß1 subunit that disrupt the α-ß1 allosteric modulation. We further characterized the electrophysiological effects of the double alanine substitution of these residues demonstrating that it decelerates inactivation and recovery from inactivation, abolishes the modulation of steady-state inactivation and induces a current rundown upon repetitive stimulation, thus causing a general loss of function. Our results contribute to delineating the process of the mammalian Na+ channel inactivation. These findings may be relevant to the design of pharmacological strategies, targeting ß subunits to treat pathologies associated to Na+ current dysfunction.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/química , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/metabolismo , Regulação Alostérica , Motivos de Aminoácidos , Animais , Fenômenos Eletrofisiológicos , Espaço Intracelular/metabolismo , Cinética , Modelos Moleculares , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Ratos
20.
Elife ; 52016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27710770

RESUMO

C-type inactivation of potassium channels fine-tunes the electrical signaling in excitable cells through an internal timing mechanism that is mediated by a hydrogen bond network in the channels' selectively filter. Previously, we used nonsense suppression to highlight the role of the conserved Trp434-Asp447 indole hydrogen bond in Shaker potassium channels with a non-hydrogen bonding homologue of tryptophan, Ind (Pless et al., 2013). Here, molecular dynamics simulations indicate that the Trp434Ind hydrogen bonding partner, Asp447, unexpectedly 'flips out' towards the extracellular environment, allowing water to penetrate the space behind the selectivity filter while simultaneously reducing the local negative electrostatic charge. Additionally, a protein engineering approach is presented whereby split intein sequences are flanked by endoplasmic reticulum retention/retrieval motifs (ERret) are incorporated into the N- or C- termini of Shaker monomers or within sodium channels two-domain fragments. This system enabled stoichiometric control of Shaker monomers and the encoding of multiple amino acids within a channel tetramer.


Assuntos
Potenciais da Membrana/fisiologia , Mutagênese Sítio-Dirigida/métodos , Canal de Sódio Disparado por Voltagem NAV1.4/química , Proteínas Recombinantes de Fusão/química , Superfamília Shaker de Canais de Potássio/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Expressão Gênica , Células HEK293 , Humanos , Ativação do Canal Iônico , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Oócitos/citologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Termodinâmica , Xenopus laevis
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