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1.
Cell ; 184(20): 5151-5162.e11, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34520724

RESUMO

The heartbeat is initiated by voltage-gated sodium channel NaV1.5, which opens rapidly and triggers the cardiac action potential; however, the structural basis for pore opening remains unknown. Here, we blocked fast inactivation with a mutation and captured the elusive open-state structure. The fast inactivation gate moves away from its receptor, allowing asymmetric opening of pore-lining S6 segments, which bend and rotate at their intracellular ends to dilate the activation gate to ∼10 Å diameter. Molecular dynamics analyses predict physiological rates of Na+ conductance. The open-state pore blocker propafenone binds in a high-affinity pose, and drug-access pathways are revealed through the open activation gate and fenestrations. Comparison with mutagenesis results provides a structural map of arrhythmia mutations that target the activation and fast inactivation gates. These results give atomic-level insights into molecular events that underlie generation of the action potential, open-state drug block, and fast inactivation of cardiac sodium channels, which initiate the heartbeat.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Animais , Arritmias Cardíacas/genética , Microscopia Crioeletrônica , Células HEK293 , Frequência Cardíaca/efeitos dos fármacos , Humanos , Ativação do Canal Iônico , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação/genética , Miocárdio , Canal de Sódio Disparado por Voltagem NAV1.5/isolamento & purificação , Canal de Sódio Disparado por Voltagem NAV1.5/ultraestrutura , Propafenona/farmacologia , Conformação Proteica , Ratos , Sódio/metabolismo , Fatores de Tempo , Água/química
2.
Cell ; 180(1): 122-134.e10, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31866066

RESUMO

Voltage-gated sodium channel Nav1.5 generates cardiac action potentials and initiates the heartbeat. Here, we report structures of NaV1.5 at 3.2-3.5 Å resolution. NaV1.5 is distinguished from other sodium channels by a unique glycosyl moiety and loss of disulfide-bonding capability at the NaVß subunit-interaction sites. The antiarrhythmic drug flecainide specifically targets the central cavity of the pore. The voltage sensors are partially activated, and the fast-inactivation gate is partially closed. Activation of the voltage sensor of Domain III allows binding of the isoleucine-phenylalanine-methionine (IFM) motif to the inactivation-gate receptor. Asp and Ala, in the selectivity motif DEKA, line the walls of the ion-selectivity filter, whereas Glu and Lys are in positions to accept and release Na+ ions via a charge-delocalization network. Arrhythmia mutation sites undergo large translocations during gating, providing a potential mechanism for pathogenic effects. Our results provide detailed insights into Nav1.5 structure, pharmacology, activation, inactivation, ion selectivity, and arrhythmias.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/ultraestrutura , Animais , Linhagem Celular , Células HEK293 , Coração/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Sódio/metabolismo , Canais de Sódio/química , Relação Estrutura-Atividade , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/ultraestrutura
3.
Cell ; 176(4): 702-715.e14, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30661758

RESUMO

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/ultraestrutura , Peptídeos/metabolismo , Venenos de Aranha/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetulus , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , Células HEK293 , Humanos , Ativação do Canal Iônico , Peptídeos/toxicidade , Domínios Proteicos , Venenos de Aranha/toxicidade , Aranhas , Bloqueadores do Canal de Sódio Disparado por Voltagem , Canais de Sódio Disparados por Voltagem/metabolismo
4.
Cell ; 178(4): 993-1003.e12, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31353218

RESUMO

Voltage-gated sodium (NaV) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaVAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.


Assuntos
Potenciais de Ação/fisiologia , Membrana Externa Bacteriana/metabolismo , Escherichia coli/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Linhagem Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Mutação , Conformação Proteica em alfa-Hélice , Sódio/metabolismo , Spodoptera/citologia , Canais de Sódio Disparados por Voltagem/química
5.
Mol Cell ; 82(13): 2427-2442.e4, 2022 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-35597238

RESUMO

The voltage-gated ion channel activity depends on both activation (transition from the resting state to the open state) and inactivation. Inactivation is a self-restraint mechanism to limit ion conduction and is as crucial to membrane excitability as activation. Inactivation can occur when the channel is open or closed. Although open-state inactivation is well understood, the molecular basis of closed-state inactivation has remained elusive. We report cryo-EM structures of human KV4.2 channel complexes in inactivated, open, and closed states. Closed-state inactivation of KV4 involves an unprecedented symmetry breakdown for pore closure by only two of the four S4-S5 linkers, distinct from known mechanisms of open-state inactivation. We further capture KV4 in a putative resting state, revealing how voltage sensor movements control the pore. Moreover, our structures provide insights regarding channel modulation by KChIP2 and DPP6 auxiliary subunits. Our findings elucidate mechanisms of closed-state inactivation and voltage-dependent activation of the KV4 channel.


Assuntos
Ativação do Canal Iônico , Canais de Potássio Shal , Humanos , Ativação do Canal Iônico/fisiologia , Cinética , Potenciais da Membrana/fisiologia , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo
6.
Mol Cell ; 81(1): 38-48.e4, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33232657

RESUMO

Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting state. The structural basis for tarantula-toxin action remains elusive because of the difficulty of capturing the functionally relevant form of the toxin-channel complex. Here, we engineered the model sodium channel NaVAb with voltage-shifting mutations and the toxin-binding site of human NaV1.7, an attractive pain target. This mutant chimera enabled us to determine the cryoelectron microscopy (cryo-EM) structure of the channel functionally arrested by tarantula toxin. Our structure reveals a high-affinity resting-state-specific toxin-channel interaction between a key lysine residue that serves as a "stinger" and penetrates a triad of carboxyl groups in the S3-S4 linker of the voltage sensor. By unveiling this high-affinity binding mode, our studies establish a high-resolution channel-docking and resting-state locking mechanism for huwentoxin-IV and provide guidance for developing future resting-state-targeted analgesic drugs.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/química , Venenos de Aranha/química , Substituição de Aminoácidos , Animais , Humanos , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Células Sf9 , Spodoptera
7.
Proc Natl Acad Sci U S A ; 121(14): e2309000121, 2024 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-38547067

RESUMO

Apneic events are frightening but largely benign events that often occur in infants. Here, we report apparent life-threatening apneic events in an infant with the homozygous SCN1AL263V missense mutation, which causes familial hemiplegic migraine type 3 in heterozygous family members, in the absence of epilepsy. Observations consistent with the events in the infant were made in an Scn1aL263V knock-in mouse model, in which apnea was preceded by a large brainstem DC-shift, indicative of profound brainstem depolarization. The L263V mutation caused gain of NaV1.1 function effects in transfected HEK293 cells. Sodium channel blockade mitigated the gain-of-function characteristics, rescued lethal apnea in Scn1aL263V mice, and decreased the frequency of severe apneic events in the patient. Hence, this study shows that SCN1AL263V can cause life-threatening apneic events, which in a mouse model were caused by profound brainstem depolarization. In addition to being potentially relevant to sudden infant death syndrome pathophysiology, these data indicate that sodium channel blockers may be considered therapeutic for apneic events in patients with these and other gain-of-function SCN1A mutations.


Assuntos
Apneia , Mutação com Ganho de Função , Bloqueadores dos Canais de Sódio , Animais , Humanos , Camundongos , Apneia/tratamento farmacológico , Apneia/genética , Tronco Encefálico , Células HEK293 , Enxaqueca com Aura/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Bloqueadores dos Canais de Sódio/uso terapêutico , Lactente , Feminino
8.
Proc Natl Acad Sci U S A ; 121(9): e2322899121, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38381792

RESUMO

Voltage-gated sodium channels (Nav) undergo conformational shifts in response to membrane potential changes, a mechanism known as the electromechanical coupling. To delineate the structure-function relationship of human Nav channels, we have performed systematic structural analysis using human Nav1.7 as a prototype. Guided by the structural differences between wild-type (WT) Nav1.7 and an eleven mutation-containing variant, designated Nav1.7-M11, we generated three additional intermediate mutants and solved their structures at overall resolutions of 2.9-3.4 Å. The mutant with nine-point mutations in the pore domain (PD), named Nav1.7-M9, has a reduced cavity volume and a sealed gate, with all voltage-sensing domains (VSDs) remaining up. Structural comparison of WT and Nav1.7-M9 pinpoints two residues that may be critical to the tightening of the PD. However, the variant containing these two mutations, Nav1.7-M2, or even in combination with two additional mutations in the VSDs, named Nav1.7-M4, failed to tighten the PD. Our structural analysis reveals a tendency of PD contraction correlated with the right shift of the static inactivation I-V curves. We predict that the channel in the resting state should have a "tight" PD with down VSDs.


Assuntos
Canais de Sódio Disparados por Voltagem , Humanos , Canais de Sódio Disparados por Voltagem/genética , Potenciais da Membrana , Mutação , Relação Estrutura-Atividade
9.
Trends Biochem Sci ; 47(2): 160-172, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34294545

RESUMO

The flagellar stator unit is an oligomeric complex of two membrane proteins (MotA5B2) that powers bi-directional rotation of the bacterial flagellum. Harnessing the ion motive force across the cytoplasmic membrane, the stator unit operates as a miniature rotary motor itself to provide torque for rotation of the flagellum. Recent cryo-electron microscopic (cryo-EM) structures of the stator unit provided novel insights into its assembly, function, and subunit stoichiometry, revealing the ion flux pathway and the torque generation mechanism. Furthermore, in situ cryo-electron tomography (cryo-ET) studies revealed unprecedented details of the interactions between stator unit and rotor. In this review, we summarize recent advances in our understanding of the structure and function of the flagellar stator unit, torque generation, and directional switching of the motor.


Assuntos
Proteínas de Bactérias , Flagelos , Bactérias/metabolismo , Proteínas de Bactérias/química , Microscopia Crioeletrônica/métodos , Flagelos/química , Flagelos/metabolismo , Flagelos/ultraestrutura , Torque
10.
Proc Natl Acad Sci U S A ; 120(14): e2219624120, 2023 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-36996107

RESUMO

Gain-of-function mutations in voltage-gated sodium channel NaV1.7 cause severe inherited pain syndromes, including inherited erythromelalgia (IEM). The structural basis of these disease mutations, however, remains elusive. Here, we focused on three mutations that all substitute threonine residues in the alpha-helical S4-S5 intracellular linker that connects the voltage sensor to the pore: NaV1.7/I234T, NaV1.7/I848T, and NaV1.7/S241T in order of their positions in the amino acid sequence within the S4-S5 linkers. Introduction of these IEM mutations into the ancestral bacterial sodium channel NaVAb recapitulated the pathogenic gain-of-function of these mutants by inducing a negative shift in the voltage dependence of activation and slowing the kinetics of inactivation. Remarkably, our structural analysis reveals a common mechanism of action among the three mutations, in which the mutant threonine residues create new hydrogen bonds between the S4-S5 linker and the pore-lining S5 or S6 segment in the pore module. Because the S4-S5 linkers couple voltage sensor movements to pore opening, these newly formed hydrogen bonds would stabilize the activated state substantially and thereby promote the 8 to 18 mV negative shift in the voltage dependence of activation that is characteristic of the NaV1.7 IEM mutants. Our results provide key structural insights into how IEM mutations in the S4-S5 linkers may cause hyperexcitability of NaV1.7 and lead to severe pain in this debilitating disease.


Assuntos
Eritromelalgia , Canais de Sódio Disparados por Voltagem , Humanos , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor/genética , Dor/metabolismo , Mutação , Eritromelalgia/genética , Eritromelalgia/metabolismo , Eritromelalgia/patologia , Canais de Sódio Disparados por Voltagem/genética , Treonina/genética
11.
Proc Natl Acad Sci U S A ; 120(26): e2220343120, 2023 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-37339196

RESUMO

In bacterial voltage-gated sodium channels, the passage of ions through the pore is controlled by a selectivity filter (SF) composed of four glutamate residues. The mechanism of selectivity has been the subject of intense research, with suggested mechanisms based on steric effects, and ion-triggered conformational change. Here, we propose an alternative mechanism based on ion-triggered shifts in pKa values of SF glutamates. We study the NavMs channel for which the open channel structure is available. Our free-energy calculations based on molecular dynamics simulations suggest that pKa values of the four glutamates are higher in solution of K+ ions than in solution of Na+ ions. Higher pKa in the presence of K+ stems primarily from the higher population of dunked conformations of the protonated Glu sidechain, which exhibit a higher pKa shift. Since pKa values are close to the physiological pH, this results in predominant population of the fully deprotonated state of glutamates in Na+ solution, while protonated states are predominantly populated in K+ solution. Through molecular dynamics simulations we calculate that the deprotonated state is the most conductive, the singly protonated state is less conductive, and the doubly protonated state has significantly reduced conductance. Thus, we propose that a significant component of selectivity is achieved through ion-triggered shifts in the protonation state, which favors more conductive states for Na+ ions and less conductive states for K+ ions. This mechanism also suggests a strong pH dependence of selectivity, which has been experimentally observed in structurally similar NaChBac channels.


Assuntos
Bactérias , Canais de Sódio Disparados por Voltagem , Íons , Bactérias/metabolismo , Simulação de Dinâmica Molecular , Glutamatos , Potássio/metabolismo
12.
J Neurosci ; 44(29)2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-38858080

RESUMO

The resurgent sodium current (INaR) activates on membrane repolarization, such as during the downstroke of neuronal action potentials. Due to its unique activation properties, INaR is thought to drive high rates of repetitive neuronal firing. However, INaR is often studied in combination with the persistent or noninactivating portion of sodium currents (INaP). We used dynamic clamp to test how INaR and INaP individually affect repetitive firing in adult cerebellar Purkinje neurons from male and female mice. We learned INaR does not scale repetitive firing rates due to its rapid decay at subthreshold voltages and that subthreshold INaP is critical in regulating neuronal firing rate. Adjustments to the voltage-gated sodium conductance model used in these studies revealed INaP and INaR can be inversely scaled by adjusting occupancy in the slow-inactivated kinetic state. Together with additional dynamic clamp experiments, these data suggest the regulation of sodium channel slow inactivation can fine-tune INaP and Purkinje neuron repetitive firing rates.


Assuntos
Potenciais de Ação , Células de Purkinje , Canais de Sódio , Animais , Camundongos , Feminino , Masculino , Potenciais de Ação/fisiologia , Células de Purkinje/fisiologia , Canais de Sódio/fisiologia , Canais de Sódio/metabolismo , Sódio/metabolismo , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Modelos Neurológicos
13.
J Biol Chem ; 300(10): 107757, 2024 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-39260690

RESUMO

Venoms are used by arthropods either to immobilize prey or as defense against predators. Our study focuses on the venom peptide, Ta3a, from the African ant species, Tetramorium africanum and its effects on voltage-gated sodium (NaV) channels, which are ion channels responsible for the generation of electrical signals in electrically excitable cells, such as neurons. Using the NaV1.7 isoform as our model NaV channel we show that Ta3a prolongs single channel active periods with increased open probability and induces non-inactivating whole-cell currents. Ta3a-affected NaV1.7 channels exhibit a leftward (hyperpolarizing) shift in activation threshold, constitutive activity even in the absence of an activating voltage stimulus, and at cell membrane voltages where channels are normally silent. Current-voltage experiments show that Ta3a shifts the voltage at which NaV current changes direction (reversal potential) by altering the local ionic concentration of permeant ions (Na+) rather than changing the channel's preference for ionic species. We propose a model where Ta3a maintains the positively charged voltage-sensing (S4) domains of the channel in the activated configuration where their electric field is exposed to the extracellular membrane surface to create an ionic bilayer comprising S4 domains and mobile anions (Cl-). This bilayer has a depolarizing effect on the cell membrane, thus reducing the amount of externally applied voltage required for channel activation.

14.
J Biol Chem ; 300(11): 107833, 2024 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-39343005

RESUMO

The voltage-gated sodium (NaV) channel is critical for cardiomyocyte function since it is responsible for action potential initiation and its propagation throughout the cell. It consists of a protein complex made of a pore forming α subunit and associated ß subunits, which regulate α subunit function and subcellular localization. We previously showed the implication of N-linked glycosylation and S-acylation of ß2 in its polarized trafficking. Here, we present evidence of ß2 dimerization. Moreover, we demonstrate the implication of the cytoplasmic tail, extracellular loop, and transmembrane domain on proper ß2 folding and export to the cell surface of polarized Madin-Darby canine kidney cells. Substantial alteration, or lack of any of these domains, leads to accumulation of ß2 in the endoplasmic reticulum, along with impaired complex N-glycosylation, which is needed for its efficient surface delivery. We also show that these alterations to ß2 affected to a certain extent NaV1.5 surface localization. Conversely, however, NaV1.5 had little or no influence on ß2 trafficking, its localization to the surface, or homodimer formation. Altogether, our data link the architecture of the ß2 domains to the establishment of its proper subcellular localization. These findings could provide valuable insights to gain a deeper comprehension of the elusive biology of ß subunits in excitable cells, such as neurons and cardiomyocytes.

15.
J Biol Chem ; 300(4): 105785, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38401845

RESUMO

The epithelial sodium channel (ENaC) is essential for mediating sodium absorption in several epithelia. Its impaired function leads to severe disorders, including pseudohypoaldosteronism type 1 and respiratory distress. Therefore, pharmacological ENaC activators have potential therapeutic implications. Previously, a small molecule ENaC activator (S3969) was developed. So far, little is known about molecular mechanisms involved in S3969-mediated ENaC stimulation. Here, we identified an S3969-binding site in human ENaC by combining structure-based simulations with molecular biological methods and electrophysiological measurements of ENaC heterologously expressed in Xenopus laevis oocytes. We confirmed a previous observation that the extracellular loop of ß-ENaC is essential for ENaC stimulation by S3969. Molecular dynamics simulations predicted critical residues in the thumb domain of ß-ENaC (Arg388, Phe391, and Tyr406) that coordinate S3969 within a binding site localized at the ß-γ-subunit interface. Importantly, mutating each of these residues reduced (R388H; R388A) or nearly abolished (F391G; Y406A) the S3969-mediated ENaC activation. Molecular dynamics simulations also suggested that S3969-mediated ENaC stimulation involved a movement of the α5 helix of the thumb domain of ß-ENaC away from the palm domain of γ-ENaC. Consistent with this, the introduction of two cysteine residues (ßR437C - γS298C) to form a disulfide bridge connecting these two domains prevented ENaC stimulation by S3969 unless the disulfide bond was reduced by DTT. Finally, we demonstrated that S3969 stimulated ENaC endogenously expressed in cultured human airway epithelial cells (H441). These new findings may lead to novel (patho-)physiological and therapeutic concepts for disorders associated with altered ENaC function.


Assuntos
Agonistas do Canal de Sódio Epitelial , Canais Epiteliais de Sódio , Indóis , Animais , Humanos , Sítios de Ligação , Agonistas do Canal de Sódio Epitelial/metabolismo , Agonistas do Canal de Sódio Epitelial/farmacologia , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/metabolismo , Simulação de Dinâmica Molecular , Oócitos/efeitos dos fármacos , Xenopus laevis , Ligação Proteica , Indóis/metabolismo , Indóis/farmacologia
16.
J Biol Chem ; 300(3): 105715, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38309503

RESUMO

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, NaV1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For NaV1.5, this process is critical as alterations in Na+ current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of NaV1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of NaV1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na+ current reduction using the NEDD4L HECT domain as cargo of a NaV1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding NaV1.5 ubiquitination.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Canal de Sódio Disparado por Voltagem NAV1.5 , Ubiquitina-Proteína Ligases Nedd4 , Ubiquitinação , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ubiquitina/metabolismo , Humanos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Células HEK293
17.
Circulation ; 150(8): 642-650, 2024 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-39159224

RESUMO

Intravenous infusion of sodium-channel blockers (SCB) with either ajmaline, flecainide, procainamide, or pilsicainide to unmask the ECG of Brugada syndrome is the drug challenge most commonly used for diagnostic purposes when investigating cases possibly related to inherited arrhythmia syndromes. For a patient undergoing an SCB challenge, the impact of a positive result goes well beyond its diagnostic implications. It is, therefore, appropriate to question who should undergo a SCB test to diagnose or exclude Brugada syndrome and, perhaps more importantly, who should not. We present a critical review of the benefits and drawbacks of the SCB challenge when performed in cardiac arrest survivors, patients presenting with syncope, family members of probands with confirmed Brugada syndrome, and asymptomatic patients with suspicious ECG.


Assuntos
Síndrome de Brugada , Eletrocardiografia , Bloqueadores dos Canais de Sódio , Humanos , Síndrome de Brugada/diagnóstico , Síndrome de Brugada/fisiopatologia , Síncope/diagnóstico , Síncope/etiologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-38976181

RESUMO

The normal functioning of every cell in the body depends on its bioelectric properties and many diseases are caused by genetic and/or epigenetic dysregulation of the underlying ion channels. Metastasis, the main cause of death from cancer, is a complex multi-stage process in which cells break away from a primary tumour, invade the surrounding tissues, enter the circulation by encountering a blood vessel and spread around the body, ultimately lodging in distant organs and reproliferating to form secondary tumours leading to devastating organ failure. Such cellular behaviours are well known to involve ion channels. The CELEX model offers a novel insight to metastasis where it is the electrical excitation of the cancer cells that is responsible for their aggressive and invasive behaviour. In turn, the hyperexcitability is underpinned by concomitant upregulation of functional voltage-gated sodium channels and downregulation of voltage-gated potassium channels. Here, we update the in vitro and in vivo evidence in favour of the CELEX model for carcinomas. The results are unequivocal for the sodium channel. The potassium channel arm is also broadly supported by existing evidence although these data are complicated by the impact of the channels on the membrane potential and consequent secondary effects. Finally, consistent with the CELEX model, we show (i) that carcinomas are indeed electrically excitable and capable of generating action potentials and (ii) that combination of a sodium channel inhibitor and a potassium channel opener can produce a strong, additive anti-invasive effect. We discuss the possible clinical implications of the CELEX model in managing cancer.

19.
Annu Rev Genomics Hum Genet ; 23: 255-274, 2022 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-35567276

RESUMO

Brugada syndrome is a heritable channelopathy characterized by a peculiar electrocardiogram (ECG) pattern and increased risk of cardiac arrhythmias and sudden death. The arrhythmias originate because of an imbalance between the repolarizing and depolarizing currents that modulate the cardiac action potential. Even if an overt structural cardiomyopathy is not typical of Brugada syndrome, fibrosis and structural changes in the right ventricle contribute to a conduction slowing, which ultimately facilitates ventricular arrhythmias. Currently, Mendelian autosomal dominant transmission is detected in less than 25% of all clinical confirmed cases. Although 23 genes have been associated with the condition, only SCN5A, encoding the cardiac sodium channel, is considered clinically actionable and disease causing. The limited monogenic inheritance has pointed toward new perspectives on the possible complex genetic architecture of the disease, involving polygenic inheritance and a polygenic risk score that can influence penetrance and risk stratification.


Assuntos
Síndrome de Brugada , Síndrome de Brugada/genética , Eletrocardiografia , Humanos , Herança Multifatorial , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canais de Sódio/genética
20.
Brain ; 147(4): 1231-1246, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-37812817

RESUMO

Dravet syndrome is an intractable developmental and epileptic encephalopathy caused by de novo variants in SCN1A resulting in haploinsufficiency of the voltage-gated sodium channel Nav1.1. We showed previously that administration of the antisense oligonucleotide STK-001, also called ASO-22, generated using targeted augmentation of nuclear gene output technology to prevent inclusion of the nonsense-mediated decay, or poison, exon 20N in human SCN1A, increased productive Scn1a transcript and Nav1.1 expression and reduced the incidence of electrographic seizures and sudden unexpected death in epilepsy in a mouse model of Dravet syndrome. Here, we investigated the mechanism of action of ASO-84, a surrogate for ASO-22 that also targets splicing of SCN1A exon 20N, in Scn1a+/- Dravet syndrome mouse brain. Scn1a +/- Dravet syndrome and wild-type mice received a single intracerebroventricular injection of antisense oligonucleotide or vehicle at postnatal Day 2. We examined the electrophysiological properties of cortical pyramidal neurons and parvalbumin-positive fast-spiking interneurons in brain slices at postnatal Days 21-25 and measured sodium currents in parvalbumin-positive interneurons acutely dissociated from postnatal Day 21-25 brain slices. We show that, in untreated Dravet syndrome mice, intrinsic cortical pyramidal neuron excitability was unchanged while cortical parvalbumin-positive interneurons showed biphasic excitability with initial hyperexcitability followed by hypoexcitability and depolarization block. Dravet syndrome parvalbumin-positive interneuron sodium current density was decreased compared to wild-type. GABAergic signalling to cortical pyramidal neurons was reduced in Dravet syndrome mice, suggesting decreased GABA release from interneurons. ASO-84 treatment restored action potential firing, sodium current density and GABAergic signalling in Dravet syndrome parvalbumin-positive interneurons. Our work suggests that interneuron excitability is selectively affected by ASO-84. This new work provides critical insights into the mechanism of action of this antisense oligonucleotide and supports the potential of antisense oligonucleotide-mediated upregulation of Nav1.1 as a successful strategy to treat Dravet syndrome.


Assuntos
Epilepsias Mioclônicas , Oligonucleotídeos Antissenso , Camundongos , Animais , Humanos , Oligonucleotídeos Antissenso/farmacologia , Parvalbuminas/metabolismo , Epilepsias Mioclônicas/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Interneurônios/metabolismo , Ácido gama-Aminobutírico , Modelos Animais de Doenças
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