67 research outputs found

    Bacterial Voltage-Gated Sodium Channels (BacNaVs) from the Soil, Sea, and Salt Lakes Enlighten Molecular Mechanisms of Electrical Signaling and Pharmacology in the Brain and Heart

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    AbstractVoltage-gated sodium channels (NaVs) provide the initial electrical signal that drives action potential generation in many excitable cells of the brain, heart, and nervous system. For more than 60years, functional studies of NaVs have occupied a central place in physiological and biophysical investigation of the molecular basis of excitability. Recently, structural studies of members of a large family of bacterial voltage-gated sodium channels (BacNaVs) prevalent in soil, marine, and salt lake environments that bear many of the core features of eukaryotic NaVs have reframed ideas for voltage-gated channel function, ion selectivity, and pharmacology. Here, we analyze the recent advances, unanswered questions, and potential of BacNaVs as templates for drug development efforts

    Novel pathomechanisms and disease associations of the voltage-gated sodium channel NaV1.4

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    Voltage-gated sodium channels initiate and shape the upstroke of the action potential, allowing fast electrical signaling between cells. Mutations in the genes encoding these channels are associated with a group of disorders known as channelopathies. This project aimed to characterize mutations in SCN4A encoding NaV1.4 associated with traditional skeletal muscle channelopathies as well as novel conditions using functional expression in Xenopus oocytes or HEK293T cells. Mutations of gating charges in the voltage sensor domain in the fourth transmembrane segment (S4), such as p.R222W or p.R222G, were found in patients with hypokalemic periodic paralysis. Another mutation, p.R222Q, was found in an individual with myotonia. I found that unlike hypoPP S4 arginine mutations causing gating pore currents, p.R222Q results in gain of function typically associated with sodium-channel myotonia. In another project, novel homozygous or compound heterozygous SCN4A mutations were found in eleven families with congenital myopathy. Each affected individual carried at least one mutation causing full loss of function. In all but one case the mutation in the opposite allele caused full or partial loss of function. The genetic and functional data are consistent with heteroallelic loss of function mutations—one of which confers full loss of function—underlying the clinical presentation by reducing the action potential amplitude in the muscle to a level insufficient to sustain normal muscle function. Some SCN4A mutations are lethal in infants when affecting muscle regulating respiration. Whole-exome sequencing of 434 cases of sudden infant death syndrome (SIDS) identified in six novel and five very rare SCN4A variants. Channel defects were found in four variants, two of which were gain of function and the other two loss of function. Dysfunctional SCN4A variants were also overrepresented in the SIDS cohort compared to controls. These results suggest pathogenic variations in SCN4A may be a genetic risk factor for SIDS

    Structural characterisation of the prokaryotic sodium channel C-terminal domain

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    Since the discovery of the first prokaryotic voltage gated sodium channel (Nav) in 2001, prokaryotic Navs have been a high priority target for structural study. Prokaryotic Navs are of interest as a model system due to their homology to eukaryotic Navs, which are high value drug development targets for their roles in pain perception and neural function. While prokaryotic Navs have function and pharmacology distinct from their eukaryotic homologues, understanding their structure holds implications for drug development and for understanding diseases stemming from neuronal dysfunction. However, Navs have historically been challenging targets for structural study, resisting attempts at crystallisation until recently. In this study, expression, purification, and characterisation of a chimera of the NavBh channel and the ligand gating RCK domain from the prokaryotic potassium channel MthK has been performed. It was hypothesised that the addition of the RCK domain would improve the channel’s crystallisation potential, and create a ligand gated Nav for functional characterisation. Electrophysiological studies demonstrated that the RCK domain was capable of gating NavBh, however the chimera had reduced solubility, indicating that this chimeric fusion was not an ideal target for structural study due to low purification yields. Following this, and in light of recent studies that suggested the structure of the prokaryotic Nav C-terminus had a role in channel function, structural analysis of the C-terminus of a prokaryotic Nav homologue cloned from Bacillus alcalophilus has been performed. Synchrotron radiation circular dichroism analysis of serial C-terminal truncations demonstrated the structure of the NsvBa C-terminus consists of a helical region connected to the channel pore by a disordered neck region, despite conflicting bioinformatics predictions. This offers further support for the hypothesis that in functional Navs, the C-terminus consists of a disordered neck region connecting a coiled-coil to the base of the pore, which acts as a spring to assist in channel gating and inactivation

    Molecular pathophysiology and pharmacology of the voltage-sensing module of neuronal ion channels

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    Voltage-gated ion channels (VGICs) are membrane proteins that switch from a closed to open state in response to changes in membrane potential, thus enabling ion fluxes across the cell membranes. The mechanism that regulate the structural rearrangements occurring in VGICs in response to changes in membrane potential still remains one of the most challenging topic of modern biophysics. Na+, Ca2+ and K+ voltage-gated channels are structurally formed by the assembly of four similar domains, each comprising six transmembrane segments. Each domain can be divided into two main regions: the Pore Module (PM) and the Voltage-Sensing Module (VSM). The PM (helices S-5 and S-6 and intervening linker) is responsible for gate opening and ion selectivity; by contrast, the VSM, comprising the first four transmembrane helices (S-1-S-4), undergoes the first conformational changes in response to membrane voltage variations. In particular, the S-4 segment of each domain, which contains several positively charged residues interspersed with hydrophobic amino acids, is located within the membrane electric field and plays an essential role in voltage sensing. In neurons, specific gating properties of each channel subtype underlie a variety of biological events, ranging from the generation and propagation of electrical impulses, to the secretion of neurotransmitters and to the regulation of gene expression. Given the important functional role played by the VSM in neuronal VGICs, it is not surprising that various VSM mutations affecting the gating process of these channels are responsible for human diseases, and that compounds acting on the VSM have emerged as important investigational tools with great therapeutic potential. In the present review we will briefly describe the most recent discoveries concerning how the VSM exerts its function, how genetically inherited diseases caused by mutations occurring in the VSM affects gating in VGICs, and how several classes of drugs and toxins selectively target the VSM

    Inhibition of voltage-dependent sodium currents by cannabidiol

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    Voltage-gated sodium channels initiate action potentials in excitable tissues. Altering these channels’ function can lead to many pathophysiological conditions. The family of voltage-gated sodium channel genes encodes 10 proteins (including Nav2.1) distributed throughout the central and peripheral nervous systems, cardiac and skeletal muscles. The SCN4A gene encodes the Nav1.4 channel, which is primarily responsible for depolarization of the skeletal muscle fibers. Many mutations in SCN4A are found and associated with the myotonic syndromes and periodic paralyses. These conditions are both considered gain-of-function and can be severely life-limiting with respect to performing everyday tasks. From a broader standpoint, hyperexcitability presents as a significant problem in other tissues besides skeletal muscles. Gain-of-function in sodium channels has been linked to a wide-range of pathophysiological conditions such as inherited erythromelalgia, epilepsy, and arrhythmias. Treating these types of pathologies requires an in-depth understanding of their underlying mechanisms. One way to gain this understanding is to investigate physiological triggers. There is also a dire need for novel ways of reducing the hyperexcitability associated with mutant sodium channels. One promising compound is the non-psychotropic component of the Cannabis sativa plant, cannabidiol. This compound has recently been shown to modulate some of the neuronal sodium channels. Although cannabidiol has shown efficacy in clinical trials, the underlying mechanism of action remains unknown. Sodium channels could be among the molecular targets for cannabidiol.In my doctoral research: 1) I studied how a single missense mutation, P1158S, in Nav1.4 causes various degrees of gain-of-function (myotonia and periodic paralysis) by using pH changes to probe P1158S gating modifications; 2) I studied the inhibitory effects of cannabidiol on voltage-dependent sodium currents; 3) I investigated the mechanism through which cannabidiol imparts inhibition. Overall, these data reveal novel insights into sodium channel hyperexcitability and pharmacologically targeting of this hyperexcitability using cannabidiol

    Structure-activity studies on the opening- and closure-mechanism of L-type calcium-channels

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    In der vorliegenden Arbeit wurden gating-sensitive Aminosäuren und Sequenzabschnitte in den S6-Segmenten des kardialen L-Typ Kanals Cav1.2 identifiziert und mit Hilfe von Homologiemodellen Hypothesen zu deren Funktion formuliert. Der Großteil dieser Arbeit befasst sich mit der Struktur-Funktionsanalyse des stark konservierten "G/A/G/A" Motivs in der "bundle crossing region" der S6-Segmente. In den Bereich dieses Motivs fällt eine bekannte, autosomal dominante, Punktmutation, die zu einer Kanalerkrankung, dem Timothy Syndrom, führt. Tritt diese Mutation G432S auf, so sind u.a. Arrhythmien, Autismus und neurologische Defekte die Folge. Dabei wird die Inaktivierung aufgehoben und durch die so verlängerte Kanalöffnungszeit strömt vermehrt Kalzium ein. Die Arbeit zeigt, dass Mutationen in allen analogen Positionen der Domänen II, III und IV starke Verschiebungen der Aktivierung auslösen. Eine Entkopplung des Aktivierungsprozesses vom Inaktivierungsprozess für Mutationen in Position 432 konnte nachgewiesen werden. Darüber hinaus wurde mittels Homologiemodell festgestellt, dass die Aminosäuren des "G/A/G/A"-Motivs wahrscheinlich einen räumlich dicht gepackten Ring in der geschlossenen Konformation des Kanals bilden. Es wird angenommen, dass dieser bei jeglicher Form der Mutation nicht mehr vollständig schließen kann. Um den Einfluss von Spannungssensoren auf das entdeckte "G/A/G/A" Motiv - und damit das gating-Verhalten - zu untersuchen, wurde jedes S4 Segment der vier Domänen neutralisiert. Durch die Neutralisierung der S4 Segmente in den Domänen I, III und IV wurde der Kanal funktionsunfähig. Interessanterweise konnte die Aktivierung des neutralisierten Segments in Domäne II (IIS4N) nicht von jener des Wildtypkanals unterschieden werden. Die Kombination von (IIS4N) mit allen "G/A/G/A" Mutanten verursachte ein Rescue-Verhalten, die Wildtyp-Aktivierung wurde also wiederhergestellt. Die Kombination von IIS4N mit den "G/A/G/A" umliegenden Positionen verursachte kein Rescue-Verhalten, was auf eine Schlüsselrolle von "G/A/G/A" für die Kanalöffnung schließen lässt. Thermodynamische Untersuchungen zeigten, dass der Spannungssensor IIS4 energetisch an den "G/A/G/A" Ring gekoppelt ist und diese Kopplung zu Konformationsänderungen der Domänen II, III und IV führt.The objective of the present study was to identify gating sensitive positions in the segments of the cardiac L-type Cav1.2 channel. This was supported by homology models that suggested structural details of certain amino acid regions. The main part of this thesis focuses on the highly conserved "G/A/G/A" motif of the bundle crossing region in the S6 segments. A well known autosomal dominant point mutation which causes a channelopathy called Timothy Syndrome is located inside this motif. This inherited disease leads to arrhythmias, autism and neurological defects. The source of this channelopathy is mutation G423S which dramatically reduces inactivation. The prolonged opening of the channel causes increased calcium influx. This work shows that mutations in all analogous positions in domains II, III and IV accelerate activation. Inactivation in Domains II-IV is not affected. Since inactivation is Domain I specific, a decoupling of the inactivation and the deactivation process can be shown. Homology modelling demonstrates that all amino acids of the "G/A/G/A" motif form a tightly packed ring in the closed conformation of the channel. We hypothesize that mutating this ring prevents complete channel closure. To investigate influence of voltage sensors on the identified "G/A/G/A" motif, all charged positions in each of the S4 segments were neutralized. Neutralization of the S4 segments in domains I, III and IV lead to a non-functional channel. Interestingly the activation shift of the neutralized segment in domain II (IIS4N) could not be distinguished from the wild-type channel. Combination of IIS4N with all "G/A/G/A" mutants caused a rescue behaviour with wild-type like activation. In contrast IIS4N in conjunction with amino acid mutations next to the "G/A/G/A" motif showed no rescue behaviour. This indicates the important function of the "G/A/G/A" motif. Thermodynamic analysis showed that the voltage sensor IIS4 is energetically coupled to the "G/A/G/A" ring. This coupling leads to conformational changes of domains II, II and IV

    Mecanismos de condução iônica em canais de sódio

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    Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2013.Canais iônicos (CIs) são proteínas transmembrânicas capazes de regular o fluxo de íons, para dentro e para fora das células, abrindo e fechando seus poros. Tal regulação se da em resposta a diferentes estímulos, como ligação de moléculas, diferenças de potencial eletrostático, estímulos mecânicos, etc. Além disso, em geral, CIs são altamente seletivos a um íon. Assim, em função do íon transportado pelo canal, estes podem ser denominados canais de Na+, K+, Ca++ ou Cl-. O funcionamento de CIs está relacionado a uma variedade de funções biológicas essenciais como: contração muscular, sinapses, geração e transmissão de impulsos nervosos, controle da pressão osmótica, secreção hormonal, percepção do ambiente e consciência, entre outros. Portanto, essas proteínas tem sido alvo de intensas investigações ao longo das ultimas décadas. Diversas estruturas cristalográficas de canais iônicos foram resolvidas nos últimos anos. Conjuntamente com avanços no poder computacional, tais estruturas possibilitam a investigação de CIs com alta resolução, via simulações de dinâmica molecular (DM). Em 2011 foi publicada a primeira estrutura cristalográfica de canal de Na+: um CI bacteriano denominado NavAb (Payandeh et al., 2011). Observa-se que o poro de CIs apresenta uma estrutura geral comum. Em contrapartida, a região responsável pela discriminação dos íons, o chamado filtro de seletividade (FS), naturalmente mostra diferenças consideráveis. Segundo pode ser observado na estrutura cristalográfica, o FS de canais de Na+ e relativamente curto e amplo, sendo portanto capaz de acomodar íons hidratados no seu interior. Além disso, e sugerido que íons dispõem de certa mobilidade no interior desse FS. Tal descrição difere significativamente do ambiente constrito e pouco hidratado observado no FS de canais de K+. Frente a isso, espera-se que os mecanismos de condução em canais de Na+ e K+ sejam diferentes. Neste trabalho, cálculos de energia livre (via metadinâmica) e simulações de DM com aplicação de potenciais eletrostáticos transmembrânicos foram empregados na investigação do mecanismo de condução de canais de Na+, através do CI NavAb. Conjuntamente, ambas metodologias mostram que os íons ligam-se essencialmente a três sítios no interior do FS, chamados HFS, CEN e IN. Mostrou-se também que o movimento de íons e água no interior do filtro e pouco restrito. Em decorrência disso, e permitida a ocupação concomitante de um único sitio do FS por mais de um íon, o que implica em um mecanismo fundamentalmente distinto daquele observado em canais de K+. Outro resultado interessante aponta para uma assimetria nos mecanismos de condução de Na+ em condições de hiperpolarização ou equilíbrio (ΔV≤0) e de despolarização (ΔV>0). O mecanismo de condução predominante a ΔV ≤ 0 envolve essencialmente a participação de dois Na+. Em contrapartida, a ΔV > 0, o mecanismo conta com três íons, sendo que a chegada do terceiro estimula a liberação de um dos íons anteriormente presentes no canal. Perspectivas desse estudo impactam algumas das questões mais desafiantes da biofísica molecular atual, tais como: identificar os fatores determinantes da seletividade iônica; compreender melhor a relação estrutura-função de canais iônicos eucariotos, particularmente canais de Na+ e Ca++; mecanismo de ação de anestésicos e aplicações nanobiotecnológicas. _______________________________________________________________________________________________________ ABSTRACTIon channels (ICs) are transmembrane (TM) protein pores, capable of regulating the flow of ions in and out of the cells by opening and closing the hydrated pathway they form. This can be done in response to various stimuli, such as binding of ligands, voltage, mechanical stimuli, etc. Moreover, most ICs are highly selective. Hence, according to the ion they selectively transport, ICs can be denominated either Na+, K+, Ca++ or Cl- channels. ICs functioning is essential to a variety of biological functions, e.g.: muscular contraction, electric signaling in neurons, control of the osmotic pressure, hormone secretion, consciousness, etc. Therefore have been the subject of intense research over the last 50 years. Several IC crystal structures have been resolved over the last few years. Along with improvements in computer processing power, such structures enable the investigation of ICs with atomic resolution, by means of molecular dynamics (MD) simulations. In 2011 the first crystal structure of a sodium channel (a bacterial channel named NavAb) was published (Payandeh et al., 2011). While ICs display a pore with overall conserved structure, significant differences can be seen in the so-called selectivity filter (SF): the pore region responsible for discriminating between different ionic species. As of its crystal structure, the Na+ channel SF is relatively short and wide, and thence can fit hydrated cations. Besides, ions and water molecules are expected to have a certain degree of mobility inside the SF. Such SF characteristics greatly differ from the narrow dehydrated environment described for the K+ channel SF. In this context, conduction mechanisms are also expected to be significantly different in these channels. Here, free energy calculations and MD simulations with an applied TM voltage were harnessed to investigate the conduction mechanisms of Na+ channels. Altogether, these methodologies indicate that Na+ ions can bind to three main sites within the SF, named HFS, CEN and IN. Because the movement of ions and water is less restricted, a side-by-side configuration of ions is permitted. This implies in a conduction mechanism fundamentally different from what is known for K+ channels. What is more, this study describes an asymmetry in the conduction mechanisms taking place under hyperpolarizing/neutral (ΔV≤0) and depolarizing (ΔV>0) conditions. Under ΔV≤0, conduction involves two Na+ ions bound to the filter. Contrastingly, under ΔV>0, 3 ions participate and the approach of the third ion triggers the conduction. Perspectives of this study can impart some of the most interesting questions in the molecular biophysics field, such as: identify the determinants of ion selectivity; better understand the structure-function interplay in eukaryotic ICs, specially Na+ and Ca++; the molecular mechanism of anesthetics action and nanobiotechnological applications

    A chemical biology approach to understanding the basis of voltage-gated sodium channel modulation

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    The voltage-gated sodium channel, Nav1.7, is involved in the propagation of pain signals from the peripheral nervous system. Genomic data from individuals with non-functional Nav1.7 expression strongly suggest it has potential to be the target of novel analgesics; loss of Nav1.7 function completely abolishes pain sensations in otherwise healthy phenotypes. The focus of this thesis is the development of chemical tools to elucidate mechanisms of Nav1.7 modulation in the cell. The design, synthesis, characterisation and potency data of photocrosslinking probes that target two distinct Nav1.7 domains is reported. Domain II is targeted by photoprobes derived from the spider venom inhibitory cystine knot peptide Huwentoxin-IV. Moreover, a photoprobe based on the novel family of Nav1.7-selective aryl sulfonamide inhibitors targets domain IV of Nav1.7. Determining the binding sites that lead to modulation of gating was firstly attempted in bacterial/hNav1.7 chimeric proteins that have been purified and used for crystallographic and biophysical studies. According to gel shift assays, certain photoprobes exhibited efficient photocrosslinking capabilities and were taken forward to proteomic mass spectrometry analysis in pursuit of photocrosslinking sites. Additionally, a series of approaches were explored in order to optimise the identification of Nav1.7 by proteomic mass spectrometry in an engineered cell line. Finally, the maturation of induced pluripotent stem cells from patients that carry a Nav1.7 mutation was followed by quantitative proteomics as an initial approach to understand Nav1.7-related mechanisms in a disease model.Open Acces

    Structures Illuminate Cardiac Ion Channel Functions in Health and in Long QT Syndrome

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    The cardiac action potential is critical to the production of a synchronized heartbeat. This electrical impulse is governed by the intricate activity of cardiac ion channels, among them the cardiac voltage-gated potassium (Kv) channels KCNQ1 and hERG as well as the voltage-gated sodium (Nav) channel encoded by SCN5A. Each channel performs a highly distinct function, despite sharing a common topology and structural components. These three channels are also the primary proteins mutated in congenital long QT syndrome (LQTS), a genetic condition that predisposes to cardiac arrhythmia and sudden cardiac death due to impaired repolarization of the action potential and has a particular proclivity for reentrant ventricular arrhythmias. Recent cryo-electron microscopy structures of human KCNQ1 and hERG, along with the rat homolog of SCN5A and other mammalian sodium channels, provide atomic-level insight into the structure and function of these proteins that advance our understanding of their distinct functions in the cardiac action potential, as well as the molecular basis of LQTS. In this review, the gating, regulation, LQTS mechanisms, and pharmacological properties of KCNQ1, hERG, and SCN5A are discussed in light of these recent structural findings
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