RESUMEN
The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel is a voltage-gated cation channel that mediates neuronal and cardiac pacemaker activity. The HCN channel exhibits reversed voltage dependence, meaning it closes with depolarization and opens with hyperpolarization. Different from Na+, Ca2+, and Kv1-Kv7 channels, the HCN channel does not have domain-swapped voltage sensors. We introduced a reversible, metal-mediated cross bridge into the voltage sensors to create the chemical equivalent of a hyperpolarized conformation and determined the structure using cryoelectron microscopy (cryo-EM). Unlike the depolarized HCN channel, the S4 helix is displaced toward the cytoplasm by two helical turns. Near the cytoplasm, the S4 helix breaks into two helices, one running parallel to the membrane surface, analogous to the S4-S5 linker of domain-swapped voltage-gated channels. These findings suggest a basis for allosteric communication between voltage sensors and the gate in this kind of channel. They also imply that voltage sensor movements are not the same in all voltage-gated channels.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/química , Activación del Canal Iónico , Animales , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Potenciales de la Membrana , Conformación Proteica en Hélice alfa , Células Sf9 , SpodopteraRESUMEN
KCNQ1 is the pore-forming subunit of cardiac slow-delayed rectifier potassium (IKs) channels. Mutations in the kcnq1 gene are the leading cause of congenital long QT syndrome (LQTS). Here, we present the cryoelectron microscopy (cryo-EM) structure of a KCNQ1/calmodulin (CaM) complex. The conformation corresponds to an "uncoupled," PIP2-free state of KCNQ1, with activated voltage sensors and a closed pore. Unique structural features within the S4-S5 linker permit uncoupling of the voltage sensor from the pore in the absence of PIP2. CaM contacts the KCNQ1 voltage sensor through a specific interface involving a residue on CaM that is mutated in a form of inherited LQTS. Using an electrophysiological assay, we find that this mutation on CaM shifts the KCNQ1 voltage-activation curve. This study describes one physiological form of KCNQ1, depolarized voltage sensors with a closed pore in the absence of PIP2, and reveals a regulatory interaction between CaM and KCNQ1 that may explain CaM-mediated LQTS.
Asunto(s)
Calmodulina/química , Canal de Potasio KCNQ1/química , Síndrome de QT Prolongado/metabolismo , Secuencia de Aminoácidos , Animales , Calmodulina/metabolismo , Microscopía por Crioelectrón , Humanos , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ1/metabolismo , Modelos Moleculares , Mutación , Alineación de Secuencia , Xenopus laevisRESUMEN
Nanoporous silver (NPS), characterized by its three-dimensional bi-continuous interpenetrating ligament channel structure, is a good candidate for surface enhanced Raman scattering (SERS), attributed to its exceptional surface-to-volume ratio and significant SERS enhancement capabilities. Here, we have successfully fabricated NPS through the dealloying ofα-terpineol (α-TPN) coated Ag55Al45alloy. The resultingα-NPS exhibits uniform ligaments and nanopore sizes, maintaining high SERS performance even after being exposed to air for more than one month. The pretreatment of precusor alloy withα-TPN is crucial not only for the formation of nanoporous structure but also for ensuring the long term stability ofα-NPS. Specifically,α-TPN functions as a surfactant, facilitating atomic diffusion to achieve a superior interconnected NPS. Furthermore, during the dealloying process, the carbonization ofα-TPN serves as a protective layer, effectively inhibiting the oxidation of silver.
RESUMEN
In the CryoEM-structure of the hSkMNaV1.4 ion channel (PDB:6AGF), the 59-residue DIS5-S6 linker peptide was omitted due to absence of electron density. This peptide is intriguing - comprised of unique sequence and found only in mammalian skeletal muscle sodium ion channels. To probe potential physiological and evolutionary significance, we constructed an homology model of the complete hSkMNaV1.4 channel. Rather than a flexible random coil potentiating drift across the channel, the linker folds into a compact configuration through self-assembling secondary structural elements. Analogous sequences from 48 mammalian organisms show hypervariability with between 40% and 100% sequence similarity. To investigate structural implications, sequences from 14 representative organisms were additionally modelled. All showed highly conserved N-and C-terminal residues closely superimposed, suggesting a critical functional role. An optimally located asparagine residue within the conserved region was investigated for N-linked glycosylation and MD simulations carried out. Results suggest a complex glycan added at this site in the linker may form electrostatic interactions with the DIV voltage sensing domain and be mechanistically involved in channel gating. The relationship of unique sequence, compact configuration, potential glycosylation and MD simulations are discussed relative to SkMNaV1.4 structure and function.
Asunto(s)
Simulación de Dinámica Molecular , Canales de Sodio , Animales , Glicosilación , Canales de Sodio/química , Canales de Sodio/metabolismo , Mamíferos/metabolismoRESUMEN
Developing new membranes with both high selectivity and permeability is critical in membrane science since conventional membranes are often limited by the trade-off between selectivity and permeability. In recent years, the emergence of advanced materials with accurate structures at atomic or molecular scale, such as metal organic framework, covalent organic framework, graphene, has accelerated the development of membranes, which benefits the precision of membrane structures. In this review, current state-of-the-art membranes are first reviewed and classified into three different types according to the structures of their building blocks, including laminar structured membranes, framework structured membranes and channel structured membranes, followed by the performance and applications for representative separations (liquid separation and gas separation) of these precisely designed membranes. Last, the challenges and opportunities of these advanced membranes are also discussed.
RESUMEN
Ion channels play important roles in fundamental biological processes, such as electric signaling in cells, muscle contraction, hormone secretion, and regulation of the immune response. Targeting ion channels with drugs represents a treatment option for neurological and cardiovascular diseases, muscular degradation disorders, and pathologies related to disturbed pain sensation. While there are more than 300 different ion channels in the human organism, drugs have been developed only for some of them and currently available drugs lack selectivity. Computational approaches are an indispensable tool for drug discovery and can speed up, especially, the early development stages of lead identification and optimization. The number of molecular structures of ion channels has considerably increased over the last ten years, providing new opportunities for structure-based drug development. This review summarizes important knowledge about ion channel classification, structure, mechanisms, and pathology with the main focus on recent developments in the field of computer-aided, structure-based drug design on ion channels. We highlight studies that link structural data with modeling and chemoinformatic approaches for the identification and characterization of new molecules targeting ion channels. These approaches hold great potential to advance research on ion channel drugs in the future.
Asunto(s)
Canales Iónicos , Enfermedades Musculares , Humanos , Canales Iónicos/metabolismo , Diseño de Fármacos , Descubrimiento de Drogas , Estructura Molecular , Computadores , Diseño Asistido por ComputadoraRESUMEN
Positively charged amino acid side-chains play important roles in anion binding and permeation through the CFTR chloride channel. One pore-lining lysine residue in particular (K95) has been shown to be indispensable for anion binding, conductance, and selectivity. Here, we use functional investigation of CFTR to show that a nearby arginine (R134) plays a functionally analogous role. Removal of this positive charge (in the R134Q mutant) drastically reduces single-channel conductance, weakens binding of both permeant and blocking anions, and abolishes the normal anion conductance selectivity pattern. Each of these functional effects was reversed by a second-site mutation (S1141K) that introduces an ectopic positive charge to a nearby pore-lining residue. Substituted cysteine accessibility experiments confirm that R134-but not nearby residues in the same transmembrane helix-is accessible within the pore lumen. These results suggest that K95 and R134, which are very close together within the inner vestibule of the pore, play analogous, important roles, and that both are required for the normal anion binding and anion conductance properties of the pore. Nevertheless, that fact that both positive charges can be "transplanted" to other sites in the inner vestibule with little effect on channel permeation properties indicates that it is the overall number of charges-rather than their exact locations-that controls pore function.
Asunto(s)
Aniones/metabolismo , Arginina/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Lisina/metabolismo , Mutación , Animales , Arginina/química , Arginina/genética , Células Cultivadas , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Lisina/química , Lisina/genética , Técnicas de Placa-Clamp , Conformación ProteicaRESUMEN
Cyclic nucleotide-gated (CNG) channels are key to the signal transduction machinery of certain sensory modalities both in vertebrate and invertebrate organisms. They translate a chemical change in cyclic nucleotide concentration into an electrical signal that can spread through sensory cells. Despite CNG and voltage-gated potassium channels sharing a remarkable amino acid sequence homology and basic architectural plan, their functional properties are dramatically different. While voltage-gated potassium channels are highly selective and require membrane depolarization to open, CNG channels have low ion selectivity and are not very sensitive to voltage. In the last few years, many high-resolution structures of intact CNG channels have been released. This wealth of new structural information has provided enormous progress toward the understanding of the molecular mechanisms and driving forces underpinning CNG channel activation. In this review, we report on the current understanding and controversies surrounding the gating mechanism in CNG channels, as well as the deep intertwining existing between gating, the ion permeation process, and its modulation by membrane voltage. While the existence of this powerful coupling was recognized many decades ago, its direct structural demonstration, and ties to the CNG channel inherent pore flexibility, is a recent achievement.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/química , Canales Catiónicos Regulados por Nucleótidos Cíclicos/fisiología , Activación del Canal Iónico/fisiología , Animales , Humanos , Conformación Proteica , Estructura Secundaria de Proteína , Células Fotorreceptoras Retinianas Bastones/química , Células Fotorreceptoras Retinianas Bastones/fisiología , Transducción de Señal/fisiologíaRESUMEN
The Kca3.1 channels, previously designated as IK1 or SK4 channels and encoded by the KCNN4 gene, are activated by a rise of the intracellular Ca2+ concentration. These K+ channels are widely expressed in many organs and involved in many pathologies. In particular, Kca3.1 channels have been studied intensively in the context of cancer. They are not only a marker and a valid prognostic tool for cancer patients, but have an important share in driving cancer progression. Their function is required for many characteristic features of the aggressive cancer cell behavior such as migration, invasion and metastasis as well as proliferation and therapy resistance. In the context of cancer, another property of Kca3.1 is now emerging. These channels can be a target for novel small molecule-based imaging probes, as it has been validated in case of fluorescently labeled senicapoc-derivatives. The aim of this review is (i) to give an overview on the role of Kca3.1 channels in cancer progression and in shaping the cancer microenvironment, (ii) discuss the potential of using Kca3.1 targeting drugs for cancer imaging, (iii) and highlight the possibility of combining molecular dynamics simulations to image inhibitor binding to Kca3.1 channels in order to provide a deeper understanding of Kca3.1 channel pharmacology. Alltogether, Kca3.1 is an attractive therapeutic target so that senicapoc, originally developed for the treatment of sickle cell anemia, should be repurposed for the treatment of cancer patients.
Asunto(s)
Acetamidas/uso terapéutico , Antineoplásicos/uso terapéutico , Calcio/metabolismo , Neoplasias/tratamiento farmacológico , Bloqueadores de los Canales de Potasio/uso terapéutico , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Compuestos de Tritilo/uso terapéutico , Antineoplásicos/química , Antidrepanocíticos/química , Antidrepanocíticos/uso terapéutico , Sitios de Unión , Señalización del Calcio , Progresión de la Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Simulación de Dinámica Molecular , Terapia Molecular Dirigida/métodos , Metástasis de la Neoplasia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Bloqueadores de los Canales de Potasio/química , Estructura Secundaria de Proteína , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/química , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genéticaRESUMEN
High-temperature electrolysis using solid oxide electrolysis cells (SOECs) provides a promising way for the storage of renewable energy into chemical fuels. During the past, nickel-based cathode-supported thin-film electrolyte configuration was widely adopted. However, such cells suffer from the serious challenge of anode delamination at high electrolysis currents due to enormous gaseous oxygen formation at the anode-electrolyte interface with insufficient adhesion caused by low sintering temperatures for ensuring high anode porosity and cathode pulverization because of potential nickel redox reaction. Here, the authors propose, fabricate, and test asymmetric thick anode-supported SOECs with firm anode-electrolyte interface and graded anode gas diffusion channel for realizing efficient and stable electrolysis at ultrahigh currents. Such a specially structured anode allows the co-sintering of anode support and electrolyte at high temperatures to form strong interface adhesion while suppressing anode sintering. The mixed oxygen-ion and electron conducting anode with graded channel structure provides a fast oxygen release pathway, large anode surface for oxygen evolution reaction, and excellent support for depositing nanocatalysts, to further improve oxygen evolution activity. As a result, the as-prepared cells demonstrate both high performance, comparable or even higher than state-of-the-art cathode-supported SOECs, and outstanding stability at a record current density of 2.5 A cm-2 .
RESUMEN
Potassium channels are the most diverse and ubiquitous family of ion channels found in cells. The Ca2+ and voltage gated members form a subfamily that play a variety of roles in both excitable and non-excitable cells and are further classified on the basis of their single channel conductance to form the small conductance (SK), intermediate conductance (IK) and big conductance (BK) K+ channels.In this chapter, we will focus on the mechanisms underlying the gating of BK channels, whose function is modified in different tissues by different splice variants as well as the expanding array of regulatory accessory subunits including ß, γ and LINGO subunits. We will examine how BK channels are modified by these regulatory subunits and describe how the channel gating is altered by voltage and Ca2+ whilst setting this in context with the recently published structures of the BK channel. Finally, we will discuss how BK and other calcium-activated channels are modulated by novel ion channel modulators and describe some of the challenges associated with trying to develop compounds with sufficient efficacy, potency and selectivity to be of therapeutic benefit.
Asunto(s)
Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Calcio/metabolismo , Cinética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismoRESUMEN
Cell survival and normal cell function require a highly coordinated and precise regulation of basal cytosolic Ca2+ concentrations. The primary source of Ca2+ entry into the cell is mediated by the Ca2+ release-activated Ca2+ (CRAC) channel. Its action is stimulated in response to internal Ca2+ store depletion. The fundamental constituents of CRAC channels are the Ca2+ sensor, stromal interaction molecule 1 (STIM1) anchored in the endoplasmic reticulum, and a highly Ca2+-selective pore-forming subunit Orai1 in the plasma membrane. The precise nature of the Orai1 pore opening is currently a topic of intensive research. This review describes how Orai1 gating checkpoints in the middle and cytosolic extended transmembrane regions act together in a concerted manner to ensure an opening-permissive Orai1 channel conformation. In this context, we highlight the effects of the currently known multitude of Orai1 mutations, which led to the identification of a series of gating checkpoints and the determination of their role in diverse steps of the Orai1 activation cascade. The synergistic action of these gating checkpoints maintains an intact pore geometry, settles STIM1 coupling, and governs pore opening. We describe the current knowledge on Orai1 channel gating mechanisms and summarize still open questions of the STIM1-Orai1 machinery.
Asunto(s)
Señalización del Calcio/genética , Calcio/metabolismo , Proteína ORAI1/genética , Molécula de Interacción Estromal 1/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Supervivencia Celular/genética , Citosol/metabolismo , Retículo Endoplásmico/genética , HumanosRESUMEN
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are widely expressed intracellular channels that release Ca2+ from the endoplasmic reticulum (ER). We review how studies of IP3Rs removed from their intracellular environment ('ex cellula'), alongside similar analyses of ryanodine receptors, have contributed to understanding IP3R behaviour. Analyses of permeabilized cells have demonstrated that the ER is the major intracellular Ca2+ store, and that IP3 stimulates Ca2+ release from this store. Radioligand binding confirmed that the 4,5-phosphates of IP3 are essential for activating IP3Rs, and facilitated IP3R purification and cloning, which paved the way for structural analyses. Reconstitution of IP3Rs into lipid bilayers and patch-clamp recording from the nuclear envelope have established that IP3Rs have a large conductance and select weakly between Ca2+ and other cations. Structural analyses are now revealing how IP3 binding to the N-terminus of the tetrameric IP3R opens the pore â¼7â nm away from the IP3-binding core (IBC). Communication between the IBC and pore passes through a nexus of interleaved domains contributed by structures associated with the pore and cytosolic domains, which together contribute to a Ca2+-binding site. These structural analyses provide evidence to support the suggestion that IP3 gates IP3Rs by first stimulating Ca2+ binding, which leads to pore opening and Ca2+ release.
Asunto(s)
Señalización del Calcio/fisiología , Retículo Endoplásmico/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Animales , Calcio/metabolismo , Humanos , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
Our molecular understanding of the cystic fibrosis transmembrane conductance regulator (CFTR)-the chloride channel that is mutated in cystic fibrosis-has been greatly enhanced by a number of recent atomic-level structures of the protein in different conformations. One surprising aspect of these structures was the finding that the eighth of CFTR's 12 membrane-spanning segments (TM8) appeared close to the channel pore. Although functional evidence supports a role for other TMs in forming the pore, such a role for TM8 has not previously been reported. Here, we use patch-clamp recording to investigate the functional role of TM8. Using substituted cysteine accessibility mutagenesis, we find that three amino acid side-chains in TM8 (Y913, Y914, and Y917) are exposed to the extracellular, but not the intracellular, solution. Cysteine cross-linking experiments suggest that Y914 and Y917 are in close proximity to L102 (TM1) and F337 (TM6), respectively, suggesting that TM8 contributes to the narrow selectivity filter region of the pore. Different amino acid substitutions suggest that Y914, and to a lesser extent Y917, play important roles in controlling anion flux through the open channel. Furthermore, substitutions that reduce side-chain volume at Y917 severely affect channel gating, resulting in a channel with an extremely unstable open state. Our results suggest that pore-lining TM8 is among the most important TMs controlling the permeation phenotype of the CFTR channel, and also that movement of TM8 may be critically involved in channel gating.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Animales , Células CHO , Línea Celular , Cricetulus , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Modelos Moleculares , Mutación Puntual , Conformación ProteicaRESUMEN
Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd2+ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.
Asunto(s)
Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Activación del Canal Iónico , Conformación Proteica , Animales , Células CHO , Cricetinae , Cricetulus , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Humanos , Metales/química , Metales/metabolismo , Modelos Moleculares , MutaciónRESUMEN
Ca2+ is a ubiquitous intracellular messenger that regulates numerous physiological activities in humans, animals, plants, and bacteria. Cytosolic Ca2+ is kept at a low level, but subcellular organelles such as the endoplasmic reticulum (ER) and Golgi apparatus maintain high-concentration Ca2+ stores. Under resting conditions, store Ca2+ homeostasis is dynamically regulated to equilibrate between active Ca2+ uptake and passive Ca2+ leak processes. The evolutionarily conserved Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) proteins mediate Ca2+ homeostasis and cell death. This review focuses on recent advances in functional and structural analysis of TMBIM proteins in regulation of the two related functions. The roles of TMBIM proteins in pathogen infection and cancer are also discussed with prospects for treatment. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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Apoptosis/fisiología , Calcio/fisiología , Homeostasis/fisiología , Proteínas del Tejido Nervioso/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Neoplasias/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas de Plantas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The renal proximal tubule achieves the majority of renal water and solute reabsorption with the help of paracellular channels which lead through the tight junction. The proteins forming such channels in the proximal tubule are claudin-2, claudin-10a, and possibly claudin-17. Claudin-2 forms paracellular channels selective for small cations like Na+ and K+. Independently of each other, claudin-10a and claudin-17 form anion-selective channels. The claudins form the paracellular "pore pathway" and are integrated, together with purely sealing claudins and other tight junction proteins, in the belt of tight junction strands surrounding the tubular epithelial cells. In most species, the proximal tubular tight junction consists of only 1-2 (pars convoluta) to 3-5 (pars recta) horizontal strands. Even so, they seal the tubule very effectively against leak passage of nutrients and larger molecules. Remarkably, claudin-2 channels are also permeable to water so that 20-25% of proximal water absorption may occur paracellularly. Although the exact structure of the claudin-2 channel is still unknown, it is clear that Na+ and water share the same pore. Already solved claudin crystal structures reveal a characteristic ß-sheet, comprising ß-strands from both extracellular loops, which is anchored to a left-handed four-transmembrane helix bundle. This allowed homology modeling of channel-forming claudins present in the proximal tubule. The surface of cation- and anion-selective claudins differ in electrostatic potentials in the area of the proposed ion channel, resulting in the opposite charge selectivity of these claudins. Presently, while models of the molecular structure of the claudin-based oligomeric channels have been proposed, its full understanding has only started.
Asunto(s)
Claudinas/metabolismo , Túbulos Renales Proximales/metabolismo , Uniones Estrechas/metabolismo , Animales , Claudinas/química , Humanos , Túbulos Renales Proximales/fisiología , Túbulos Renales Proximales/ultraestructura , Uniones Estrechas/ultraestructuraRESUMEN
Voltage-gated sodium channels initiate action potentials in brain neurons. In the 1970s, much was known about the function of sodium channels from measurements of ionic currents using the voltage clamp method, but there was no information about the sodium channel molecules themselves. As a postdoctoral fellow and staff scientist at the National Institutes of Health, I developed neurotoxins as molecular probes of sodium channels in cultured neuroblastoma cells. During those years, Bruce Ransom and I crossed paths as members of the laboratories of Marshall Nirenberg and Philip Nelson and shared insights about sodium channels in neuroblastoma cells from my work and electrical excitability and synaptic transmission in cultured spinal cord neurons from Bruce's pioneering electrophysiological studies. When I established my laboratory at the University of Washington in 1977, my colleagues and I used those neurotoxins to identify the protein subunits of sodium channels, purify them, and reconstitute their ion conductance activity in pure form. Subsequent studies identified the molecular basis for the main functions of sodium channels-voltage-dependent activation, rapid and selective ion conductance, and fast inactivation. Bruce Ransom and I re-connected in the 1990s, as ski buddies at the Winter Conference on Brain Research and as faculty colleagues at the University of Washington when Bruce became our founding Chair of Neurology and provided visionary leadership of that department. In the past decade my work on sodium channels has evolved into structural biology. Molecular modeling and X-ray crystallographic studies have given new views of sodium channel function at atomic resolution. Sodium channels are also the molecular targets for genetic diseases, including Dravet Syndrome, an intractable pediatric epilepsy disorder with major co-morbidities of cognitive deficit, autistic-like behaviors, and premature death that is caused by loss-of-function mutations in the brain sodium channel NaV1.1. Our work on a mouse genetic model of this disease has shown that its multi-faceted pathophysiology and co-morbidities derive from selective loss of electrical excitability and action potential firing in GABAergic inhibitory neurons, which disinhibits neural circuits throughout the brain and leads directly to the epilepsy, premature death and complex co-morbidities of this disease. It has been rewarding for me to use our developing knowledge of sodium channels to help understand the pathophysiology and to suggest potential therapeutic approaches for this devastating childhood disease.
Asunto(s)
Epilepsia/metabolismo , Canales de Sodio/química , Canales de Sodio/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Neurotoxinas/metabolismo , Neurotoxinas/farmacología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/fisiologíaRESUMEN
Oxidation is an important biochemical defense mechanism, but it also elicits toxicity; therefore, oxidation must be under strict control. In phagocytotic events in neutrophils, the voltage-gated H(+) (Hv) channel is a key regulator of the production of reactive oxygen species against invading bacteria. The cytoplasmic domain of the Hv channel forms a dimeric coiled coil underpinning a dimerized functional unit. Importantly, in the alignment of the coiled-coil core, a conserved cysteine residue forms a potential intersubunit disulfide bond. In this study, we solved the crystal structures of the coiled-coil domain in reduced, oxidized, and mutated (Cys â Ser) states. The crystal structures indicate that a pair of Cys residues forms an intersubunit disulfide bond dependent on the redox conditions. CD spectroscopy revealed that the disulfide bond increases the thermal stability of the coiled-coil protein. We also reveal that two thiol modifier molecules are able to bind to Cys in a redox-dependent manner without disruption of the dimeric coiled-coil assembly. Thus, the biochemical properties of the cytoplasmic coiled-coil domain in the Hv channel depend on the redox condition, which may play a role in redox sensing in the phagosome.
Asunto(s)
Canales Iónicos/química , Canales Iónicos/fisiología , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Western Blotting , Dicroismo Circular , Cristalografía por Rayos X , Disulfuros/química , Disulfuros/metabolismo , Células HEK293 , Humanos , Canales Iónicos/genética , Macrófagos/metabolismo , Potenciales de la Membrana , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oxidación-Reducción , Técnicas de Placa-Clamp , Fagosomas/metabolismo , Multimerización de Proteína , Homología de Secuencia de Aminoácido , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Voltage-gated sodium channels (Navs) are an important family of transmembrane ion channel proteins and Nav drug discovery is an exciting field. Pharmaceutical investment in Navs for pain therapeutics has expanded exponentially due to genetic data such as SCN10A mutations and an improved ability to establish an effective screen sequence for example IonWorks Barracuda®, Synchropatch® and Qube®. Moreover, emerging clinical data (AZD-3161, XEN402, CNV1014802, PF-05089771, PF-04531083) combined with recent breakthroughs in Nav structural biology pave the way for a future of fruitful prospective Nav drug discovery.