scholarly journals Batrachotoxin acts as a stent to hold open homotetrameric prokaryotic voltage-gated sodium channels

2018 ◽  
Vol 151 (2) ◽  
pp. 186-199 ◽  
Author(s):  
Rocio K. Finol-Urdaneta ◽  
Jeffrey R. McArthur ◽  
Marcel P. Goldschen-Ohm ◽  
Rachelle Gaudet ◽  
Denis B. Tikhonov ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Molecular Model ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Nav Channels ◽  
Single Polypeptide ◽  
Nerve Muscle

Batrachotoxin (BTX), an alkaloid from skin secretions of dendrobatid frogs, causes paralysis and death by facilitating activation and inhibiting deactivation of eukaryotic voltage-gated sodium (Nav) channels, which underlie action potentials in nerve, muscle, and heart. A full understanding of the mechanism by which BTX modifies eukaryotic Nav gating awaits determination of high-resolution structures of functional toxin–channel complexes. Here, we investigate the action of BTX on the homotetrameric prokaryotic Nav channels NaChBac and NavSp1. By combining mutational analysis and whole-cell patch clamp with molecular and kinetic modeling, we show that BTX hinders deactivation and facilitates activation in a use-dependent fashion. Our molecular model shows the horseshoe-shaped BTX molecule bound within the open pore, forming hydrophobic H-bonds and cation-π contacts with the pore-lining helices, leaving space for partially dehydrated sodium ions to permeate through the hydrophilic inner surface of the horseshoe. We infer that bulky BTX, bound at the level of the gating-hinge residues, prevents the S6 rearrangements that are necessary for closure of the activation gate. Our results reveal general similarities to, and differences from, BTX actions on eukaryotic Nav channels, whose major subunit is a single polypeptide formed by four concatenated, homologous, nonidentical domains that form a pseudosymmetric pore. Our determination of the mechanism by which BTX activates homotetrameric voltage-gated channels reveals further similarities between eukaryotic and prokaryotic Nav channels and emphasizes the tractability of bacterial Nav channels as models of voltage-dependent ion channel gating. The results contribute toward a deeper, atomic-level understanding of use-dependent natural and synthetic Nav channel agonists and antagonists, despite their overlapping binding motifs on the channel proteins.

scholarly journals Spider Knottin Pharmacology at Voltage-Gated Sodium Channels and Their Potential to Modulate Pain Pathways

Toxins ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 626 ◽  
Author(s):  
Yashad Dongol ◽  
Fernanda Caldas Cardoso ◽  
Richard J Lewis
Keyword(s):  
Chronic Pain ◽  
Sodium Channels ◽  
Structural Features ◽  
Voltage Gated ◽  
Subtype Selectivity ◽  
Voltage Gated Sodium Channels ◽  
Neuronal Signalling ◽  
Pain Pathways ◽  
Nav Channels ◽  
Chronic Pain Conditions

Voltage-gated sodium channels (NaVs) are a key determinant of neuronal signalling. Neurotoxins from diverse taxa that selectively activate or inhibit NaV channels have helped unravel the role of NaV channels in diseases, including chronic pain. Spider venoms contain the most diverse array of inhibitor cystine knot (ICK) toxins (knottins). This review provides an overview on how spider knottins modulate NaV channels and describes the structural features and molecular determinants that influence their affinity and subtype selectivity. Genetic and functional evidence support a major involvement of NaV subtypes in various chronic pain conditions. The exquisite inhibitory properties of spider knottins over key NaV subtypes make them the best lead molecules for the development of novel analgesics to treat chronic pain.

scholarly journals Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Paul G DeCaen ◽  
Yuka Takahashi ◽  
Terry A Krulwich ◽  
Masahiro Ito ◽  
David E Clapham
Keyword(s):  
Divalent Cations ◽  
Ion Selectivity ◽  
Resting Membrane Potential ◽  
Na Channel ◽  
Ph Homeostasis ◽  
Ionic Selectivity ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Nav Channels ◽  
Alkaliphilic Bacillus

Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na+ to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na+ channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na+, Ca2+ and K+ ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na+ or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

scholarly journals Subtype Specificity of β-Toxin Tf1a from Tityus fasciolatus in Voltage Gated Sodium Channels

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 339 ◽  
Author(s):  
Daniel Mata ◽  
Diogo Tibery ◽  
Leandro Campos ◽  
Thalita Camargos ◽  
Steve Peigneur ◽  
...  
Keyword(s):  
Sodium Channels ◽  
Complex Mixture ◽  
Normal Function ◽  
Structural Function ◽  
Scorpion Toxins ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Subtype Specificity ◽  
Nav Channels ◽  
Predictive Strength

Scorpion venoms are a complex mixture of components. Among them the most important are peptides, which presents the capacity to interact and modulate several ion channel subtypes, including voltage-gated sodium channels (NaV). Screening the activity of scorpion toxins on different subtypes of NaV reveals the scope of modulatory activity and, in most cases, low channel selectivity. Until now there are approximately 60 scorpion toxins experimentally assayed on NaV channels. However, the molecular bases of interaction between scorpion toxins and NaV channels are not fully elucidated. The activity description of new scorpion toxins is crucial to enhance the predictive strength of the structural–function correlations of these NaV modulatory molecules. In the present work a new scorpion toxin (Tf1a) was purified from Tityus fasciolatus venom by RP-HPLC, and characterized using electrophysiological experiments on different types of voltage-gated sodium channels. Tf1a was able to modify the normal function of NaV tested, showing to be a typical β-NaScTx. Tf1a also demonstrated an unusual capability to alter the kinetics of NaV1.5.

scholarly journals Distribution of TTX-sensitive voltage-gated sodium channels in primary sensory endings of mammalian muscle spindles

2017 ◽  
Vol 117 (4) ◽  
pp. 1690-1701 ◽  
Author(s):  
Dario I. Carrasco ◽  
Jacob A. Vincent ◽  
Timothy C. Cope
Keyword(s):  
Sodium Channels ◽  
Molecular Mechanisms ◽  
Mammalian Species ◽  
Muscle Spindles ◽  
Tonic Firing ◽  
Muscle Stretch ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Nav Channels ◽  
Sensory Endings

Knowledge of the molecular mechanisms underlying signaling of mechanical stimuli by muscle spindles remains incomplete. In particular, the ionic conductances that sustain tonic firing during static muscle stretch are unknown. We hypothesized that tonic firing by spindle afferents depends on sodium persistent inward current (INaP) and tested for the necessary presence of the appropriate voltage-gated sodium (NaV) channels in primary sensory endings. The NaV1.6 isoform was selected for both its capacity to produce INaP and for its presence in other mechanosensors that fire tonically. The present study shows that NaV1.6 immunoreactivity (IR) is concentrated in heminodes, presumably where tonic firing is generated, and we were surprised to find NaV1.6 IR strongly expressed also in the sensory terminals, where mechanotransduction occurs. This spatial pattern of NaV1.6 IR distribution was consistent for three mammalian species (rat, cat, and mouse), as was tonic firing by primary spindle afferents. These findings meet some of the conditions needed to establish participation of INaP in tonic firing by primary sensory endings. The study was extended to two additional NaV isoforms, selected for their sensitivity to TTX, excluding TTX-resistant NaV channels, which alone are insufficient to support firing by primary spindle endings. Positive immunoreactivity was found for NaV1.1, predominantly in sensory terminals together with NaV1.6 and for NaV1.7, mainly in preterminal axons. Differential distribution in primary sensory endings suggests specialized roles for these three NaV isoforms in the process of mechanosensory signaling by muscle spindles. NEW & NOTEWORTHY The molecular mechanisms underlying mechanosensory signaling responsible for proprioceptive functions are not completely elucidated. This study provides the first evidence that voltage-gated sodium channels (NaVs) are expressed in the spindle primary sensory ending, where NaVs are found at every site involved in transduction or encoding of muscle stretch. We propose that NaVs contribute to multiple steps in sensory signaling by muscle spindles as it does in other types of slowly adapting sensory neurons.

scholarly journals Hypoxic Conditions Promote Rhythmic Contractile Oscillations Mediated by Voltage-Gated Sodium Channels Activation in Human Arteries

2021 ◽  
Vol 22 (5) ◽  
pp. 2570
Author(s):  
Anne Virsolvy ◽  
Aurélie Fort ◽  
Lucie Erceau ◽  
Azzouz Charrabi ◽  
Maurice Hayot ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Sodium Channels ◽  
Rhythmic Activity ◽  
Voltage Gated ◽  
Hypoxic Conditions ◽  
Voltage Gated Sodium Channels ◽  
Arterial Tissue ◽  
Nav Channels ◽  
Human Arteries

Arterial smooth muscle exhibits rhythmic oscillatory contractions called vasomotion and believed to be a protective mechanism against tissue hypoperfusion or hypoxia. Oscillations of vascular tone depend on voltage and follow oscillations of the membrane potential. Voltage-gated sodium channels (Nav), responsible for the initiation and propagation of action potentials in excitable cells, have also been evidenced both in animal and human vascular smooth muscle cells (SMCs). For example, they contribute to arterial contraction in rats, but their physiopathological relevance has not been established in human vessels. In the present study, we investigated the functional role of Nav in the human artery. Experiments were performed on human uterine arteries obtained after hysterectomy and on SMCs dissociated from these arteries. In SMCs, we recorded a tetrodotoxin (TTX)-sensitive and fast inactivating voltage-dependent INa current. Various Nav genes, encoding -subunit isoforms sensitive (Nav 1.2; 1.3; 1.7) and resistant (Nav 1.5) to TTX, were detected both in arterial tissue and in SMCs. Nav channels immunostaining showed uniform distribution in SMCs and endothelial cells. On arterial tissue, we recorded variations of isometric tension, ex vivo, in response to various agonists and antagonists. In arterial rings placed under hypoxic conditions, the depolarizing agent KCl and veratridine, a specific Nav channels agonist, both induced a sustained contraction overlaid with rhythmic oscillations of tension. After suppression of sympathetic control either by blocking the release of catecholamine or by antagonizing the target adrenergic response, rhythmic activity persisted while the sustained contraction was abolished. This rhythmic activity of the arteries was suppressed by TTX but, in contrast, only attenuated by antagonists of calcium channels, Na+/Ca2+ exchanger, Na+/K+-ATPase and the cardiac Nav channel. These results highlight the role of Nav as a novel key element in the vasomotion of human arteries. Hypoxia promotes activation of Nav channels involved in the initiation of rhythmic oscillatory contractile activity.

scholarly journals Intrinsic Gating Behavior of Voltage-Gated Sodium Channels Predetermines Regulation by Auxiliary β-subunits

2021 ◽  
Author(s):  
Niklas Brake ◽  
Adamo S Mancino ◽  
Yuhao Yan ◽  
Takushi Shimomura ◽  
Heika Silveira ◽  
...  
Keyword(s):  
Excitable Cells ◽  
Voltage Sensing ◽  
Auxiliary Subunits ◽  
Voltage Gated ◽  
Closed State ◽  
Channel Function ◽  
Voltage Gated Sodium Channels ◽  
Nav Channels ◽  
Regulatory Effects ◽  
Electrical Signaling

AbstractVoltage-gated sodium (Nav) channels mediate rapid millisecond electrical signaling in excitable cells. Auxiliary subunits, β1-β4, are thought to regulate Nav channel function through covalent and/or polar interactions with the channel’ s voltage-sensing domains. How these interactions translate into the diverse and variable regulatory effects of β-subunits remains unclear. Here, we find that the intrinsic movement order of the voltage-sensing domains during channel gating is unexpectedly variable across Nav channel isoforms. This movement order dictates the channel’ s propensity for closed-state inactivation, which in turn modulates the actions of β1 and β3. We show that the differential regulation of skeletal muscle, cardiac, and neuronal Nav channels is explained by their variable levels of closed-state inactivation. Together, this study provides a unified mechanism for the regulation of all Nav channel isoforms by β1 and β3, which explains how the fixed structural interactions of auxiliary subunits can paradoxically exert variable effects on channel function.

scholarly journals Biophysical characterization of the honeybee DSC1 orthologue reveals a novel voltage-dependent Ca2+ channel subfamily: CaV4

2016 ◽  
Vol 148 (2) ◽  
pp. 133-145 ◽  
Author(s):  
Pascal Gosselin-Badaroudine ◽  
Adrien Moreau ◽  
Louis Simard ◽  
Thierry Cens ◽  
Matthieu Rousset ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Expression System ◽  
Selectivity Filter ◽  
Inactivation Kinetics ◽  
Na Channel ◽  
Biophysical Characterization ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Nav Channels

Bilaterian voltage-gated Na+ channels (NaV) evolved from voltage-gated Ca2+ channels (CaV). The Drosophila melanogaster Na+ channel 1 (DSC1), which features a D-E-E-A selectivity filter sequence that is intermediate between CaV and NaV channels, is evidence of this evolution. Phylogenetic analysis has classified DSC1 as a Ca2+-permeable Na+ channel belonging to the NaV2 family because of its sequence similarity with NaV channels. This is despite insect NaV2 channels (DSC1 and its orthologue in Blatella germanica, BSC1) being more permeable to Ca2+ than Na+. In this study, we report the cloning and molecular characterization of the honeybee (Apis mellifera) DSC1 orthologue. We reveal several sequence variations caused by alternative splicing, RNA editing, and genomic variations. Using the Xenopus oocyte heterologous expression system and the two-microelectrode voltage-clamp technique, we find that the channel exhibits slow activation and inactivation kinetics, insensitivity to tetrodotoxin, and block by Cd2+ and Zn2+. These characteristics are reminiscent of CaV channels. We also show a strong selectivity for Ca2+ and Ba2+ ions, marginal permeability to Li+, and impermeability to Mg2+ and Na+ ions. Based on current ion channel nomenclature, the D-E-E-A selectivity filter, and the properties we have uncovered, we propose that DSC1 homologues should be classified as CaV4 rather than NaV2. Indeed, channels that contain the D-E-E-A selectivity sequence are likely to feature the same properties as the honeybee’s channel, namely slow activation and inactivation kinetics and strong selectivity for Ca2+ ions.

scholarly journals Bidirectional Modulation of the Voltage-Gated Sodium (Nav1.6) Channel by Rationally Designed Peptidomimetics

Molecules ◽  
2020 ◽  
Vol 25 (15) ◽  
pp. 3365
Author(s):  
Nolan M. Dvorak ◽  
Paul A. Wadsworth ◽  
Pingyuan Wang ◽  
Haiying Chen ◽  
Jia Zhou ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Functional Evaluation ◽  
Complex Assembly ◽  
Voltage Gated ◽  
Whole Cell Patch Clamp ◽  
Protective Groups ◽  
Nav Channels ◽  
Current Densities ◽  
Patch Clamp Electrophysiology ◽  
Structural Region

Disruption of protein:protein interactions (PPIs) that regulate the function of voltage-gated Na+ (Nav) channels leads to neural circuitry aberrations that have been implicated in numerous channelopathies. One example of this pathophysiology is mediated by dysfunction of the PPI between Nav1.6 and its regulatory protein fibroblast growth factor 14 (FGF14). Thus, peptides derived from FGF14 might exert modulatory actions on the FGF14:Nav1.6 complex that are functionally relevant. The tetrapeptide Glu-Tyr-Tyr-Val (EYYV) mimics surface residues of FGF14 at the β8–β9 loop, a structural region previously implicated in its binding to Nav1.6. Here, peptidomimetics derived from EYYV (6) were designed, synthesized, and pharmacologically evaluated to develop probes with improved potency. Addition of hydrophobic protective groups to 6 and truncation to a tripeptide (12) produced a potent inhibitor of FGF14:Nav1.6 complex assembly. Conversely, addition of hydrophobic protective groups to 6 followed by addition of an N-terminal benzoyl substituent (19) produced a potentiator of FGF14:Nav1.6 complex assembly. Subsequent functional evaluation using whole-cell patch-clamp electrophysiology confirmed their inverse activities, with 12 and 19 reducing and increasing Nav1.6-mediated transient current densities, respectively. Overall, we have identified a negative and positive allosteric modulator of Nav1.6, both of which could serve as scaffolds for the development of target-selective neurotherapeutics.

scholarly journals An unconventional conductance is required for pacemaking of nigral dopamine neurons

2020 ◽  
Author(s):  
Kevin Jehasse ◽  
Laurent Massotte ◽  
Sebastian Hartmann ◽  
Romain Vitello ◽  
Sofian Ringlet ◽  
...  
Keyword(s):  
Substantia Nigra ◽  
Sodium Channels ◽  
Dopamine Neurons ◽  
Substantia Nigra Pars Compacta ◽  
Voltage Gated ◽  
Molecular Identity ◽  
Voltage Gated Sodium Channels ◽  
Voltage Dependent ◽  
Ionic Mechanisms ◽  
Voltage Gated Channels

ABSTRACTAlthough several ionic mechanisms are known to control rate and regularity of the pacemaker in dopamine (DA) neurons from the substantia nigra pars compacta (SNc), a conductance essential for pacing has yet to be defined. Here we provide pharmacological evidence that pacemaking of SNc DA neurons is enabled by an unconventional conductance. We found that 1-(2,4-xylyl)guanidine (XG), an established blocker of gating pore currents in mutant voltage gated sodium channels, selectively stops pacemaking of DA SNc neurons and is without effect on the main pore of their voltage-gated channels. We isolated a voltage-dependent, non-inactivating XG-sensitive current of 20-25 pA which operates in the relevant subthreshold range and is carried by both Na+ and Cl- ions. While the molecular identity of this conductance remains to be determined, we show that this XG-sensitive current is crucial to sustain pacemaking in these neurons.

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