Evidence of Cytolysin A nanopore incorporation in mammalian cells assessed by a graphical user interface.

Technologies capable of assessing cellular metabolites with high precision and temporal resolution are currently limited. Recent developments in the field of nanopore sensors allow the non-stochastic quantification of metabolites, where a nanopore is acting as an electrical transducer for selective substrate binding proteins (SBPs). Here we show that incorporation of the pore-forming toxin Cytolysin A (ClyA) into the plasma membrane of Chinese hamster ovary cells (CHO-K1) results in the appearance of single-channel conductance amenable to multiplexed automated patch-clamp (APC) electrophysiology. In CHO-K1 cells, SBPs modify the ionic current flowing though ClyA nanopores, thus demonstrating its potential for metabolite sensing of living cells. Moreover, we developed a graphical user interface for the analysis of the complex signals resulting from multiplexed APC recordings. This system lays the foundation to bridge the gap between recent advances in the nanopore field (e.g., proteomic and transcriptomic) and potential cellular applications.

A previously unrecognized superfamily of macro-conotoxins includes an inhibitor of the sensory neuron calcium channel Cav2.3.

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.

Automated Patch Clamp Screening of Amiloride and 5-N,N-Hexamethyleneamiloride Analogs Identifies 6-Iodoamiloride as a Potent Acid-Sensing Ion Channel Inhibitor.

Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K+-sparing diuretic amiloride is a moderate nonspecific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch clamp protocol and identified 6-iodoamiloride as a potent ASIC1 inhibitor. Follow-up IC50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 94 (hASIC1 94 IC50 = 88 nM, cf. amiloride 11 IC50 = 1.7 µM). A similar improvement in activity was observed in ASIC3-mediated currents from rat dorsal root ganglion neurons (rDRG single-concentration 94 IC50 = 230 nM, cf. 11 IC50 = 2.7 µM). 6-Iodoamiloride represents the amiloride analog of choice for studying the effects of ASIC inhibition on cell physiology.

Investigation of CACNA1I Cav3.3 Dysfunction in Hemiplegic Migraine.

Familial hemiplegic migraine (FHM) is a severe neurogenetic disorder for which three causal genes, CACNA1A, SCN1A, and ATP1A2, have been implicated. However, more than 80% of referred diagnostic cases of hemiplegic migraine (HM) are negative for exonic mutations in these known FHM genes, suggesting the involvement of other genes. Using whole-exome sequencing data from 187 mutation-negative HM cases, we identified rare variants in the CACNA1I gene encoding the T-type calcium channel Cav3.3. Burden testing of CACNA1I variants showed a statistically significant increase in allelic burden in the HM case group compared to gnomAD (OR = 2.30, P = 0.00005) and the UK Biobank (OR = 2.32, P = 0.0004) databases. Dysfunction in T-type calcium channels, including Cav3.3, has been implicated in a range of neurological conditions, suggesting a potential role in HM. Using patch-clamp electrophysiology, we compared the biophysical properties of five Cav3.3 variants (p.R111G, p.M128L, p.D302G, p.R307H, and p.Q1158H) to wild-type (WT) channels expressed in HEK293T cells. We observed numerous functional alterations across the channels with Cav3.3-Q1158H showing the greatest differences compared to WT channels, including reduced current density, right-shifted voltage dependence of activation and inactivation, and slower current kinetics. Interestingly, we also found significant differences in the conductance properties exhibited by the Cav3.3-R307H and -Q1158H variants compared to WT channels under conditions of acidosis and alkalosis. In light of these data, we suggest that rare variants in CACNA1I may contribute to HM etiology.

Analgesic α-Conotoxin Binding Site on the Human GABAB Receptor.

The analgesic α-conotoxins Vc1.1, RgIA, and PeIA attenuate nociceptive transmission via activation of G protein-coupled GABAB receptors (GABABRs) to modulate N-type calcium channels in primary afferent neurons and recombinantly coexpressed human GABABR and Cav2.2 channels in human embryonic kidney 293T cells. Here, we investigate the effects of analgesic α-conotoxins following the mutation of amino acid residues in the Venus flytrap (VFT) domains of the GABABR subunits predicted through computational peptide docking and molecular dynamics simulations. Our docking calculations predicted that all three of the α-conotoxins form close contacts with VFT residues in both B1 and B2 subunits, comprising a novel GABABR ligand-binding site. The effects of baclofen and α-conotoxins on the peak Ba2+ current (IBa) amplitude were investigated on wild-type and 15 GABABR mutants individually coexpressed with human Cav2.2 channels. Mutations at the interface of the VFT domains of both GABABR subunits attenuated baclofen-sensitive IBa inhibition by the analgesic α-conotoxins. In contrast, mutations located outside the putative peptide-binding site (D380A and R98A) did not. The key GABABR residues involved in interactions with the α-conotoxins are K168 and R207 on the B2 subunit and S130, S153, R162, E200, F227, and E253 on the B1 subunit. The double mutant, S130A + S153A, abolished inhibition by both baclofen and the α-conotoxins. Depolarization-activated IBa mediated by both wild-type and all GABABR mutants were inhibited by the selective GABABR antagonist CGP 55845. This study identifies specific residues of GABABR involved in the binding of the analgesic α-conotoxins to the VFT domains of the GABABR. SIGNIFICANCE STATEMENT: This study defines the binding site of the analgesic α-conotoxins Vc1.1, RgIA, and PeIA on the human GABAB receptor to activate Gi/o proteins and inhibit Cav2.2 channels. Computational docking and molecular dynamics simulations of GABABR identified amino acids of the Venus flytrap (VFT) domains with which the α-conotoxins interact. GABABR alanine mutants attenuated baclofen-sensitive Cav2.2 inhibition by the α-conotoxins. We identify an allosteric binding site at the interface of the VFT domains of the GABABR subunits for the analgesic α-conotoxins.

µ-Theraphotoxin Pn3a inhibition of CaV3.3 channels reveals a novel isoform-selective drug binding site.

Low voltage-activated calcium currents are mediated by T-type calcium channels CaV3.1, CaV3.2, and CaV3.3, which modulate a variety of physiological processes including sleep, cardiac pace-making, pain, and epilepsy. CaV3 isoforms' biophysical properties, overlapping expression, and lack of subtype-selective pharmacology hinder the determination of their specific physiological roles in health and disease. We have identified µ-theraphotoxin Pn3a as the first subtype-selective spider venom peptide inhibitor of CaV3.3, with >100-fold lower potency against the other T-type isoforms. Pn3a modifies CaV3.3 gating through a depolarizing shift in the voltage dependence of activation thus decreasing CaV3.3-mediated currents in the normal range of activation potentials. Paddle chimeras of KV1.7 channels bearing voltage sensor sequences from all four CaV3.3 domains revealed preferential binding of Pn3a to the S3-S4 region of domain II (CaV3.3DII). This novel T-type channel pharmacological site was explored through computational docking simulations of Pn3a, site-directed mutagenesis, and full domain II swaps between CaV3 channels highlighting it as a subtype-specific pharmacophore. This research expands our understanding of T-type calcium channel pharmacology and supports the suitability of Pn3a as a molecular tool in the study of the physiological roles of CaV3.3 channels.

Multitarget nociceptor sensitization by a promiscuous peptide from the venom of the King Baboon spider.

The King Baboon spider, Pelinobius muticus, is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus, but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including NaV1.8, KV2.1, and tetrodotoxin-sensitive NaV channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms.

Analgesic α-conotoxins modulate native and recombinant GIRK1/2 channels via activation of GABAB receptors and reduce neuroexcitability.

BACKGROUND AND PURPOSE: Activation of GIRK channels via G protein-coupled GABAB receptors has been shown to attenuate nociceptive transmission. The analgesic α-conotoxin Vc1.1 activates GABAB receptors resulting in inhibition of Cav 2.2 and Cav 2.3 channels in mammalian primary afferent neurons. Here, we investigated the effects of analgesic α-conotoxins on recombinant and native GIRK-mediated K+ currents and on neuronal excitability. EXPERIMENTAL APPROACH: The effects of analgesic α-conotoxins, Vc1.1, RgIA, and PeIA, were investigated on inwardly-rectifying K+ currents in HEK293T cells recombinantly co-expressing either heteromeric human GIRK1/2 or homomeric GIRK2 subunits, with GABAB receptors. The effects of α-conotoxin Vc1.1 and baclofen were studied on GIRK-mediated K+ currents and the passive and active electrical properties of adult mouse dorsal root ganglion neurons. KEY RESULTS: Analgesic α-conotoxins Vc1.1, RgIA, and PeIA potentiate inwardly-rectifying K+ currents in HEK293T cells recombinantly expressing human GIRK1/2 channels and GABAB receptors. GABAB receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen occurs via a pertussis toxin-sensitive G protein and is inhibited by the selective GABAB receptor antagonist CGP 55845. In adult mouse dorsal root ganglion neurons, GABAB receptor-dependent GIRK channel potentiation by Vc1.1 and baclofen hyperpolarizes the cell membrane potential and reduces excitability. CONCLUSIONS AND IMPLICATIONS: This is the first report of GIRK channel potentiation via allosteric α-conotoxin Vc1.1-GABAB receptor agonism, leading to decreased neuronal excitability. Such action potentially contributes to the analgesic effects of Vc1.1 and baclofen observed in vivo.

Pathophysiological metabolic changes associated with disease modify the proarrhythmic risk profile of drugs with potential to prolong repolarisation.

BACKGROUND AND PURPOSE: Hydroxychloroquine, chloroquine and azithromycin are three drugs that were proposed to treat coronavirus disease 2019 (COVID-19). While concern already existed around their proarrhythmic potential, there are little data regarding how altered physiological states encountered in patients such as febrile state, electrolyte imbalances or acidosis might change their risk profiles. EXPERIMENTAL APPROACH: Potency of human ether-à-go-go related gene (hERG) block was measured using high-throughput electrophysiology in the presence of variable environmental factors. These potencies informed simulations to predict population risk profiles. Effects on cardiac repolarisation were verified in human induced pluripotent stem cell-derived cardiomyocytes from multiple individuals. KEY RESULTS: Chloroquine and hydroxychloroquine blocked hERG with IC50 of 1.47 ± 0.07 and 3.78 ± 0.17 µM, respectively, indicating proarrhythmic risk at concentrations effective against severe acute respiratory syndrome-coronovirus-2 (SARS-CoV-2) in vitro. Hypokalaemia and hypermagnesaemia increased potency of chloroquine and hydroxychloroquine, indicating increased proarrhythmic risk. Acidosis significantly reduced potency of all drugs, whereas increased temperature decreased potency of chloroquine and hydroxychloroquine against hERG but increased potency for azithromycin. In silico simulations demonstrated that proarrhythmic risk was increased by female sex, hypokalaemia and heart failure and identified specific genetic backgrounds associated with emergence of arrhythmia. CONCLUSION AND IMPLICATIONS: Our study demonstrates how proarrhythmic risk can be exacerbated by metabolic changes and pre-existing disease. More broadly, the study acts as a blueprint for how high-throughput in vitro screening, combined with in silico simulations, can help guide both preclinical screening and clinical management of patients in relation to drugs with potential to prolong repolarisation.

Small-molecule mimicry hunting strategy in the imperial cone snail, Conus imperialis.

Venomous animals hunt using bioactive peptides, but relatively little is known about venom small molecules and the resulting complex hunting behaviors. Here, we explored the specialized metabolites from the venom of the worm-hunting cone snail, Conus imperialis Using the model polychaete worm Platynereis dumerilii, we demonstrate that C. imperialis venom contains small molecules that mimic natural polychaete mating pheromones, evoking the mating phenotype in worms. The specialized metabolites from different cone snails are species-specific and structurally diverse, suggesting that the cones may adopt many different prey-hunting strategies enabled by small molecules. Predators sometimes attract prey using the prey's own pheromones, in a strategy known as aggressive mimicry. Instead, C. imperialis uses metabolically stable mimics of those pheromones, indicating that, in biological mimicry, even the molecules themselves may be disguised, providing a twist on fake news in chemical ecology.

In vivo survival and differentiation of Friedreich ataxia iPSC-derived sensory neurons transplanted in the adult dorsal root ganglia.

Friedreich ataxia (FRDA) is an autosomal recessive disease characterized by degeneration of dorsal root ganglia (DRG) sensory neurons, which is due to low levels of the mitochondrial protein Frataxin. To explore cell replacement therapies as a possible approach to treat FRDA, we examined transplantation of sensory neural progenitors derived from human embryonic stem cells (hESC) and FRDA induced pluripotent stem cells (iPSC) into adult rodent DRG regions. Our data showed survival and differentiation of hESC and FRDA iPSC-derived progenitors in the DRG 2 and 8 weeks post-transplantation, respectively. Donor cells expressed neuronal markers, including sensory and glial markers, demonstrating differentiation to these lineages. These results are novel and a highly significant first step in showing the possibility of using stem cells as a cell replacement therapy to treat DRG neurodegeneration in FRDA as well as other peripheral neuropathies.

Molecular and Functional Characterization of Neurogenin-2 Induced Human Sensory Neurons.

Sensory perception is fundamental to everyday life, yet understanding of human sensory physiology at the molecular level is hindered due to constraints on tissue availability. Emerging strategies to study and characterize peripheral neuropathies in vitro involve the use of human pluripotent stem cells (hPSCs) differentiated into dorsal root ganglion (DRG) sensory neurons. However, neuronal functionality and maturity are limited and underexplored. A recent and promising approach for directing hPSC differentiation towards functionally mature neurons involves the exogenous expression of Neurogenin-2 (NGN2). The optimized protocol described here generates sensory neurons from hPSC-derived neural crest (NC) progenitors through virally induced NGN2 expression. NC cells were derived from hPSCs via a small molecule inhibitor approach and enriched for migrating NC cells (66% SOX10+ cells). At the protein and transcript level, the resulting NGN2 induced sensory neurons (NGN2iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, NGN2iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of NGN2iSN excitability revealed functional expression of ion channels associated with the excitability of primary afferent neurons, such as Nav1.7, Nav1.8, Kv1.2, Kv2.1, BK, Cav2.1, Cav2.2, Cav3.2, ASICs and HCN among other ion channels, for which we provide functional and transcriptional evidence. Our characterization of stem cell-derived sensory neurons sheds light on the molecular basis of human sensory physiology and highlights the suitability of using hPSC-derived sensory neurons for modeling human DRG development and their potential in the study of human peripheral neuropathies and drug therapies.

Marine Toxins Targeting Kv1 Channels: Pharmacological Tools and Therapeutic Scaffolds.

Toxins from marine animals provide molecular tools for the study of many ion channels, including mammalian voltage-gated potassium channels of the Kv1 family. Selectivity profiling and molecular investigation of these toxins have contributed to the development of novel drug leads with therapeutic potential for the treatment of ion channel-related diseases or channelopathies. Here, we review specific peptide and small-molecule marine toxins modulating Kv1 channels and thus cover recent findings of bioactives found in the venoms of marine Gastropod (cone snails), Cnidarian (sea anemones), and small compounds from cyanobacteria. Furthermore, we discuss pivotal advancements at exploiting the interaction of κM-conotoxin RIIIJ and heteromeric Kv1.1/1.2 channels as prevalent neuronal Kv complex. RIIIJ's exquisite Kv1 subtype selectivity underpins a novel and facile functional classification of large-diameter dorsal root ganglion neurons. The vast potential of marine toxins warrants further collaborative efforts and high-throughput approaches aimed at the discovery and profiling of Kv1-targeted bioactives, which will greatly accelerate the development of a thorough molecular toolbox and much-needed therapeutics.

Ketamine inhibits synaptic transmission and nicotinic acetylcholine receptor-mediated responses in rat intracardiac ganglia in situ.

The intravenous anaesthetic ketamine, has been demonstrated to inhibit nicotinic acetylcholine receptor (nAChR)-mediated currents in dissociated rat intracardiac ganglion (ICG) neurons (Weber et al., 2005). This effect would be predicted to depress synaptic transmission in the ICG and would account for the inhibitory action of ketamine on vagal transmission to the heart (Inoue and König, 1988). This investigation was designed to examine the activity of ketamine on (i) postsynaptic responses to vagal nerve stimulation, (ii) the membrane potential, and (iii) membrane current responses evoked by exogenous application of ACh and nicotine in ICG neurons in situ. Intracellular recordings were made using sharp intracellular microelectrodes in a whole mount ICG preparation. Preganglionic nerve stimulation and recordings in current- and voltage-clamp modes were used to assess the action of ketamine on ganglionic transmission and nAChR-mediated responses. Ketamine attenuated the postsynaptic responses evoked by nerve stimulation. This reduction was significant at clinically relevant concentrations at high frequencies. The excitatory membrane potential and current responses to focal application of ACh and nicotine were inhibited in a concentration-dependent manner by ketamine. In contrast, ketamine had no effect on either the directly-evoked action potential or excitatory responses evoked by focal application of γ-aminobutyric acid (GABA). Taken together, ketamine inhibits synaptic transmission and nicotine- and ACh-evoked currents in adult rat ICG. Ketamine inhibition of synaptic transmission and nAChR-mediated responses in the ICG contributes significantly to its attenuation of the bradycardia observed in response to vagal stimulation in the mammalian heart.

Spider Venom Peptide Pn3a Inhibition of Primary Afferent High Voltage-Activated Calcium Channels.

Despite potently inhibiting the nociceptive voltage-gated sodium (Nav) channel, Nav1.7, µ-theraphotoxin Pn3a is antinociceptive only upon co-administration with sub-therapeutic opioid agonists, or by itself at doses >3,000-fold greater than its Nav1.7 IC 50 by a yet undefined mechanism. Nav channels are structurally related to voltage-gated calcium (Cav) channels, Cav1 and Cav2. These channels mediate the high voltage-activated (HVA) calcium currents (I Ca ) that orchestrate synaptic transmission in nociceptive dorsal root ganglion (DRG) neurons and are fine-tuned by opioid receptor (OR) activity. Using whole-cell patch clamp recording, we found that Pn3a (10 µM) inhibits ∼55% of rat DRG neuron HVA-I Ca and 60-80% of Cav1.2, Cav1.3, Cav2.1, and Cav2.2 mediated currents in HEK293 cells, with no inhibition of Cav2.3. As a major DRG I Ca component, Cav2.2 inhibition by Pn3a (IC 50 = 3.71 ± 0.21 µM) arises from an 18 mV hyperpolarizing shift in the voltage dependence of inactivation. We observed that co-application of Pn3a and µ-OR agonist DAMGO results in enhanced HVA-I Ca inhibition in DRG neurons whereas co-application of Pn3a with the OR antagonist naloxone does not, underscoring HVA channels as shared targets of Pn3a and opioids. We provide evidence that Pn3a inhibits native and recombinant HVA Cavs at previously reportedly antinociceptive concentrations in animal pain models. We show additive modulation of DRG HVA-I Ca by sequential application of low Pn3a doses and sub-therapeutic opioids ligands. We propose Pn3a's antinociceptive effects result, at least in part, from direct inhibition of HVA-I Ca at high Pn3a doses, or through additive inhibition by low Pn3a and mild OR activation.

Structural basis of the potency and selectivity of Urotoxin, a potent Kv1 blocker from scorpion venom.

Urotoxin (α-KTx 6), a peptide from venom of the Australian scorpion Urodacus yaschenkoi, is the most potent inhibitor of Kv1.2 described to date (IC50 = 160 pM). The native peptide also inhibits Kv1.1, Kv1.3 and KCa3.1 with nanomolar affinity but its low abundance in venom precluded further studies of its actions. Here we produced recombinant Urotoxin (rUro) and characterized the molecular determinants of Kv1 channel inhibition. The 3D structure of rUro determined using NMR spectroscopy revealed a canonical cysteine-stabilised α/ß (CSα/ß) fold. Functional assessment of rUro using patch-clamp electrophysiology revealed the importance of C-terminal amidation for potency against Kv1.1-1.3 and Kv1.5. Neutralization of the putative pore-blocking K25 residue in rUro by mutation to Ala resulted in a major decrease in rUro potency against all Kv channels tested, without perturbing the toxin's structure. Reciprocal mutations in the pore of Uro-sensitive Kv1.2 and Uro-resistant Kv1.5 channels revealed a direct interaction between Urotoxin and the Kv channel pore. Our experimental work supports postulating a mechanism of action in which occlusion of the permeation pathway by the K25 residue in Urotoxin is the basis of its Kv1 inhibitory activity. Docking analysis was consistent with occlusion of the pore by K25 and the requirement of a small, non-charged amino acid in the Kv1 channel vestibule to facilitate toxin-channel interactions. Finally, computational studies revealed key interactions between the amidated C-terminus of Urotoxin and a conserved Asp residue in the turret of Kv1 channels, offering a potential rationale for potency differences between native and recombinant Urotoxin.

Extremely Potent Block of Bacterial Voltage-Gated Sodium Channels by µ-Conotoxin PIIIA.

µ-Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide µ-conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10-12 to 10-5 M), we estimated an IC50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

Analgesic transient receptor potential vanilloid-1-active compounds inhibit native and recombinant T-type calcium channels.

BACKGROUND AND PURPOSE: T-type calcium (Cav 3) and transient receptor potential vanilloid-1 (TRPV1) channels play central roles in the control of excitability in the peripheral nervous system and are regarded as potential therapeutic pain targets. Modulators that either activate or inhibit TRPV1-mediated currents display analgesic properties in various pain models despite opposing effects on their connate target, TRPV1. We explored the effects of TRPV1-active compounds on Cav 3-mediated currents. EXPERIMENTAL APPROACH: Whole-cell patch clamp recordings were used to examine the effects of TRPV1-active compounds on rat dorsal root ganglion low voltage-activated calcium currents and recombinant Cav 3 isoforms in expression systems. KEY RESULTS: The classical TRPV1 agonist capsaicin as well as TRPV1 antagonists A-889425, BCTC, and capsazepine directly inhibited Cav 3 channels. These compounds altered the voltage-dependence of activation and inactivation of Cav 3 channels and delayed their recovery from inactivation, leading to a concomitant decrease in T-type current availability. The TRPV1 antagonist capsazepine potently inhibited Cav 3.1 and 3.2 channels (KD  < 120 nM), as demonstrated by its slow off rate. In contrast, neither the TRPV1 agonists, Palvanil and resiniferatoxin, nor the TRPV1 antagonist AMG9810 modulated Cav 3-mediated currents. CONCLUSIONS AND IMPLICATIONS: Analgesic TRPV1-active compounds inhibit Cav 3 currents in native and heterologous systems. Hence, their analgesic effects may not be exclusively attributed to their actions on TRPV1, which has important implications in the current understanding of nociceptive pathways. Importantly, our results highlight the need for attention in the experimental design used to address the analgesic properties of Cav 3 channel inhibitors.

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

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.

NMR Structure of µ-Conotoxin GIIIC: Leucine 18 Induces Local Repacking of the N-Terminus Resulting in Reduced NaV Channel Potency.

µ-Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of µ-conotoxin GIIIC was determined using 2D NMR spectroscopy and simulated annealing calculations. Despite high sequence similarity, GIIIC adopts a three-dimensional structure that differs from the previously observed conformation of µ-conotoxins GIIIA and GIIIB due to the presence of a bulky, non-polar leucine residue at position 18. The side chain of L18 is oriented towards the core of the molecule and consequently the N-terminus is re-modeled and located closer to L18. The functional characterization of GIIIC defines it as a canonical µ-conotoxin that displays substantial selectivity towards skeletal muscle sodium channels (NaV), albeit with ~2.5-fold lower potency than GIIIA. GIIIC exhibited a lower potency of inhibition of NaV1.4 channels, but the same NaV selectivity profile when compared to GIIIA. These observations suggest that single amino acid differences that significantly affect the structure of the peptide do in fact alter its functional properties. Our work highlights the importance of structural factors, beyond the disulfide pattern and electrostatic interactions, in the understanding of the functional properties of bioactive peptides. The latter thus needs to be considered when designing analogues for further applications.