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Brevetoxins (PbTx) and brevenal are marine ladder-frame polyethers. PbTx binds to and activates voltage-gated sodium (Nav) channels in native tissues, whereas brevenal antagonizes these actions. However, the effects of PbTx and brevenal on recombinant Nav channel function have not been systematically analyzed. In this study, the PbTx-3 and brevenal modulation of tissue-representative Nav channel subtypes Nav1.2, Nav1.4, Nav1.5, and Nav1.7 were examined using automated patch-clamp. While PbTx-3 and brevenal elicit concentration-dependent and subtype-specific modulatory effects, PbTx-3 is >1000-fold more potent than brevenal. Consistent with effects observed in native tissues, Nav1.2 and Nav1.4 channels were PbTx-3- and brevenal-sensitive, whereas Nav1.5 and Nav1.7 appeared resistant. Interestingly, the incorporation of brevenal in the intracellular solution caused Nav channels to become less sensitive to PbTx-3 actions. Furthermore, we generated a computational model of PbTx-2 bound to the lipid-exposed side of the interface between domains I and IV of Nav1.2. Our results are consistent with competitive antagonism between brevetoxins and brevenal, setting a basis for future mutational analyses of Nav channels’ interaction with brevetoxins and brevenal. Our findings provide valuable insights into the functional modulation of Nav channels by brevetoxins and brevenal, which may have implications for the development of new Nav channel modulators with potential therapeutic applications.

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.

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.

Familial hemiplegic migraine (FHM) is a severe neurogenetic disorder for which three causal genes

Significance Pain development and discomfort are universal features of spider envenomation, yet severe pain arising from bites by Old World spiders is poorly understood. Molecular analyses of the venom of the King Baboon spider revealed abundant expression of the inhibitory cystine knot peptide Pm1a. Synthetic Pm1a induces pain in mice while simultaneously enhancing proexcitatory sodium currents and decreasing inhibitory potassium currents. These concomitant effects promote hyperexcitability in pain-sensing neurons that can be reversed by pharmacological inhibition of voltage-gated sodium channels. The coordinated modulation of excitatory and inhibitory ion channels involved in pain propagation may represent an economical and effective defense strategy in pain-inducing defensive venoms.

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. Notably, CaV3.3’s contribution to normal and pathophysiological function has remained largely unexplored. We have identified 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 into all T-type channel isoforms highlighting it as subtype-specific pharmacophore with therapeutic potential. 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.

Low voltage-activated calcium currents are mediated by T-type calcium channels Ca

Activation of GIRK channels via G protein-coupled GABAThe effects of analgesic α-conotoxins, Vc1.1, RgIA, and PeIA, were investigated on inwardly-rectifying KAnalgesic α-conotoxins Vc1.1, RgIA, and PeIA potentiate inwardly-rectifying KThis is the first report of GIRK channel potentiation via allosteric α-conotoxin Vc1.1-GABA

Batrachotoxin (BTX) causes paralysis and death by activating “pseudosymmetric” eukaryotic sodium (Nav) channels. Finol-Urdaneta et al. investigate its action on the prokaryotic homotetrameric homologues NaChBac and NavSp1, revealing use-dependent facilitation of activation, and inhibition of deactivation, caused by BTX binding to the symmetric pore., 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.

Venomous molluscs (Superfamily Conoidea) comprise a substantial fraction of tropical marine biodiversity (>15,000 species). Prior characterization of cone snail venoms established that bioactive venom components used to capture prey, defend against predators and for competitive interactions were relatively small, structured peptides (10–35 amino acids), most with multiple disulfide crosslinks. These venom components (“conotoxins, conopeptides”) have been widely studied in many laboratories, leading to pharmaceutical agents and probes. In this review, we describe how it has recently become clear that to varying degrees, cone snail venoms also contain bioactive non-peptidic small molecule components. Since the initial discovery of genuanine as the first bioactive venom small molecule with an unprecedented structure, a broad set of cone snail venoms have been examined for non-peptidic bioactive components. In particular, a basal clade of cone snails (Stephanoconus) that prey on polychaetes produce genuanine and many other small molecules in their venoms, suggesting that this lineage may be a rich source of non-peptidic cone snail venom natural products. In contrast to standing dogma in the field that peptide and proteins are predominantly used for prey capture in cone snails, these small molecules also contribute to prey capture and push the molecular diversity of cone snails beyond peptides. The compounds so far characterized are active on neurons and thus may potentially serve as leads for neuronal diseases. Thus, in analogy to the incredible pharmacopeia resulting from studying venom peptides, these small molecules may provide a new resource of pharmacological agents.

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., This study is the first to describe transplantation of dorsal root ganglia (DRG) sensory neural progenitors derived from human pluripotent stem cells (hPSC) into adult rodent DRG tissue. Donor cells differentiated to neurons and glia in vivo and expressed DRG sensory markers. These findings are highly significant in supporting stem cell‐based therapies for treating peripheral DRG neuropathies, including Friedreich ataxia. Figure created using BioRender.com

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.

Activation of G protein-coupled inwardly rectifying potassium (GIRK or Kir3) channels leads to membrane hyperpolarization and dampening of neuronal excitability. Here we show that the analgesic α-conotoxin Vc1.1 potentiates inwardly rectifying K+currents (IKir) mediated through native and recombinant GIRK1/2 channels by activation of the G protein-coupled GABABreceptor (GABABR) via aPertussistoxin (PTX)-sensitive G protein. Recombinant co-expression of human GIRK1/2 subunits and GABABR in HEK293T cells resulted in a Ba2+-sensitive IKirpotentiated by baclofen and Vc1.1 which was inhibited by PTX, intracellular GDP-β-S, or the GABABR-selective antagonist CGP 55845. In adult mouse DRG neurons, GABABR-dependent GIRK channel potentiation by Vc1.1 and baclofen hyperpolarizes the cell resting membrane potential with concomitant reduction of excitability consistent with Vc1.1 and baclofen analgesic effectsin vivo. This study provides new insight into Vc1.1 as an allosteric agonist for GABABR-mediated potentiation of GIRK channels and may aid in the development of novel non-opioid treatments for chronic pain.

The imperial cone uses small-molecule mimicry, imitating the pheromones of its worm prey in a hunting strategy., 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.

16 p.-7 fig.-1 tab., Background:Drug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment., Methods:Vincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey’s post hoc test or a two-tailed paired t test was used to determine statistical significance., Results: CEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated sodium channel function than those treated with Vinca alkaloids, strongly supportive of continued action potential firing., Conclusions:The N-alkylisatins are able to retain cytotoxicity towards ALL cell lines with functionally distinct drug resistance mechanisms and show potential for reduced neurotoxicity. As such they pose as promising candidates for future implementation into anticancer regimes for ALL. Further in vivo studies are therefore warranted., European Union H2020-MSCA-ITN-ETN/0582 ITN TubInTrain to J.F. Diaz is gratefully acknowledged.

Background and purpose P2X4 receptors are emerging therapeutic targets for treating chronic pain and cardiovascular disease. Dogs are well-recognised natural models of human disease, but information regarding P2X4 receptors in dogs is lacking. To aid the development and validation of P2X4 receptor ligands, we have characterised and compared canine and human P2X4 receptors. Experimental approach Genomic DNA was extracted from whole blood samples from 101 randomly selected dogs and sequenced across the P2RX4 gene to identify potential missense variants. Recombinant canine and human P2X4 receptors tagged with Emerald GFP were expressed in 1321N1 and HEK293 cells and analysed by immunoblotting and confocal microscopy. In these cells, receptor pharmacology was characterised using nucleotide-induced Fura-2 AM measurements of intracellular Ca2+ and known P2X4 receptor antagonists. P2X4 receptor-mediated inward currents in HEK293 cells were assessed by automated patch clamp. Key results No P2RX4 missense variants were identified in any canine samples. Canine and human P2X4 receptors were localised primarily to lysosomal compartments. ATP was the primary agonist of canine P2X4 receptors with near identical efficacy and potency at human receptors. 2'(3')-O-(4-benzoylbenzoyl)-ATP, but not ADP, was a partial agonist with reduced potency for canine P2X4 receptors compared to the human orthologues. Five antagonists inhibited canine P2X4 receptors, with 1-(2,6-dibromo-4-isopropyl-phenyl)-3-(3-pyridyl)urea displaying reduced sensitivity and potency at canine P2X4 receptors. Conclusion and implications P2X4 receptors are highly conserved across dog pedigrees and display expression patterns and pharmacological profiles similar to human receptors, supporting validation and use of therapeutic agents for P2X4 receptor-related disease onset and management in dogs and humans.

Somatosensory neurons have historically been classified by a variety of approaches, including structural, anatomical, and genetic markers; electrophysiological properties; pharmacological sensitivities; and more recently, transcriptional profile differentiation. These methodologies, used separately, have yielded inconsistent classification schemes. Here, we describe phenotypic differences in response to pharmacological agents as measured by changes in cytosolic calcium concentration for the rapid classification of neurons in vitro; further analysis with genetic markers, whole-cell recordings, and single-cell transcriptomics validated these findings in a functional context. Using this general approach, which we refer to as tripartite constellation analysis (TCA), we focused on large-diameter dorsal-root ganglion (L-DRG) neurons with myelinated axons. Divergent responses to the K-channel antagonist, κM-conopeptide RIIIJ (RIIIJ), reliably identified six discrete functional cell classes. In two neuronal subclasses (L1 and L2), block with RIIIJ led to an increase in [Ca](i). Simultaneous electrophysiology and calcium imaging showed that the RIIIJ-elicited increase in [Ca](i) corresponded to different patterns of action potentials (APs), a train of APs in L1 neurons, and sporadic firing in L2 neurons. Genetically labeled mice established that L1 neurons are proprioceptors. The single-cell transcriptomes of L1 and L2 neurons showed that L2 neurons are Aδ–low-threshold mechanoreceptors. RIIIJ effects were replicated by application of the K(v)1.1 selective antagonist, Dendrotoxin-K, in several L-DRG subclasses (L1, L2, L3, and L5), suggesting the presence of functional K(v)1.1/K(v)1.2 heteromeric channels. Using this approach on other neuronal subclasses should ultimately accelerate the comprehensive classification and characterization of individual somatosensory neuronal subclasses within a mixed population.

Additional file 1: Figure S1. Growth properties of CEM, CEM-VCR R and CEM/2ME2-28.8R cell lines. Figure S2. SH-SY5Y cell viability after treatment with HMB-isatin, VCR and VBL. Figure S3. Dose dependent effect of HMB-isatin and VCR on RA-differentiated SH-SY5Y neurite morphology. Table S1. Electrophysiological parameters of endogenous sodium currents. Table S2. Current density estimates from 3 independent experiments.

Hille’s (1971) seminal study of organic cation selectivity of eukaryotic voltage-gated sodium channels showed a sharp size cut-off for ion permeation, such that no ion possessing a methyl group was permeant. Using the prokaryotic channel, NaChBac, we found some similarity and two peculiar differences in the selectivity profiles for small polyatomic cations. First, we identified a diverse group of minimally permeant cations for wildtype NaChBac, ranging in sizes from ammonium to guanidinium and tetramethylammonium; and second, for both ammonium and hydrazinium, the charge-conserving selectivity filter mutation (E191D) yielded substantial increases in relative permeability (PX/PNa). The relative permeabilities varied inversely with relative Kd calculated from 1D Potential of Mean Force profiles (PMFs) for the single cations traversing the channel. Several of the cations bound more strongly than Na+, and hence appear to act as blockers, as well as charge carriers. Consistent with experimental observations, the E191D mutation had little impact on Na+ binding to the selectivity filter, but disrupted the binding of ammonium and hydrazinium, consequently facilitating ion permeation across the NaChBac-like filter. We concluded that for prokaryotic sodium channels, a fine balance among filter size, binding affinity, occupancy, and flexibility seems to contribute to observed functional differences.

&micro, Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide &micro, 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&minus, 12 to 10&minus, 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.

BACKGROUND AND PURPOSE: T‐type calcium (Ca(v)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 Ca(v)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 Ca(v)3 isoforms in expression systems. KEY RESULTS: The classical TRPV1 agonist capsaicin as well as TRPV1 antagonists A‐889425, BCTC, and capsazepine directly inhibited Ca(v)3 channels. These compounds altered the voltage‐dependence of activation and inactivation of Ca(v)3 channels and delayed their recovery from inactivation, leading to a concomitant decrease in T‐type current availability. The TRPV1 antagonist capsazepine potently inhibited Ca(v)3.1 and 3.2 channels (K (D)

The vast complexity of native heteromeric K+ channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K+ channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this “zone of ignorance” in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets “asymmetric” Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit with 100-fold higher apparent affinity compared with homomeric Kv1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional Kv1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related Kv1 family to Kv1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K+ channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric Kv1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric Kv channel complexes that operate in native tissues.

The vast complexity of native heteromeric K(+) channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K(+) channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this “zone of ignorance” in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets “asymmetric” K(v) channels composed of three K(v)1.2 subunits and one K(v)1.1 or K(v)1.6 subunit with 100-fold higher apparent affinity compared with homomeric K(v)1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional K(v)1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative K(v)1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related K(v)1 family to K(v)1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K(+) channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric K(v)1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric K(v) channel complexes that operate in native tissues.

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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

&mu, Conotoxins are potent and highly specific peptide blockers of voltage-gated sodium channels. In this study, the solution structure of &mu, 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 &mu, 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 &mu, 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.

Conotoxins (conopeptides) are a diverse group of peptides isolated from the venom of marine cone snails. Conus peptides modulate pain by interacting with voltage-gated ion channels and G protein-coupled receptors (GPCRs). Opiate drugs targeting GPCRs have long been used, nonetheless, many undesirable side effects associated with opiates have been observed including addiction. Consequently, alternative avenues to pain management are a largely unmet need. It has been shown that various voltage-gated calcium channels (VGCCs) respond to GPCR modulation. Thus, regulation of VGCCs by GPCRs has become a valuable alternative in the management of pain. In this review, we focus on analgesic conotoxins that exert their effects via GPCR-mediated inhibition of ion channels involved in nociception and pain transmission. Specifically, α-conotoxin Vc1.1 activation of GABAB receptors and inhibition of voltage-gated calcium channels as a novel mechanism for reducing the excitability of dorsal root ganglion neurons is described. Vc1.1 and other α-conotoxins have been shown to be analgesic in different animal models of chronic pain. This review will outline the functional effects of conopeptide modulation of GPCRs and how their signalling is translated to downstream components of the pain pathways. Where available we present the proposed signalling mechanisms that couples metabotropic receptor activation to their downstream effectors to produce analgesia. This article is part of the Special Issue entitled 'Venom-derived Peptides as Pharmacological Tools.'

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

The molecular strategy for alkali cation selectivity by a bacterial sodium channel resembles those of eukaryotic calcium and potassium channels, rather than those of eukaryotic sodium channels., Striking structural differences between voltage-gated sodium (Nav) channels from prokaryotes (homotetramers) and eukaryotes (asymmetric, four-domain proteins) suggest the likelihood of different molecular mechanisms for common functions. For these two channel families, our data show similar selectivity sequences among alkali cations (relative permeability, Pion/PNa) and asymmetric, bi-ionic reversal potentials when the Na/K gradient is reversed. We performed coordinated experimental and computational studies, respectively, on the prokaryotic Nav channels NaChBac and NavAb. NaChBac shows an “anomalous,” nonmonotonic mole-fraction dependence in the presence of certain sodium–potassium mixtures; to our knowledge, no comparable observation has been reported for eukaryotic Nav channels. NaChBac’s preferential selectivity for sodium is reduced either by partial titration of its highly charged selectivity filter, when extracellular pH is lowered from 7.4 to 5.8, or by perturbation—likely steric—associated with a nominally electro-neutral substitution in the selectivity filter (E191D). Although no single molecular feature or energetic parameter appears to dominate, our atomistic simulations, based on the published NavAb crystal structure, revealed factors that may contribute to the normally observed selectivity for Na over K. These include: (a) a thermodynamic penalty to exchange one K+ for one Na+ in the wild-type (WT) channel, increasing the relative likelihood of Na+ occupying the binding site; (b) a small tendency toward weaker ion binding to the selectivity filter in Na–K mixtures, consistent with the higher conductance observed with both sodium and potassium present; and (c) integrated 1-D potentials of mean force for sodium or potassium movement that show less separation for the less selective E/D mutant than for WT. Overall, tight binding of a single favored ion to the selectivity filter, together with crucial inter-ion interactions within the pore, suggests that prokaryotic Nav channels use a selective strategy more akin to those of eukaryotic calcium and potassium channels than that of eukaryotic Nav channels.

Voltage-gated sodium channels (Nav) play an essential role in the generation and propagation of action potentials. Mammalian Nav alpha subunits are large single polypeptide chains organized in four different domains (DI-DIV). Each domain is composed of 6 transmembrane segments (S1-S6) in which S1-S4 constitute the voltage sensing domain (VSD) and S5 and S6 form the pore. Although Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully elucidated. Recently, the crystal structures of several prokaryotic sodium channels have been solved, but they are homo-tetrameric proteins in contrast to the mammalian Navs and these studies only provide a single snapshot of the channels in one of many possible conformational states. Therefore, techniques that provide dynamic structural information are needed. In this work we have used Lanthanide-based resonance energy transfer (LRET) to obtain structural information and conformational dynamics of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4). We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which binds a lanthanide (Tb3+) ion with high affinity, inserted in several strategic places at the top of the S4 segment in each domain. Two new conotoxin analogs were synthesized to function as LRET acceptors: KIIIA-Bodipy and GIIIA-Bodipy. We calculated multiple distances from each one of the domains to the pore region where the labeled toxin binds in both resting and slow inactivated states in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs, which remained functionally active. The results give a geometrical map of the S4 positions of each domain in the mammalian Nav channels and provide evidence for voltage-dependent structural changes. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

Voltage-dependent potassium (Kv) channels provide the repolarizing power that shapes the action potential duration and helps control the firing frequency of neurons. The K(+) permeation through the channel pore is controlled by an intracellularly located bundle-crossing (BC) gate that communicates with the voltage-sensing domains (VSDs). During prolonged membrane depolarizations, most Kv channels display C-type inactivation that halts K(+) conduction through constriction of the K(+) selectivity filter. Besides triggering C-type inactivation, we show that in Shaker and Kv1.2 channels (expressed in Xenopus laevis oocytes), prolonged membrane depolarizations also slow down the kinetics of VSD deactivation and BC gate closure during the subsequent membrane repolarization. Measurements of deactivating gating currents (reporting VSD movement) and ionic currents (BC gate status) showed that the kinetics of both slowed down in two distinct phases with increasing duration of the depolarizing prepulse. The biphasic slowing in VSD deactivation and BC gate closure was strongly correlated in time and magnitude. Simultaneous recordings of ionic currents and fluorescence from a probe tracking VSD movement in Shaker directly demonstrated that both processes were synchronized. Whereas the first slowing originates from a stabilization imposed by BC gate opening, the subsequent slowing reflects the rearrangement of the VSD toward its relaxed state (relaxation). The VSD relaxation was observed in the Ciona intestinalis voltage-sensitive phosphatase and in its isolated VSD. Collectively, our results show that the VSD relaxation is not kinetically related to C-type inactivation and is an intrinsic property of the VSD. We propose VSD relaxation as a general mechanism for depolarization-induced slowing of BC gate closure that may enable Kv1.2 channels to modulate the firing frequency of neurons based on the depolarization history.

Conus snail (Conus) venoms are a valuable source of pharmacologically active compounds; some of the peptide toxin families from the snail venoms are known to interact with potassium channels. We report the purification, synthesis, and characterization of κM-conotoxin RIIIJ from the venom of a fish-hunting species, Conus radiatus. This conopeptide, like a previously characterized peptide in the same family, κM-RIIIK, inhibits the homotetrameric human Kv1.2 channels. When tested in Xenopus oocytes, κM-RIIIJ has an order of magnitude higher affinity (IC50 = 33 nm) to Kv1.2 than κM-RIIIK (IC50 = 352 nm). Chimeras of RIIIK and RIIIJ tested on the human Kv1.2 channels revealed that Lys-9 from κM-RIIIJ is a determinant of its higher potency against hKv1.2. However, when compared in a model of ischemia/reperfusion, κM-RIIIK (100 μg/kg of body weight), administered just before reperfusion, significantly reduces the infarct size in rat hearts in vivo without influencing hemodynamics, providing a potential compound for cardioprotective therapeutics. In contrast, κM-RIIIJ does not exert any detectable cardioprotective effect. κM-RIIIJ shows more potency for Kv1.2-Kv1.5 and Kv1.2-Kv1.6 heterodimers than κM-RIIIK, whereas the affinity of κM-RIIIK to Kv1.2-Kv1.7 heterodimeric channels is higher (IC50 = 680 nm) than that of κM-RIIIJ (IC50 = 3.15 μm). Thus, the cardioprotection seems to correlate to antagonism to heteromultimeric channels, involving the Kv1.2 α-subunit rather than antagonism to Kv1.2 homotetramers. Furthermore, κM-RIIIK and κM-RIIIJ provide a valuable set of probes for understanding the underlying mechanism of cardioprotection.

The actions of alcohols and anesthetics on ion channels are poorly understood. Controversy continues about whether bilayer restructuring is relevant to the modulatory effects of these surface active agents (SAAs). Some voltage-gated K channels (Kv), but not KvAP, have putative low affinity alcohol-binding sites, and because KvAP structures have been determined in bilayers, KvAP could offer insights into the contribution of bilayer mechanics to SAA actions. We monitored KvAP unitary conductance and macroscopic activation and inactivation kinetics in PE:PG/decane bilayers with and without exposure to classic SAAs (short-chain 1-alkanols, cholesterol, and selected anesthetics: halothane, isoflurane, chloroform). At levels that did not measurably alter membrane specific capacitance, alkanols caused functional changes in KvAP behavior including lowered unitary conductance, modified kinetics, and shifted voltage dependence for activation. A simple explanation is that the site of SAA action on KvAP is its entire lateral interface with the PE:PG/decane bilayer, with SAA-induced changes in surface tension and bilayer packing order combining to modulate the shape and stability of various conformations. The KvAP structural adjustment to diverse bilayer pressure profiles has implications for understanding desirable and undesirable actions of SAA-like drugs and, broadly, predicts that channel gating, conductance and pharmacology may differ when membrane packing order differs, as in raft versus nonraft domains.

Ion channels are membrane-spanning proteins that allow ions to permeate at high rates. The kinetic characteristics of the channels present in a cell determine the cell signaling profile and therefore cell function in many different physiological processes. We found that Kv1.7 channels from mouse heart muscle have two putative translation initiation start sites that generate two channel isoforms with different functional characteristics, mKv1.7L (489 aa) and a shorter mKv1.7S (457 aa). The electrophysiological analysis of mKv1.7L and mKv1.7S channels revealed that the two channel isoforms have different inactivation kinetics. The channel resulting from the longer protein (L) inactivates faster than the shorter channels (S). Our data supports the hypothesis that mKv1.7L channels inactivate predominantly due to an N-type related mechanism, which is impaired in the mKv1.7S form. Furthermore, only the longer version mKv1.7L is regulated by the cell redox state, whereas the shorter form mKv1.7S is not. Thus, expression starting at each translation initiation site results in significant functional divergence. Our data suggest that the redox modulation of mKv1.7L may occur through a site in the cytoplasmic N-terminal domain that seems to encompass a metal coordination motif resembling those found in many redox-sensitive proteins. The mRNA expression profile and redox modulation of mKv1.7 kinetics identify these channels as molecular entities of potential importance in cellular redox-stress states such as hypoxia.

Batrachotoxin (BTX) is an excitatory component in the skin secretions of dendrobatid frogs, which advertise their armament with the gaudy colors of their lethal skin. BTX is a hydrophobic alkaloid, which crosses cell membranes and activates voltage-gated sodium (Nav) channels of muscle, nerve and heart. BTX binding causes a shift of the activation curve toward more negative voltages, and suppresses fast and slow inactivation resulting in a massive increase in excitability of the tissue and ultimately causing paralysis. BTX is thought to act by binding in the cytoplasmic cavity of the ion-conducting pore, of pseudo-symmetric eukaryotic Nav channels. This binding site is near, or overlapping, receptor sites for amphiphylic inhibitor drugs including local anesthetics, anticonvulsants and anti-arrhythmics.The four homologous domains of eukaryotic Navs parallel the homo-tetrameric arrangement of BacNav channels, which have yielded high-resolution crystal structures. Despite the absence of a “ball-and chain” or “hinged-lid” fast inactivation mechanism, BacNavs display a “pore based” inactivation, that in some cases is quite rapid. Thus, they offer an opportunity to explore BTX actions on a molecular system of gating that is amenable to structural verification.The BacNav channels NaChBac and NavSp1 both show dramatic modification by BTX, with negative shifts of activation and inhibition of inactivation. These actions are enhanced by phenylalanine substitutions of pore domain (S6) residues, at positions which align with Phe residues that are important for BTX activation in eukaryotic Nav channels.

Hille's (1971) seminal study of the organic cation selectivity of voltage-gated sodium channels, showed a striking, sharp size cut-off for ion permeation. No ion species with an aliphatic moiety having at least the diameter of a methyl group was measurably permeant. Here, we show that, despite similar alkali cation permeability sequences for pro- and eu-karyotic channels (Finol-Urdaneta et al 2014 J Gen Physiol), relative permeabilities among organic ions (ammonium, hydrazinium, guanidinium and tetramethylammonium) are different. Using molecular dynamics simulations, we have asked to what extent 1d-pmfs based on the NavAb crystal structure might suggest a basis for NavBac selectivity among organic ions. Unlike eukaryotic channels, NavBac permeabilities do not show a size cut-off based on minimal cross-section, but are correlated with apparent binding energies in 1-dimensional potentials of mean force from simulations of single ions moving through the channel. In contrast, earlier analyses for eukaryotic Nav1s, suggest that differences among ion entry barriers are sufficient to account for relative permeabilities (Hille 1975 J Gen Physiol). We surmise that multi-ion occupancy and structural flexibility of Bac-Navs may also contribute to the observed and simulated functional differences. Meanwhile, we examined the consistency of our PMFs based on Zhu and Hummer's paper (Zhu & Hummer, 2011 J Comput Chem). The results indicate that it needs at least 10ns for particular windows to get accurate PMFs even for small molecules like ammonium.