Bert Billen

Multisite Binding of a General Anesthetic to the Prokaryotic Pentameric Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC)

Background: Pentameric ligand-gated ion channels are modulated by general anesthetics. Results: The crystal structure of ELIC in complex with bromoform reveals anesthetic binding in the channel pore and in novel sites in the transmembrane and extracellular domain. Conclusion: General anesthetics allosterically modulate channel function via multisite binding. Significance: Our data reveal detailed insight into multisite recognition of general anesthetics at the structural level. Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABAA/C receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7–β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.

Animal Peptides Targeting Voltage-Activated Sodium Channels

Throughout millions of years of evolution, nature has supplied various organisms with a massive arsenal of venoms to defend themselves against predators or to hunt prey. These venoms are rich cocktails of diverse bioactive compounds with divergent functions, extremely effective in immobilizing or killing the recipient. In fact, venom peptides from various animals have been shown to specifically act on ion channels and other cellular receptors, and impair their normal functioning. Because of their key role in the initiation and propagation of electrical signals in excitable tissue, it is not very surprising that several isoforms of voltage-activated sodium channels are specifically targeted by many of these venom peptides. Therefore, these peptide toxins provide tremendous opportunities to design drugs with a higher efficacy and fewer undesirable side effects. This review puts venom peptides from spiders, scorpions and cone snails that target voltage-activated sodium channels in the spotlight, and addresses their potential therapeutical applications.

A bifunctional sea anemone peptide with Kunitz type protease and potassium channel inhibiting properties.

Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APEKTx1 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 63 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APEKTx1 is a new member of the type 2 sea anemone peptides targeting voltage-gated potassium channels (K(V)s), which also include the kalicludines from Anemonia sulcata. Similar to the kalicludines, APEKTx1 shares structural homology with both the basic pancreatic trypsin inhibitor (BPTI), a very potent Kunitz-type protease inhibitor, and dendrotoxins which are powerful blockers of voltage-gated potassium channels. In this study, APEKTx1 has been subjected to a screening on a wide range of 23 ion channels expressed in Xenopus laevis oocytes: 13 cloned voltage-gated potassium channels (K(V)1.1-K(V)1.6, K(V)1.1 triple mutant, K(V)2.1, K(V)3.1, K(V)4.2, K(V)4.3, hERG, the insect channel Shaker IR), 2 cloned hyperpolarization-activated cyclic nucleotide-sensitive cation non-selective channels (HCN1 and HCN2) and 8 cloned voltage-gated sodium channels (Na(V)1.2-Na(V)1.8 and the insect channel DmNa(V)1). Our data show that APEKTx1 selectively blocks K(V)1.1 channels in a very potent manner with an IC(50) value of 0.9nM. Furthermore, we compared the trypsin inhibitory activity of this toxin with BPTI. APEKTx1 inhibits trypsin with a dissociation constant of 124nM. In conclusion, this study demonstrates that APEKTx1 has the unique feature to combine the dual functionality of a potent and selective blocker of K(V)1.1 channels with that of a competitive inhibitor of trypsin.

Molecular actions of smoking cessation drugs at α4β2 nicotinic receptors defined in crystal structures of a homologous binding protein

Partial agonists of the α4β2 nicotinic acetylcholine receptor (nAChR), such as varenicline, are therapeutically used in smoking cessation treatment. These drugs derive their therapeutic effect from fundamental molecular actions, which are to desensitize α4β2 nAChRs and induce channel opening with higher affinity, but lower efficacy than a full agonist at equal receptor occupancy. Here, we report X-ray crystal structures of a unique acetylcholine binding protein (AChBP) from the annelid Capitella teleta, Ct-AChBP, in complex with varenicline or lobeline, which are both partial agonists. These structures highlight the architecture for molecular recognition of these ligands, indicating the contact residues that potentially mediate their molecular actions in α4β2 nAChRs. We then used structure-guided mutagenesis and electrophysiological recordings to pinpoint crucial interactions of varenicline with residues on the complementary face of the binding site in α4β2 nAChRs. We observe that residues in loops D and E are molecular determinants of desensitization and channel opening with limited efficacy by the partial agonist varenicline. Together, this study analyzes molecular recognition of smoking cessation drugs by nAChRs in a structural context.

Solution structure and alanine scan of a spider toxin that affects the activation of mammalian voltage-gated sodium channels

Magi 5, from the hexathelid spider Macrothele gigas, is a 29-residue polypeptide containing three disulfide bridges. It binds specifically to receptor site 4 on mammalian voltage-gated sodium channels and competes with scorpion β-toxins, such as Css IV from Centruroides suffusus suffusus. As a consequence, Magi 5 shifts the activation voltage of the mammalian rNav1.2a channel to more hyperpolarized voltages, whereas the insect channel, DmNav1, is not affected. To gain insight into toxin-channel interactions, Magi 5 and 23 analogues were synthesized. The three-dimensional structure of Magi 5 in aqueous solution was determined, and its voltage-gated sodium channel-binding surfaces were mapped onto this structure using data from electrophysiological measurements on a series of Ala-substituted analogues. The structure clearly resembles the inhibitor cystine knot structural motif, although the triple-stranded β-sheet typically found in that motif is partially distorted in Magi 5. The interactive surface of Magi 5 toward voltage-gated sodium channels resembles in some respects the Janus-faced atracotoxins, with functionally important charged residues on one face of the toxin and hydrophobic residues on the other. Magi 5 also resembles the scorpion β-toxin Css IV, which has distinct nonpolar and charged surfaces that are critical for channel binding and has a key Glu involved in voltage sensor trapping. These two distinct classes of toxin, with different amino acid sequences and different structures, may utilize similar groups of residues on their surface to achieve the common end of modifying voltage-gated sodium channel function.

Synthesis, Solution Structure, and Phylum Selectivity of a Spider δ-Toxin That Slows Inactivation of Specific Voltage-gated Sodium Channel Subtypes

Magi 4, now renamed δ-hexatoxin-Mg1a, is a 43-residue neurotoxic peptide from the venom of the hexathelid Japanese funnel-web spider (Macrothele gigas) with homology to δ-hexatoxins from Australian funnel-web spiders. It binds with high affinity to receptor site 3 on insect voltage-gated sodium (NaV) channels but, unlike δ-hexatoxins, does not compete for the related site 3 in rat brain despite being previously shown to be lethal by intracranial injection. To elucidate differences in NaV channel selectivity, we have undertaken the first characterization of a peptide toxin on a broad range of mammalian and insect NaV channel subtypes showing that δ-hexatoxin-Mg1a selectively slows channel inactivation of mammalian NaV1.1, NaV1.3, and NaV1.6 but more importantly shows higher affinity for insect NaV1 (para) channels. Consequently, δ-hexatoxin-Mg1a induces tonic repetitive firing of nerve impulses in insect neurons accompanied by plateau potentials. In addition, we have chemically synthesized and folded δ-hexatoxin-Mg1a, ascertained the bonding pattern of the four disulfides, and determined its three-dimensional solution structure using NMR spectroscopy. Despite modest sequence homology, we show that key residues important for the activity of scorpion α-toxins and δ-hexatoxins are distributed in a topologically similar manner in δ-hexatoxin-Mg1a. However, subtle differences in the toxin surfaces are important for the novel selectivity of δ-hexatoxin-Mg1a for certain mammalian and insect NaV channel subtypes. As such, δ-hexatoxin-Mg1a provides us with a specific tool with which to study channel structure and function and determinants for phylum- and tissue-specific activity.

Unique Bell-shaped Voltage-dependent Modulation of Na+ Channel Gating by Novel Insect-selective Toxins from the Spider Agelena orientalis

Spider venoms provide a highly valuable source of peptide toxins that act on a wide diversity of membrane-bound receptors and ion channels. In this work, we report isolation, biochemical analysis, and pharmacological characterization of a novel family of spider peptide toxins, designated β/δ-agatoxins. These toxins consist of 36–38 amino acid residues and originate from the venom of the agelenid funnel-web spider Agelena orientalis. The presented toxins show considerable amino acid sequence similarity to other known toxins such as μ-agatoxins, curtatoxins, and δ-palutoxins-IT from the related spiders Agelenopsis aperta, Hololena curta, and Paracoelotes luctuosus. β/δ-Agatoxins modulate the insect NaV channel (DmNaV1/tipE) in a unique manner, with both the activation and inactivation processes being affected. The voltage dependence of activation is shifted toward more hyperpolarized potentials (analogous to site 4 toxins) and a non-inactivating persistent Na+ current is induced (site 3-like action). Interestingly, both effects take place in a voltage-dependent manner, producing a bell-shaped curve between −80 and 0 mV, and they are absent in mammalian NaV channels. To the best of our knowledge, this is the first detailed report of peptide toxins with such a peculiar pharmacological behavior, clearly indicating that traditional classification of toxins according to their binding sites may not be as exclusive as previously assumed.

Two novel sodium channel inhibitors from Heriaeus melloteei spider venom differentially interacting with mammalian channel's isoforms.

Two new polypeptide toxins named Hm-1 and Hm-2 were isolated from the venom of the crab spider Heriaeus melloteei. These toxins consist of 37 and 40 amino acid residues, respectively, contain three intramolecular disulfide bonds, and presumably adopt the inhibitor cystine knot motif. Hm-1 is C-terminally amidated and shows a low degree of homology to spider toxins agelenin and micro-agatoxin-II, whereas Hm-2 has no relevantly related peptide sequences. Hm-1 and Hm-2 were found to act on mammalian voltage-gated Na(+) channels. Both toxins caused a strong decrease of Na(+) current peak amplitude, with IC(50) values of 336.4 and 154.8 nM, respectively, on Na(V)1.4. Hm-1 and Hm-2 did not shift the voltage-dependence of activation, nor did they change the kinetics of fast inactivation of the Na(+) currents. Interestingly, both toxins negatively shifted the steady-state inactivation process, which might have important functional consequences in vivo. However, this hyperpolarizing shift cannot by itself explain the observed inhibition of the Na(+) current, indicating that the two presented toxins could provide important structural information about the interaction of polypeptide inhibitors with voltage-gated Na(+) channels.

Phyla- and Subtype-Selectivity of CgNa, a Na+ Channel Toxin from the Venom of the Giant Caribbean Sea Anemone Condylactis Gigantea

Because of their prominent role in electro-excitability, voltage-gated sodium (NaV) channels have become the foremost important target of animal toxins. These toxins have developed the ability to discriminate between closely related NaV subtypes, making them powerful tools to study NaV channel function and structure. CgNa is a 47-amino acid residue type I toxin isolated from the venom of the Giant Caribbean Sea Anemone Condylactis gigantea. Previous studies showed that this toxin slows the fast inactivation of tetrodotoxin-sensitive NaV currents in rat dorsal root ganglion neurons. To illuminate the underlying NaV subtype-selectivity pattern, we have assayed the effects of CgNa on a broad range of mammalian isoforms (NaV1.2–NaV1.8) expressed in Xenopus oocytes. This study demonstrates that CgNa selectively slows the fast inactivation of rNaV1.3/β1, mNaV1.6/β1 and, to a lesser extent, hNaV1.5/β1, while the other mammalian isoforms remain unaffected. Importantly, CgNa was also examined on the insect sodium channel DmNaV1/tipE, revealing a clear phyla-selectivity in the efficacious actions of the toxin. CgNa strongly inhibits the inactivation of the insect NaV channel, resulting in a dramatic increase in peak current amplitude and complete removal of fast and steady-state inactivation. Together with the previously determined solution structure, the subtype-selective effects revealed in this study make of CgNa an interesting pharmacological probe to investigate the functional role of specific NaV channel subtypes. Moreover, further structural studies could provide important information on the molecular mechanism of NaV channel inactivation.

Voltage-gated sodium channels are targets for toxins from the venom of the spider Heriaeus melloteei

Three novel peptides were isolated from the venom of the spider Heriaeus melloteei (Thomisidae) and characterized. The peptides named Hm-1, 2 and 3 blocked voltage-gated Na+ channels at concentrations in the order of 100 nM. Activity of the purified peptides was investigated in Na+ channel isoforms of mammals and insects. Hm-1 and 2 appeared to act as pore blockers, whereas Hm-3 modulated the channel activation process. The toxins described exhibit minor similarity with other known peptides and may therefore constitute new groups of Na+ channel ligands.