Victor I. Tsetlin

Crystal Structure of Nicotinic Acetylcholine Receptor Homolog Achbp in Complex with an Alpha-Conotoxin Pnia Variant

Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup α-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 Å of α-Ctx PnIA (A10L D14K), a potent blocker of the α7-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. α-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.

Snake and snail toxins acting on nicotinic acetylcholine receptors: fundamental aspects and medical applications

This review covers recent data on interactions of nicotinic acetylcholine receptors (AChR) with snake venom proteins (α‐ and κ‐neurotoxins, ‘weak’ toxins recently shown to act on AChRs), as well as with peptide α‐conotoxins from Conus snails. Mutations of AChRs and toxins, X‐ray/nuclear magnetic resonance structures of α‐neurotoxin bound to AChR fragments, and the X‐ray structure of the acetylcholine‐binding protein were used by several groups to build models for the α‐neurotoxin–AChR complexes. Application of snake toxins and α‐conotoxins for pharmacological distinction of muscle, neuronal and neuronal‐like AChR subtypes and for other medical purposes is briefly discussed.

A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors.

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT3R.

An intramembrane aromatic network determines pentameric assembly of Cys-loop receptors

Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that mediate fast synaptic transmission. Here functional pentameric assembly of truncated fragments comprising the ligand-binding N-terminal ectodomains and the first three transmembrane helices, M1–M3, of both the inhibitory glycine receptor (GlyR) α1 and the 5HT3A receptor subunits was found to be rescued by coexpressing the complementary fourth transmembrane helix, M4. Alanine scanning identified multiple aromatic residues in M1, M3 and M4 as key determinants of GlyR assembly. Homology modeling and molecular dynamics simulations revealed that these residues define an interhelical aromatic network, which we propose determines the geometry of M1–M4 tetrahelical packing such that nascent pLGIC subunits must adopt a closed fivefold symmetry. Because pLGIC ectodomains form random nonstoichiometric oligomers, proper pentameric assembly apparently depends on intersubunit interactions between extracellular domains and intrasubunit interactions between transmembrane segments.

Mitochondria Express α7 Nicotinic Acetylcholine Receptors to Regulate Ca2+ Accumulation and Cytochrome c Release: Study on Isolated Mitochondria

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate synaptic transmission in the muscle and autonomic ganglia and regulate transmitter release in the brain. The nAChRs composed of α7 subunits are also expressed in non-excitable cells to regulate cell survival and proliferation. Up to now, functional α7 nAChRs were found exclusively on the cell plasma membrane. Here we show that they are expressed in mitochondria and regulate early pro-apoptotic events like cytochrome c release. The binding of α7-specific antibody with mouse liver mitochondria was revealed by electron microscopy. Outer membranes of mitochondria from the wild-type and β2−/− but not α7−/− mice bound α7 nAChR-specific antibody and toxins: FITC-labeled α-cobratoxin or Alexa 555-labeled α-bungarotoxin. α7 nAChR agonists (1 µM acetylcholine, 10 µM choline or 30 nM PNU-282987) impaired intramitochondrial Ca2+ accumulation and significantly decreased cytochrome c release stimulated with either 90 µM CaCl2 or 0.5 mM H2O2. α7-specific antagonist methyllicaconitine (50 nM) did not affect Ca2+ accumulation in mitochondria but attenuated the effects of agonists on cytochrome c release. Inhibitor of voltage-dependent anion channel (VDAC) 4,4′-diisothio-cyano-2,2′-stilbene disulfonic acid (0.5 µM) decreased cytochrome c release stimulated with apoptogens similarly to α7 nAChR agonists, and VDAC was co-captured with the α7 nAChR from mitochondria outer membrane preparation in both direct and reverse sandwich ELISA. It is concluded that α7 nAChRs are expressed in mitochondria outer membrane to regulate the VDAC-mediated Ca2+ transport and mitochondrial permeability transition.

EPR and fluorescence study of interaction of Naja naja oxiana neurotoxin II and its derivatives with acetylcholine receptor protein from Torpedo marmorata.

Snake venom neurotoxins that specifically interact with the nicotinic acetylcholine receptor (AchR) of the postsynaptic membrane and thereby prevent the binding of acetylcholine and block neural transmission, have been successfully used to obtain highly purified preparations of the receptor, and to study its properties. Though much information on AchR and neurotoxins is available [l-3], the structural aspects of their interaction remain unclear. In particular, there are no direct data as to the sites of the neurotoxin molecule which bind to the AchR. We attacked this problem by using EPR and fluorescence spectroscopy for monitoring the binding of selectively spinand fluorescence-labeled derivatives of the short (61 residues) neurotoxin II of N@z najz oxiana with AchR from the electric organ of Torpedo marmorata. The respective reporter groups were attached to lysine e-amino groups, since upon acylation of the

Assembly of nicotinic and other Cys-loop receptors

J. Neurochem. (2011) 116, 734–741.

Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors.

At present the cryo-electron microscopy structure at 4A resolution is known for the Torpedo marmorata nicotinic acetylcholine receptor (nAChR), and high-resolution X-ray structures have been recently determined for bacterial ligand-gated ion channels which have the same type of spatial organization. Together all these structures provide the basis for better understanding functioning of muscle-type and neuronal nAChRs, as well as of other Cys-loop receptors: 5HT3-, glycine-, GABA-A and some other. Detailed information about the ligand-binding sites in nAChRs, necessary both for understanding the receptor functioning and for rational drug design, became available when the X-ray structures were solved for the acetylcholine-binding proteins (AChBP), excellent models for the ligand-binding domains of all Cys-loop receptors. Of special value in this respect are the X-ray structures of AChBP complexes with agonists and antagonists. Among the latter are the complexes with polypeptide and peptide antagonists, that is with protein neurotoxins from snake venoms and peptide neurotoxins (alpha-conotoxins) from poisonous marine snails of Conus genus. The role of a bridge between the AChBP and nAChRs is played by the X-ray structure of the ligand-binding domain of alpha1 subunit of nAChR in the complex with alpha-bungarotoxin. The purpose of this review is to show the role of well-known and new polypeptide and peptide neurotoxins, from the earlier days of nAChRs research until present time, in identification of different nAChR subtypes and mapping their binding sites.

Naturally Occurring Disulfide-bound Dimers of Three-fingered Toxins A PARADIGM FOR BIOLOGICAL ACTIVITY DIVERSIFICATION

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of α-cobratoxin (a long-chain α-neurotoxin) and heterodimers formed by α-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26–30 in polypeptide loop II of α-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the α-cobratoxin capacity to compete with α-bungarotoxin for binding to Torpedo and α7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that α-cobratoxin dimer not only interacts with α7 nAChR but, in contrast to α-cobratoxin monomer, also blocks α3β2 nAChR. In the latter activity it resembles κ-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric α3β2 nAChRs.

NMR spatial structure of α-conotoxin ImI reveals a common scaffold in snail and snake toxins recognizing neuronal nicotinic acetylcholine receptors1

A 600 MHz NMR study of α‐conotoxin ImI from Conus imperialis, targeting the α7 neuronal nicotinic acetylcholine receptor (nAChR), is presented. ImI backbone spatial structure is well defined basing on the NOEs, spin‐spin coupling constants, and amide protons hydrogen‐deuterium exchange data: rmsd of the backbone atom coordinates at the 2–12 region is 0.28 Å in the 20 best structures. The structure is described as a type I β‐turn (positions 2–5) followed bya distorted helix (positions 5–11). Similar structural psattern can be found in all neuronal‐specific α‐conotoxins. Highly mobile side chains of the Asp‐5, Arg‐7 and Trp‐10 residues form a single site for ImI binding to the α7 receptor. When depicted with opposite directions of the polypeptide chains, the ImI helix and the tip of the central loop of long chain snake neurotoxins demonstrate a common scaffold and similar positioning of the functional side chains, both of these structural elements appearing essential for binding to the neuronal nAChRs.