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 alpha-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 A of alpha-Ctx PnIA (A10L D14K), a potent blocker of the alpha(7)-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. Alpha-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.

Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake ␣-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and threefinger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 M, ws-LYNX1 competed with 125 I-␣-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing ␣7 nAChRs to 1 M ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on ␣4␤2 and ␣3␤2 nAChRs. Increasing ws-LYNX1 concentration to 10 M caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.Endogenous "prototoxins" like LYNX1, LYNX2, SLURP-1, and SLURP-2, belonging to the Ly6 protein family, modulate nicotinic acetylcholine receptors (nAChRs) 3 (1-8). In the central nervous system, LYNX1 and LYNX2 regulate nAChR activity, preventing excessive excitation (3, 4). Gene deletion of LYNX1 or LYNX2 indicates that these modulators are critical for nAChR function in the brain. LYNX1 knock-out mice demonstrated enhanced performance in specific tests of learning ability and memory, whereas loss of LYNX2 results in increased anxiety-related behaviors (3, 4). Prototoxins have also been shown to affect cell growth in lung carcinoma (9), are involved in skin diseases (6, 7), and are related to prostate stem cell antigen (10).LYNX1 and LYNX2 are tethered to the membrane by a GPI anchor, which considerably complicates in vitro studies. LYNX1 is co-localized in the brain with ␣4␤2 and ␣7 nAChRs (1-3), and its modulatory activity on ␣4␤2 nAChR was shown in experiments on Xenopus oocytes (1, 3). It was reported that soluble form of LYNX1 (not containing a GPI anchor) potentiates ␣4␤2 receptor (1), but the concentration at which it acts remains unknown. A secreted water-soluble protein SLURP-1 expressed in palmoplantar skin acts on ␣7 nAChR and regulates keratinocyte proliferation (5).It was predicted that the prototoxins should have a spatial structure similar to that of snake venom ␣-neurotoxins, effective competitive inhibitors of nAChR (1). ␣-Neurotoxins are characterized by a three-finger fold formed by three adjacent loops arising from a small globular hydrophobic core, crosslinked by four conserved disulfide bonds (11-13). Nicotinic acetylcholine receptors are ta...

SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,—non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to ‘metabotropic’ signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.

Acetylcholine binding proteins (AChBPs) are unique spatial homologs of the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), and they reproduce some pharmacological properties of nAChRs. X-ray crystal structures of AСhBP in complex with α-conotoxins provide important insights into the interactions of α-conotoxins with distinct nAChR subtypes. Although considerable efforts have been made to understand why α-conotoxin GIC is strongly selective for α3β2 nAChR, this question has not yet been solved. Here we present the structure of α-conotoxin GIC in complex with Aplysia californica AChBP (Ac-AChBP) at a resolution of 2.1 Å. Based on this co-crystal structure complemented with molecular docking data, we suggest the key residues of GIC in determining its high affinity and selectivity for human α3β2 vs α3β4 nAChRs. These suggestions were checked by radioligand and electrophysiology experiments, which confirmed the functional role of detected contacts for GIC interactions with Ac-AChBP and α3β2 nAChR subtypes. While GIC elements responsible for its high affinity binding with Ac-AChBP and α3β2 nAChR were identified, our study also showed the limitations of computer modelling in extending the data from the X-ray structures of the AChBP complexes to all nAChR subtypes.

Background: Information was not available about prototoxin LYNX1 amino acid residues involved in binding to muscle and neuronal nicotinic receptors. Results: A series of water-soluble LYNX1 (ws-LYNX1) mutants was obtained and their interaction with nicotinic receptors was analyzed. Conclusion: There are both common and selective ws-LYNX1 residues recognizing distinct receptor types. Significance: For the first time, several functionally important residues in ws-LYNX1 are identified.

Background: Different snake venom three-finger toxins interact with various receptors, channels, and membranes. Results: Here, we demonstrate that GABA A receptors are inhibited by ␣-cobratoxin, other long chain ␣-neurotoxins, nonconventional toxin from Naja kaouthia, and ␣-conotoxin ImI. Conclusion: Some toxin blockers of nicotinic acetylcholine receptors also inhibit GABA A receptors. Significance: Three-finger toxins offer new scaffolds for the design of GABA A receptor effectors.

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of ␣-cobratoxin (a long-chain ␣-neurotoxin) and heterodimers formed by ␣-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 ␣-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 ␣-cobratoxin capacity to compete with ␣-bungarotoxin for binding to Torpedo and ␣7 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 ␣-cobratoxin dimer not only interacts with ␣7 nAChR but, in contrast to ␣-cobratoxin monomer, also blocks ␣3␤2 nAChR. In the latter activity it resembles -bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric ␣3␤2 nAChRs. Three-fingered toxins (TFTs)2 are the main components of the Elapidae snake venoms. TFTs consist of one polypeptide chain, their spatial structure being characterized by a hydrophobic core stabilized by four disulfide bridges, which confine three polypeptide loops (fingers). In cobra venom TFTs are represented mainly by ␣-neurotoxins and cytotoxins. So-called short-chain ␣-neurotoxins (60 -62 amino acid residues, 4 intramolecular disulfides) effectively block nicotinic acetylcholine receptors (nAChRs) of muscle-type, and long-chain ␣-neurotoxins (65-75 residues with an additional disulfide in central loop II) block neuronal homopentameric ␣7 nAChR as well (1, 2). These toxins are widely used as tools in the nAChR studies. Cytotoxins, structurally related to short-chain ␣-neurotoxins, manifest another activity; they non-selectively disrupt cell membranes and, thus, kill the cells (3).Another example of TFT interacting with nAChR are -bungarotoxins (-Bgts), minor components of the krait (Elapidae) venom (4, 5). All -Bgts consist of 66 amino acid residues and, similar to long-chain ␣-neurotoxins, contain five disulfide bonds. However, in contrast to ␣-neurotoxins, -Bgts practically do not block muscle-type nAChRs and only weakly act on ␣7 nAChR but with high efficiency interact with ␣3␤2 neuronal receptors (6). Despite the vast array of data on structure-activity relationship for ␣-neurotoxins and -Bgts, it is not yet clear what are the main structural features determining the specificity of a toxin to the particular receptor type. Recently, based on the x-ray structure of ␣-cobratoxin (␣-CT) with acetylcholinebinding protein, Bourne et al. (7) suggested that Lys-29 is the main residue determining the difference in specificity between ␣-neurotoxins and -bungarotoxins. However, A29K mut...

J. Neurochem. (2011) 116, 734–741. Abstract The Cys‐loop receptor family consists of nicotinic acetylcholine receptors (nAChR), glycine receptor, GABA‐A and some other receptors. They fulfill a plethora of functions, whereas their malfunctioning is associated with many diseases. All three domains – extracellular ligand‐binding, membrane and cytoplasmic – of these ligand‐gated ion channels play important roles in the receptor assembly, delivery to the membrane surface and functional activity. In this study, we discuss the role of these domains in the assembly of the Cys‐loop receptors, most comprehensively for the nAChRs. Heterologous expression and mutations of large N‐terminal fragments of various subunits demonstrated their leading role in the assembly, although getting an isolated well‐structured pentameric ligand‐binding domain is still a problem. The long intracellular loop between transmembrane fragments M3 and M4 participates in modulating the receptor function and in clusterization of the receptor complexes because of interactions with the intracellular proteins. The transmembrane fragments play different functional roles: M2 fragments outline the channel, M4 fragments, the most remote from the channel, modulate the channel function and contact the lipid environment. The interactions of aromatic residues in the M1 and M3 fragments with those of M4 are important for the correct assembly of glycine receptor α1 subunit and for the formation of functional pentaoligomer. The role of the three receptor domains is discussed in the light of electron microscopy structure of the Torpedo nAChR, X‐ray structures of agonist and antagonist complexes with the acetylcholine‐binding proteins and the X‐ray structures of the prokaryotic Cys‐loop receptors.