Cross-linking of sites involved with alcohol action between transmembrane segments 1 and 3 of the glycine receptor following activation

The glycine receptor is a member of the Cys-loop, ligand-gated ion channel family and is responsible for inhibition in the CNS. We examined the orientation of amino acids I229 in transmembrane 1 (TM1) and A288 in TM3, which are both critical for alcohol and volatile anesthetic action. We mutated these two amino acids to cysteines either singly or in double mutants and expressed the receptors in Xenopus laevis oocytes. We tested whether disulfide bonds could form between A288C in TM3 paired with M227C, Y228C, I229C, or S231C in TM1. Application of cross-linking (mercuric chloride) or oxidizing (iodine) agents had no significant effect on the glycine response of wild-type receptors or the single mutants. In contrast, the glycine response of the I229C/A288C double mutant was diminished after application of either mercuric chloride or iodine only in the presence of glycine, indicating that channel gating causes I229C and A288C to fluctuate to be within 6 Å apart and form a disulfide bond. Molecular modeling was used to thread the glycine receptor sequence onto a nicotinic acetylcholine receptor template, further demonstrating that I229 and A288 are near-neighbors that can cross-link and providing evidence that these residues contribute to a single binding cavity.     

Molecular dynamics simulations of ethanol binding to the transmembrane domain of the glycine receptor: implications for the channel potentiation mechanism

The glycine receptor (GlyR) is potentiated by ethanol and other anesthetics. The potentiation mechanism at the molecular level is unknown and remains elusive, but mutagenic studies have shown that ethanol and other volatile anesthetics bind to a pocket between TM1, TM2, and TM3. The present study extends previous studies (Cheng et al., Proteins 2007;68:581-593) wherein we conducted homology modeling and molecular dynamics (MD) simulations to construct models of the homopentameric alpha1 subunits of the GlyR transmembrane domain in open and closed states. To understand the potentiation of GlyR by ethanol we compare the binding of ethanol molecules to the channel in these different states. We observe that ethanol stably resides inside solvent-accessible cavities found in the open state of GlyR that are formed by I229 (of TM1) in one subunit and S267 and A288 (of TM2 and TM3, respectively) in the adjacent subunit. The volume of these putative binding pockets is state-dependent. Selective binding to the open states of receptors has been proposed to explain the potentiating actions of this class of anesthetics. In accordance with this model, our MD simulations suggest that the potentiation of ethanol on GlyR may be effected through preferential binding of ethanol molecules to an inter-subunit binding pocket in the open state.     

The matrix metalloproteinase stromelysin-3 cleaves laminin receptor at two distinct sites between the transmembrane domain and laminin binding sequence within the extracellular domain

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However,like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites,distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demonstrated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior observed during development and pathogenesis.     

Homology modeling and molecular dynamics simulations of the alpha1 glycine receptor reveals different states of the channel

Homology modeling is used to build initial models of the transmembrane domain of the human alpha1 glycine receptor (GlyR) based on the most recently published refined structure of nAChR (PDB ID: 2BG9). Six preliminary GlyR models are constructed using two different approaches. In one approach, five different homopentamers are built by symmetric assembly of alpha1 GlyR subunits using only one of the five unique chains of nAChR as a template. In a second approach, each nAChR subunit serves as a template for an alpha1 GlyR subunit. All six initial GlyR constructs are then embedded into a hydrated POPC lipid bilayer and subjected to molecular dynamics simulation for at least six nanoseconds. Each model is stable throughout the simulation, and the final models fall into three distinct categories. Homopentameric GlyR bundles using a single alpha nAChR subunit as a template appear to be in an open conformation. Under an applied external potential, permeation of Cl(-) ions is observed within several ns in a channel built on an alpha chain. Model channels built on non-alpha chains have a constriction either near the intracellular mouth or more centrally located in the pore domain, both of which may be narrow enough to close the channel and whose locations correspond to putative gates observed in nicotinicoid receptors. The differences between these three general models suggest that channel closure may be effected by either rotation or tangential tilting of TM2.     

Homology modeling and molecular dynamics simulations of the glycine receptor ligand binding domain

We present a homology based model of the ligand binding domain (LBD) of the homopentameric alpha1 glycine receptor (GlyR). The model is based on multiple sequence alignment with other members of the nicotinicoid ligand gated ion channel superfamily and two homologous acetylcholine binding proteins (AChBP) from the freshwater (Lymnaea stagnalis) and saltwater (Aplysia californica) snails with known high resolution structure. Using two template proteins with known structure to model three dimensional structure of a target protein is especially advantageous for sequences with low homology as in the case presented in this paper. The final model was cross-validated by critical evaluation of experimental and published mutagenesis, functional and other biochemical studies. In addition, a complex structure with strychnine antagonist in the putative binding site is proposed based on docking simulation using Autodock program. Molecular dynamics (MD) simulations with simulated annealing protocol are reported on the proposed LBD of GlyR, which is stable in 5 ns simulation in water, as well as for a deformed LBD structure modeled on the corresponding domain determined in low-resolution cryomicroscopy structure of the alpha subunit of the full-length acetylcholine receptor (AChR). Our simulations demonstrate that the beta-sandwich central core of the protein monomer is fairly rigid in the simulations and resistant to deformations in water.     

Targets for ethanol action and antagonism in loop 2 of the extracellular domain of glycine receptors

The present studies used increased atmospheric pressure in place of a traditional pharmacological antagonist to probe the molecular sites and mechanisms of ethanol action in glycine receptors (GlyRs). Based on previous studies, we tested the hypothesis that physical–chemical properties at position 52 in extracellular domain Loop 2 of α1GlyRs, or the homologous α2GlyR position 59, determine sensitivity to ethanol and pressure antagonism of ethanol. Pressure antagonized ethanol in α1GlyRs that contain a non-polar residue at position 52, but did not antagonize ethanol in receptors with a polar residue at this position. Ethanol sensitivity in receptors with polar substitutions at position 52 was significantly lower than GlyRs with non-polar residues at this position. The α2T59A mutation switched sensitivity to ethanol and pressure antagonism in the WTα2GlyR, thereby making it α1-like. Collectively, these findings indicate that (i) polarity at position 52 plays a key role in determining sensitivity to ethanol and pressure antagonism of ethanol; (ii) the extracellular domain in α1- and α2GlyRs is a target for ethanol action and antagonism and (iii) there is structural-functional homology across subunits in Loop 2 of GlyRs with respect to their roles in determining sensitivity to ethanol and pressure antagonism of ethanol. These findings should help in the development of pharmacological agents that antagonize ethanol.     

Role of charged residues in coupling ligand binding and channel activation in the extracellular domain of the glycine receptor

The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor alpha1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.     

Characterization of ligand binding,DNA binding and phosphorylation of progesterone receptor by two novel progesterone receptor antagonist ligands

In order to gain a better understanding of the distinctive mechanisms of the various types of antiprogestins, we have characterized in vitro ligand binding, specific DNA binding and phosphorylation of progesterone receptor (PR) from T47D cells after treatment of cells with progestins (progesterone, R5020) and antiprogestins (RU486, ZK98299, Org 31806 and Org 31710). Treatment of the cells with R5020 or PR antagonists, with the exception of ZK98299, resulted in a quantitative upshift of PR-A and PR-B indicative of ligand/DNA-induced phosphorylation of PR. Treatment of cells with RU486, Org 31710 or Org 31806, but not R5020 or ZK98299 resulted in detectable PR-progesterone response element complexes (PR-PREc) as assessed by gel mobility shift assay. Although treatment of cells with ZK98299, a type I PR antagonist, did not induce phosphorylation, the antiprogestins, Org 31806 and Org 31710, in a manner identical to RU486, did. Our data suggest that Org 31806 and Org 31710 affect propertie s of PR from T47D cells that are similar to RU486. (Mol Cell Biochem 175: 205–212, 1997)     

Characterization of a glycine receptor domain that controls the binding and gating mechanisms of the β-amino acid agonist, taurine

The beta-amino acid, taurine, is a full agonist of the human glycine receptor alpha1 subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed in Xenopus oocytes. Several residues in the Ala101-Thr112 domain have previously been identified as determinants of beta-amino acid binding and gating mechanisms in Xenopus oocyte-expressed receptors. The present study used the substituted cysteine accessibility method to investigate the role of this domain in controlling taurine-specific binding and gating mechanisms of glycine receptors recombinantly expressed in mammalian cells. Asn102 and Glu103 are identified as taurine and glycine binding sites, whereas Ala101 is eliminated as a possible binding site. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist- and antagonist-bound conformations of beta-amino acids. The effects of mutations from Lys104-Thr112 indicate that the mechanism by which this domain controls beta-amino acid-specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only domain element in mammalian cell-expressed GlyRs which was demonstrated to discriminate between glycine and taurine.     

Delineation of ligand binding and receptor signaling activities of purified P2Y receptors reconstituted with heterotrimeric G proteins

P2Y receptors are G protein coupled receptors that respond to extracellular nucleotides to promote a multitude of signaling events. Our laboratory has purified several P2Y receptors with the goal of providing molecular insight into their: (1) ligand binding properties, (2) G protein signaling selectivities, and (3) regulation by RGS proteins and other signaling cohorts. The human P2Y₁ receptor and the human P2Y₁₂ receptor, both of which are intimately involved in ADP-mediated platelet aggregation, were purified to near homogeneity and studied in detail. After high-level expression from recombinant baculovirus infection of Sf9 insect cells, approximately 50% of the receptors were successfully extracted with digitonin. Purification of nearly homogeneous epitope-tagged P2Y receptor was achieved using metal-affinity chromatography followed by other traditional chromatographic steps. Yields of purified P2Y receptors range from 10 to 100 g/l of infected cells. Once purified, the receptors were reconstituted in model lipid vesicles along with their cognate G proteins to assess receptor function. Agonist-promoted increases in steady-state GTPase assays demonstrated the functional activity of the reconstituted purified receptor. We have utilized this reconstitution system to assess the action of various nucleotide agonists and antagonists, the relative G protein selectivity, and the influence of other proteins, such as phospholipase C, on P2Y receptor-promoted signaling. Furthermore, we have identified the RGS expression profile of platelets and have begun to assess the action of these RGS proteins in a reconstituted P2Y receptor/G protein platelet model.     

Distinct second extracellular loop structures of the brain cannabinoid CB(1) receptor: implication in ligand binding and receptor function

The G-protein-coupled receptor (GPCR) second extracellular loop (E2) is known to play an important role in receptor structure and function. The brain cannabinoid (CB(1)) receptor is unique in that it lacks the interloop E2 disulfide linkage to the transmembrane (TM) helical bundle, a characteristic of many GPCRs. Recent mutation studies of the CB(1) receptor, however, suggest the presence of an alternative intraloop disulfide bond between two E2 Cys residues. Considering the oxidation state of these Cys residues, we determine the molecular structures of the 17-residue E2 in the dithiol form (E2(dithiol)) and in the disulfide form (E2(disulfide)) of the CB(1) receptor in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer, using a combination of simulated annealing and molecular dynamics simulation approaches. We characterize the CB(1) receptor models with these two E2 forms, CB(1)(E2(dithiol)) and CB(1)(E2(disulfide)), by analyzing interaction energy, contact number, core crevice, and cross correlation. The results show that the distinct E2 structures interact differently with the TM helical bundle and uniquely modify the TM helical topology, suggesting that E2 of the CB(1) receptor plays a critical role in stabilizing receptor structure, regulating ligand binding, and ultimately modulating receptor activation. Further studies on the role of E2 of the CB(1) receptor are warranted, particularly comparisons of the ligand-bound form with the present ligand-free form.     

MTSEA prevents ligand binding to the human melanocortin-4 receptor by modification of cysteine 130 in transmembrane helix 3

We have investigated the effect of the sulfhydryl-reactive reagent, methyl thiosulfonate ethylammonium (MTSEA), on ligand binding to the human melanocortin-4 (MC4) receptor stably expressed in HEK-293 cells. MTSEA inhibited binding of the agonist, 125I-NDPalpha-MSH, and the antagonist, 125I-SHU9119, in a concentration-dependent manner. Pre-incubation of cells with either the agonist or antagonist protected from subsequent MTSEA inhibition of radioligand binding. Mutation of Cys130 in transmembrane helix 3 to alanine, whilst not affecting ligand binding, led to a complete loss of the inhibitory effect of MTSEA. Since other types of sulfhydryl-reactive reagents had no effect on ligand binding, we conclude that covalent modification of Cys130 by MTSEA disrupts ligand binding by neutralising a close-by negative charge, most likely on Asp126.     

Mapping of ligand binding sites of the cholecystokinin-B/gastrin receptor with lipo-gastrin peptides and molecular modeling

Double-tailed lipo-tetragastrin derivatives of increasing fatty acid chain length were used to identify the minimum size of the fatty acid moieties (≥C10) that restricts the access to the CCK-B/gastrin (CCK: cholecystokinin) receptor via a membrane-bound pathway. Then dimyristoyl-mercaptoglycerol/maleoyl-gastrin adducts of increasing peptide chain length were synthesized to define the minimal peptide size required for receptor binding affinities comparable to those of underivatized gastrin peptides despite anchorage of the lipid tails in the membrane bilayer. The experimental results indicated that most of the little-gastrin sequence, i.e., 2–17, is needed for optimal interaction of the molecule with the binding cleft of the receptor. From these data experimentally based restraints could be derived for docking of lipo-gastrin onto a CCK-B/gastrin receptor model applying molecular dynamics simulations and energy minimizations. In the receptor-bound state some of the secondary structure elements of gastrin as determined by nmr analysis of gastrin-peptides in low dielectric constant media are retained. The N-terminal gastrin portion interacts in a more or less extended conformation with the receptor surface, and upon a sharp kink at the Ala-Tyr dipeptide portion the C-terminal pentapeptide amide part inserts deeply into the helix bundle. Besides Arg-57 on top of helix 1 of the receptor, for which no potential interaction with the ligand could be detected, the other amino acid residues identified by mutagenesis studies as involved in gastrin recognition were found to interact with the C-terminal portion of gastrin. Even taking into account the strong limitations of such a model system, it represents an interesting tool for rationalizing the experimental results of the extensive structure-function studies performed previously on gastrin and to delineate more precisely the putative ligand binding site on the extracellular face of the receptor. © 1997 John Wiley & Sons, Inc. Biopoly 41: 799–817, 1997     

The role of stacking interactions in the binding of the NMDA receptor glycine site with antagonists and 3-hydroxykynurenine

Ab initio quantum chemical calculations of the benzene dimer, benzene dimer 5,7-chlorination of one aromatic ring, 3-hydroxykynurenine, and kynurenic acid molecules located above the Phe484 aromatic ring of a fragment of the receptor binding site were performed to study the role of stacking interaction in the binding of agonists and antagonists with the glycine binding site of the NR1 subunit of the NMDA receptor. The GAMESS 6.4 software in the 6–31G** basis set with complete optimization of the geometry and with account of electron correlation within the second-order Moller-Plesset perturbation theory was used for all calculations. It was shown that parallel shifted conformations of the benzene dimer were the most favorable in energy. Successive substitution of chlorine atoms for protons of one aromatic ring at positions 7 and 5 led to an increase in the stacking-interaction energy and mutual displacement of aromatic rings. In the case of kynurenic acid and its chlorinated derivatives, which are NMDA receptor antagonists, the increase in the stacking interaction energy further suppressed the ion channel, whereas 3-hydroxykynurenine was neither an agonist nor an antagonist of the glycine site because of steric constraints.     

Synthesis of 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides and their evaluation as ligands for NMDA receptor glycine binding site

A series of 2-substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides were synthesized and evaluated for their affinity to the glycine binding site of the N-methyl-d-aspartate (NMDA) receptor. The binding affinity was determined by the displacement of radioligand [³H]MDL-105,519 from rat cortical membrane preparations. The most attractive structures in the search for prospective NMDA receptor ligands were identified to be 2-arylcarbonylmethyl substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides. It has been demonstrated for the first time that the replacement of NH group in the ligand by sp³ CH₂ is tolerated. This finding may pave the way for previously unexplored approaches for designing new ligands of the NMDA receptor.     

Involvement of 5-HT receptors in the development and expression of methamphetamine-induced behavioral sensitization: 5-HT receptor channel and binding study

Methamphetamine (MAP) is one of the most commonly abused drugs in Asia, and previous studies suggest that serotonin 3 receptors (5-HT(3)) are involved in MAP-induced locomotion and reward. However, little is known about the role of 5-HT(3) receptors in MAP-induced behavioral sensitization. Here, we measured the effects of MDL 72222, a 5-HT(3) antagonist, and SR 57227 A, a 5-HT(3) agonist, on the development and expression of MAP-induced behavioral sensitization, and alternations of 5-HT(3) receptor binding labeled with the 5-HT(3)-selective antagonist, [(3)H]GR65630, in mice. In addition, we investigated the effects of MAP on 5-HT(3A) receptor channel activity in Xenopus laevis oocytes expressing 5-HT(3A) receptors. We found that MDL 72222 attenuated both the development and expression of behavioral sensitization to MAP (1.0 mg/kg, i.p.), and that this attenuating effect of MDL 72222 was reversed by pre-treatment with SR 57227 A. In oocytes expressing 5-HT(3A) receptor, MAP exhibited a dual modulation of 5-HT(3A) receptor channel activity, i.e. pre-treatment with a low dose of MAP (0.1 microm) enhanced 5-HT-induced inward peak current (I(5-HT)) but a high dose of MAP (100 microm) inhibited I(5-HT). The acute administration of MDL 72222 with MAP decreased [(3)H]GR65630 binding versus MAP alone in the mouse striatum. Our results suggest that MDL 72222 attenuates MAP-induced behavioral sensitization via 5-HT(3) receptors in the caudate putamen, and that 5-HT(3) receptor antagonists like MDL 72222 have potential as novel anti-psychotic agents for the treatment of MAP dependence and psychosis.     

The metal-binding sites of the zinc-transporting P-type ATPase of Escherichia coli. Lys and Asp in the seventh and eighth transmembrane segments of ZntA contribute to the coupling of metal binding and ATPase activity

ZntA is a P-type ATPase which transports Zn²⁺, Pb²⁺ and Cd²⁺ out of the cell. Two cysteine-containing motifs, CAAC near the N-terminus and CPC in transmembrane helix 6, are involved in binding of the translocated metal. We have studied these motifs by mutating the cysteines to serines. The roles of two other possible metal-binding residues, K⁶⁹³ and D⁷¹⁴, in transmembrane helices 7 and 8, were also addressed. The mutation CAAC → SAAS reduces the ATPase activity by 50%. The SAAS mutant is phosphorylated with ATP almost as efficiently as the wild type. However, its phosphorylation with P_i is poorer than that of the wild type and its dephosphorylation rate is faster than that of the wild type ATPase. The CPC → SPS mutant is inactive but residual phosphorylation with ATP could still be observed. The most important findings of this work deal with the prospective metal-binding residues K⁶⁹³ and D⁷¹⁴: the substitution K693N eliminates the Zn²⁺-stimulated ATPase activity completely, although significant Zn²⁺-dependent phosphorylation by ATP remains. The K693N ATPase is hyperphosphorylated by P_i. ZntA carrying the change D714M has strong metal-independent ATPase activity and is very weakly phosphorylated both by ATP and P_i. In conclusion, K⁶⁹³ and D⁷¹⁴ are functionally essential and appear to contribute to the metal specificity of ZntA, most probably by being parts of the metal-binding site made up by the CPC motif.     

Binding site and interlobe interactions of the ionotropic glutamate receptor GluK3 ligand binding domain revealed by high resolution crystal structure in complex with (S)-glutamate

Ionotropic glutamate receptors (iGluRs) are involved in excitatory signal transmission throughout the central nervous system and their malfunction is associated with various health disorders. GluK3 is a subunit of iGluRs, belonging to the subfamily of kainate receptors (GluK1–5). Several crystal structures of GluK1 and GluK2 ligand binding domains have been determined in complex with agonists and antagonists. However, little is known about the molecular mechanisms underlying GluK3 ligand binding properties and no compounds displaying reasonable selectivity towards GluK3 are available today. Here, we present the first X-ray crystal structure of the ligand binding domain of GluK3 in complex with glutamate, determined to 1.6 Å resolution. The structure reveals a conserved glutamate binding mode, characteristic for iGluRs, and a water molecule network in the glutamate binding site similar to that seen in GluK1. In GluK3, a slightly lower degree of domain closure around glutamate is observed compared to most other kainate receptor structures with glutamate. The volume of the GluK3 glutamate binding cavity was found to be of intermediate size between those of GluK1 and GluK2. The residues in GluK3 contributing to the subfamily differences in the binding sites are primarily: Thr520, Ala691, Asn722, Leu736 and Thr742. The GluK3 ligand binding domain seems to be less stabilized through interlobe interactions than GluK1 and this may contribute to the faster desensitization kinetics of GluK3.     

Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor

The high toxicity of clostridial neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A–G (BoNT/A–G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion.