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The long term use of many insecticides is continually threatened by the ability of insects to evolve resistance mechanisms that render the chemicals ineffective. Such resistance poses a serious threat to insect pest control both in the UK and worldwide. Resistance may result from either an increase in the ability of the insect to detoxify the insecticide or by changes in the target protein with which the insecticide interacts. DDT, the pyrethrins and the synthetic pyrethroids (the latter currently accounting for around 17% of the world insecticide market), act on the voltage-gated sodium channel proteins found in insect nerve cell membranes. The correct functioning of these channels is essential for normal transmission of nerve impulses and this process is disrupted by binding of the insecticides, leading to paralysis and eventual death. Some insect pest populations have evolved modifications of the sodium channel protein which prevent the binding of the insecticide and result in the insect developing resistance. Here we review some of the work (done at Rothamsted Research and elsewhere) that has led to the identification of specific residues on the sodium channel that may constitute the DDT and pyrethroid binding sites.

DDT inhibits Na channel inactivation and deactivation, promotes Na channel activation and reduces the resting potential of Xenopus oocytes expressing the Drosophila para Na channel. These changes are only marginally influenced by the single mutation M918T (super-kdr) but are reduced similar to 10-fold by either the single mutation L1014F (kdr) or the double mutation L1014F + M918T, both of which confer resistance to the pyre-throids permethrin and deltamethrin. We conclude that DDT binds either to or in the region of L1014 on IIS6 but only weakly to M918 on the IIS4-S5 linker, which is part of a high-affinity binding site for permethrin and deltamethrin. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Philanthotoxin-433 (PhTX-433) is a polyamine wasp toxin that antagonizes certain ionotropic receptors noncompetitively. Four analogues of PhTX-433, C-methylated in the polyamine chain, were synthesized from (RS)-1,3-butanediol, two diamine building blocks, and an activated/protected tyrosine derivative. Use of a phosphane reagent more bulky than trimethylphosphane gave a high intramolecular selectivity between primary and secondary hydroxy groups in the Fukuyama−Mitsunobu reaction. Thus, trimethylphosphane proved to be the only phosphane reagent that enabled alkylation of 2-nitrobenzenesulfonamides with a wide range of secondary alcohols, whereas tributylphosphane was selective for primary alcohol groups. This selectivity was utilized to obtain orthogonally protected, asymmetric, branched tetraamines, employed for solution-phase synthesis of philanthotoxin analogues. The branched philanthotoxin analogues thus obtained were tested in an electrophysiological assay using rat brain ionotropic glutamate receptors expressed in Xenopus laevis oocytes. Their potencies proved to be similar to the corresponding nonbranched analogues. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Two subunits from Xenopus, XenNR1G and the "short" subunit XenU1, have previously been coexpressed to form a unitary (NMDA/non-NMDA type) glutamate receptor. We now show that an antibody to XenNR1G or an antibody to XenU1 precipitates the binding sites of both XenNR1G and XenU1, with the recombinant subunits or with solubilised Xenopus brain membranes, i.e., the combination occurs in vivo. The expressed XenU1 subunits are in the cell membrane and oriented correctly. XenU1 binds not only kainate with high affinity (K(D) 1.2 nM at 25 degrees C), but also the glycine site antagonist 5,7-dichlorokynurenic acid (DCKA). DCKA, GTP, or GTPgammaS displaces competitively all of the bound [3H]kainate, but glycine has no effect. The results suggest that a common binding site for kainate, DCKA, and GTP can exist on XenU1. In the XenNR1G/XenU1 complex, the kainate affinity is lowered eightfold, whereas the DCKA affinity is considerably increased (K(D) 147 nM). Only 18% of the binding to the complex has the properties of the NMDA receptor glycine site, the rest being due to switching of the high-affinity kainate site of XenU1 (low-affinity DCKA) to a high-affinity DCKA (low-affinity kainate) conformation. Surprisingly, a mammalian NR2 subunit can also combine with XenU1, and this introduces similar reciprocal changes in the binding of kainate and DCKA. The combined evidence suggests a common basic mode of agonist site formation in different subunit types of the ionotropic glutamate receptors.

Recent progress in the cloning of alpha (para) and beta (TipE) Na channel sub-units from Drosophila melanogaster (fruit fly) and Musca domestica (housefly) have facilitated functional expression studies of insect Na channels in Xenopus laevis oocytes, assayed by voltage clamp techniques. The effects of Type I and Type II pyrethroids on the biophysical properties of these channels are critically reviewed. Pyrethroid resistance mutations (termed kdr and super-kdr) that reduce the sensitivity of the insect Na channel to pyrethroids have been identified in a range of insect species. Some of these mutations (eg L1014F, M918T and T929I) have been incorporated into the para Na channel of Drosophila, either individually or in combination, to investigate their effects on the sensitivity of this channel to pyrethroids. The kdr mutation (L1014F) shifts the voltage dependence of both activation and steady-state inactivation by similar to5 mV towards more positive potentials and facilitates Na channel inactivation. Incorporation of the super-kdr mutation (M918T) into the Drosophila Na channel also increases channel inactivation and causes a > 100-fold reduction in deltamethrin sensitivity. These effects are shared by T929I, an alternative mutation that confers super-kdr-like resistance. Parallel studies have been undertaken using the rat IIA Na channel to investigate the molecular basis for the low sensitivity of mammalian brain Na channels to pyrethroids. Pat IIA channels containing the mutation L1014F exhibit a shift in their mid-point potential for Na activation, but their overall sensitivity to permethrin remains similar to that of the wild-type rat channel (ie both are 1000-fold less sensitive than the wild-type insect channel). Mammalian neuronal Na channels have an isoleucine rather than a methionine at the position (874) corresponding to the super-kdr (M918) residue of the insect channel. Replacement of the isoleucine of the wild-type rat IIA Na channel with a methionine (I874M) increases deltamethrin sensitivity 100-fold. In this way, studies of wild-type and mutant Na channels of insects and mammals are providing a molecular understanding of kdr and super-kdr resistance in insects, and of the low pyrethroid sensitivity of most mammalian Na channels. They are also giving valuable insights into the binding sites for pyrethroids on these channels. (C) 2001 Society of Chemical Industry.

The modular structure of philanthotoxins was exploited for construction of the first combinatorial library of these compounds using solid-phase parallel synthesis. (S)-Tyrosine and (S)-3-hydroxyphenylalanine were used as amino acid components, spermine, 1,12-dodecanediamine, and 4,9-dioxa-1,12-dodecanediamine as amine components, and butanoyl, phenylacetyl, and cyclohexylacetyl as N-acyl groups. Following automated preparative HPLC, the resulting 18 compounds were isolated as the S-forms in 40-70% yields. The purity of the products was determined by HPLC with evaporative light scattering detection and by (1)H and (13)C NMR. The thus obtained philanthotoxins were tested electrophysiologically for their antagonist properties on human muscle-type nicotinic acetylcholine receptors (nAChR) expressed in TE671 cells and on rat brain non-NMDA glutamate receptors (non-NMDAR) expressed in Xenopus oocytes. 4-Hydroxy analogues lacking the secondary amino groups (PhTX-12 and 4,9-dioxa-PhTX-12 and their analogues) were inactive on non-NMDAR, whereas the potency of the spermine derivatives (PhTX-343 and its analogues) increased with steric bulk of the N-acyl group. The analogue of PhTX-343 in which the N-butanoyl group was replaced by phenylacetyl group had IC(50) of 15 +/- 4 nM on non-NMDAR. Increasing the steric bulk of the N-acyl group was not advantageous for activity at nAChR, and a sharp decrease in potency with increased steric bulk was observed with the derivatives of PhTX-12. 3-Hydroxy analogues generally exhibited lower activity and different response to alterations of the N-acyl groups as compared to the 4-hydroxy analogues. Since the acyl group alterations in PhTX-343 and 4,9-dioxa-PhTX-12 have a similar effect on potency, which is distinctly different from that observed for PhTX-12, the two former compounds may bind to nAChR in a similar fashion but differently from that of PhTX-12. The combinatorial library approach described in this work represents a prototype methodology for future exploration of structure-activity relationships of philanthotoxins.

Ionotropic receptors are modulated allosterically by natural polyamines, such as spermine, and by polyamine derivatives, such as polyamine amides (e.g. philanthotoxin-343) and polymethylene tetraamines (e.g. methoctramine. Modulation can be either positive (potentiation) or negative (non-competitive antagonism of either open or closed channel receptor conformation). Photoaffinity labelling studies have identified a site close to the channel lumen on the nicotinic acetylcholine receptor Torpedo electroplax that is probably the allosteric site responsible for antagonism of the closed channel conformation of this receptor.

kdr and super-kdr are mutations in houseflies and other insects that confer 30- and 500-fold resistance to the pyrethroid deltamethrin. They correspond to single (L1014F) and double (L1014F+M918T) mutations in segment IIS6 and linker II(S4–S5) of Na channels. We expressed Drosophila para Na channels with and without these mutations and characterized their modification by deltamethrin. All wild-type channels can be modified by

PhTX-343 and PhTX-12, analogues of the natural polyamine wasp toxin PhTX-433, were synthesised in 40-60% yields as pure enantiomers using solid phase synthesis techniques. Capillary electrophoresis procedures were developed for chiral separation and determination of enantiomeric purity (ee) of the enantiomers of PhTX-343 and PhTX-12. The methods were optimised with respect to chiral selector, buffer pH, and temperature around the capillary. Thus, rac-PhTX-343 was resolved using a separation buffer containing 30 mM heptakis-(2, 6-di-O-methyl)-beta-cyclodextrin in 50 mM 6-aminocarproic acid (pH 4. 0) at 15 degrees C. rac-PhTX-12 was not resolvable in this system, but could be resolved using a separation buffer containing 10% w/v of dextrin 10, a linear maltodextrin, in 50 mM 6-aminocaproic acid (pH 4.0) at 15 degrees C. Using these methods, the optical purity of the synthetic enantiomers was determined to be ee > 99%. The enantiomers were also characterised by chiroptical methods. The antagonist potency of the enantiomers was tested on nicotinic acetylcholine receptors (human muscle-type nAChR) expressed in TE671 cells, ionotropic glutamate receptors in Xenopus laevis oocytes (expressing recombinant GluR1flop receptors), and locust muscle ionotropic glutamate receptors sensitive to quisqualate (qGluR). The potencies of each pair of enantiomers were similar (eudismic ratio close to 1).

Several wasp venoms contain philanthotoxins (PhTXs), natural polyamine amides, which act as noncompetitive inhibitors (NCIs) on the nicotinic acetylcholine receptor (nAChR). Effects of varying the structure of PhTXs and poly(methylene tetramine)s on the binding affinity have been investigated. Using the fluorescent NCI ethidium in a displacement assay Kapp values of these compounds have been determined. We found that an increase in size of the PhTX's hydrophobic head group significantly increased the binding affinity, while inserting positive charge almost completely destroyed it. Elongating the PhTX polyamine chain by introducing an additional aminomethylene group decreased the binding affinity, whereas a terminal lysine improved it. In general, poly(methylene tetramine)s showed higher binding affinities than PhTX analogues. The stoichiometry of PhTX binding was determined to be two PhTX molecules per receptor monomer. PhTXs appeared to bind to a single class of nonallosterically interacting binding sites and bound PhTX was found to be completely displaced by well-characterized luminal NCIs. To elucidate the site of PhTX binding, a photolabile, radioactive PhTX derivative was photocross-linked to the nAChR in its closed channel conformation resulting in labeling yields for the two alpha and the beta, gamma and delta subunits of 10.4, 11.1, 4.0 and 7.4%, respectively. Based on these findings we suggest that PhTXs and poly(methylene tetramine)s enter the receptor's ionic channel from the extracellular side. The hydrophobic head groups most likely bind to the high-affinity NCI site, while the positively charged polyamine chains presumably interact with the negatively charged selectivity filter located deep in the channel lumen.

The mechanism of inhibition of a nicotinic acetylcholine receptor (nAChR) in BC(3)H1 muscle cells by philanthotoxin-343 (PhTX-343), a synthetic analogue of philanthotoxin-433, a wasp toxin, was investigated using a laser-pulse photolysis technique with microsecond time resolution and in a carbamoylcholine concentration range of 20-100 microM and PhTX-343 concentration range of 0-200 microM. The rate constant for nAChR channel opening determined by the chemical kinetic techniques decreased with increasing PhTX-343 concentration, whereas there was no significant effect on the rate constant for channel closing. The resulting decrease in the channel-opening equilibrium constant accounted quantitatively for the inhibition of the receptor by the toxin. Single-channel current measurements suggest an additional step in which the open channel:inhibitor complex isomerizes to a nonconducting receptor form. Cell-flow experiments with a time resolution of 10 ms indicate that this isomerization step is only important at very high inhibitor concentrations. The inhibitor binds to the open-channel receptor form, with an affinity that is at least 5 times smaller than that for the closed-channel form. This indicates that receptor inhibition mainly involves the binding of PhTX-343 to the closed-channel form of the receptor. PhTX-343, and an analogue of this polyamine, had no effect when applied to the inside of the cell membrane. However, there was significant inhibition of the nAChR when these compounds were applied to the outside of the cell membrane, indicating an extracellular site for inhibition. Furthermore, increasing the transmembrane potential results in a decrease in the ability of PhTX-343 to inhibit the receptor. This observation is related to the voltage dependence of the effect of PhTX-343 on the rate constant for nAChR channel opening with increasing transmembrane voltage (-60 to 50 mV). This suggests that the voltage dependence of inhibition mainly reflects the effect of transmembrane voltage on the rate constant of channel opening and, therefore, the channel-opening equilibrium constant. PhTX-343 competes with cocaine and procaine for its binding site. The finding that this toxin, which binds to a common inhibitory site with compounds such as cocaine, exerts its effect by decreasing the channel-opening equilibrium constant suggests an approach for the development of therapeutic agents. A compound that binds to this regulatory site but does not affect the channel-opening equilibrium constant may be developed. Such a compound can displace an abused drug such as cocaine and thereby alleviate the toxic effect of this compound on the organism.

The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of -latroinsectotoxin (-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. -LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of -LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (130 kDa) encoded in the -LIT gene differs from that of native -LIT purified from BWSV (110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native -LIT revealed a molecular ion with m/z+ of 110916 ± 100, indicating that the native -LIT is 991 amino acids in length. When the full-length -LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native -LIT have a high Ca permeability, whereas those formed by truncated, recombinant protein do not.

We have previously described the isolation of a complementary DNA (cDNA) from the freshwater mollusc Lymnaea stagnalis encoding a polypeptide that exhibits approximately 50% identity to the beta-subunits of vertebrate gamma-aminobutyric acid (GABA) type A (GABAA) receptor. When expressed in Xenopus laevis oocytes from in vitro-transcribed RNA, the snail subunit forms functional homo-oligomeric receptors possessing chloride-selective ion channels. In recordings from voltage-clamped oocytes held at -60 mV, GABA induced an inward current, whereas application of the chloride-channel blocker picrotoxin (in the absence of agonist) elicited an apparent outward current. Single channel recordings obtained from cell-attached patches have revealed a single population of approximately 20 pS channels, with an open probability greater than 90% (at a pipette potential of -100 mV) in the absence of GABA. The relationship between single channel current and pipette potential was linear over the studied range (-100 mV to +60 mV), but the open probability was less for hyperpolarizations than for depolarizations. The spontaneous channel openings were blocked by micromolar concentrations of picrotoxin. Functional hetero-oligomeric receptors were formed when the molluscan subunit was co-expressed in oocytes with the bovine GABAA receptor alpha 1-subunit, but the channels gated by these receptors did not open spontaneously.

The synthesis of the non-competitive glutamate receptor antagonist, argiotoxin-636 is described. Furthermore, synthetic routes are presented for the preparation of structural analogs including argiotoxin-philanthotoxin hybrids. Biological activities on glutamate receptors are discussed.

Several wasp venoms contain philanthotoxins (PhTXs) that act as noncompetitive inhibitors (NCIs) on cation-selective ion channels including the nicotinic acetylcholine receptor (nAChR). In the search for a ligand with high affinity and specificity for the nAChR we tested a series of newly developed PhTX analogues. Modulation of the structural elements of PhTXs can significantly influence their binding affinities. This approach resulted in the development of the photolabile compound MR44. In photoaffinity labelling studies 125 I-MR44 was used to map the ligand-binding site at the Torpedo californica nAChR. Upon UV irradiation of the receptor–ligand complex, 125 I-MR44 was mainly incorporated into the receptor α-subunit. Proteolytic mapping and microsequencing identified the site of 125 I-MR44 cross-linking within the sequence αHis-186 to αLeu-199 that in its C-terminal region partially overlaps with the agonist-binding site. Since bound agonists had only minor influence on 125 I-MR44 photocross-linking, the site where the hydrophobic head group of 125 I-MR44 binds must be located outside the zone that is sterically influenced by agonists bound at the nAChR. A possible site of interaction of 125 I-MR44 would be the N-terminal region of the labelled sequence, in which aromatic amino-acid residues are accumulated. We suggest that the polyamine moiety of 125 I-MR44 interacts with the high affinity non-competitive inhibitor site deep in the ion channel, while the aromatic ring of this compound binds in the vestibule of the nAChR to a hydrophobic region on the α-subunit that is located close to the agonist binding site.

The effects of extracellularly-applied synthetic philanthotoxin-343 (PhTX-343) on transmission and long-term potentiation (LTP) at Schaffer-collateral/commissural-CA1 synapses were investigated. PhTX-343 was ineffective in antagonizing CA1 field-EPSPs mediated by AMPA/kainate receptors. However, when a micromolar concentration of the toxin was present during tetanization, the induction of LTP was suppressed. In contrast, when PhTX-343 was applied either immediately after or long after tetanization no effect on LTP could be found. It appears that the synaptic, non-NMDA receptors of the CA1-region are insensitive to PhTX-343. Suppression of LTP induction could result from antagonism of postsynaptic NMDA receptors, but the results do not rule out other possibilities such as presynaptic block.

We have purified and characterized two vertebrate excitatory amino acid ionotropic receptors from the Xenopus central nervous system. Each is a unitary receptor (i.e., having more than one class of excitatory amino acid agonist specificity within one protein oligomer). The first is a unitary non-N-methyl-D-aspartate (non-NMDA) receptor and the second is a unitary NMDA/non-NMDA receptor. The specific agonist-activated channel activity and pharmacology of each type were recognized by patch-clamping lipid bilayers in which the isolated protein was reconstituted. In the second case, the NMDA and the non-NMDA sites could not be physically separated and exhibited functional interaction. Parallel evidence for this was obtained when poly(A) RNA from Xenopus brain was translated in oocytes: a noncompetitive inhibition of the response to L-kainate is produced by NMDA to a maximum depression of 30% at 1 mM NMDA. Each isolated oligomer contains 42-kDa subunits of the non-NMDA ligand binding type, but the second type has an additional NMDA-receptor-specific 100-kDa subunit. Thus, a subunit-exchange hypothesis can account for the known multiplicity of excitatory amino acid receptor types.

Synopsis:The polyamine amides comprise a newly-discovered class of compounds which exhibits considerable potential for the development of selective pharmacological tools and pharmaceutical agents. In mammals and other vertebrates, they are selective, non-competitive antagonists of ionotropic glutamate receptors, but they also interact with other ionotropic receptors (e.g. nicotinic acetylcholine receptors). Thus, they are channel blockers which are selective for cation channels. We report on synthetic studies undertaken to produce hybrid analogues of these toxins based upon the argiotoxins of spider venoms and the philanthotoxins of parasitic, predatory wasp venom. The synthesis and characterisation of a mono-acylated spermine is also described. In addition, an account of current views on the many possible sites and modes of actions of the polyamine amides is presented and their potential for therapeutic neurochemistry, e.g. for the possible treatment of ischaemic damage to the nervous system, is highlighted.

DDT and the pyrethroid class of insecticides target voltage-gated sodium channels. Their binding stabilises the channel open state, inducing prolonged tail currents associated with insect paralysis (‘knockdown’) and death. Target-site mutations conferring resistance, while a challenge for pest control programs, have provided valuable information on the location of the elusive insecticide-binding site. Homology modelling of the housefly sodium channel and automated ligand docking studies have identified a binding site consistent with resistance-associated mutagenesis data, structure-activity relationships of insecticides and their state-dependent binding activity [O'Reilly et al (2006) Biochem. J. 396:255-263]. The putative binding site, delimited by the domain II S4-S5 linker, S5 & S6 helices and domain III S6 helix, interfaces the lipid bilayer and is therefore accessible to lipid-soluble insecticide ligands. The model is supported by recent experimental results from voltage-clamp electrophysiology studies on mutant fruitfly sodium channels [Usherwood et al (2007) FEBS Letters 581:5485-5492]. The mutation T929I, predicted to inhibit DDT binding through steric hindrance, abolishes the effects of DDT on channel activity. M918, a residue predicted to form a binding contact with pyrethroids but not DDT, decreased deltamethrin potency without effecting DDT potency when mutated. We have developed methods based on circular dichroism spectroscopy to identify ligand binding to a non-insect channel system, namely the voltage-gated sodium channel NaChBac from Bacillus halodurans [Nurani et al (2008) Biochemistry 31:8114-8121], which will be used as a further test of the binding of various insecticides as predicted by the model. (Supported by grants from the BBSRC)

Single channel analysis was used to compare the electrophysiological properties of wild-type (WT) and I874M mutant (M874) rat Na(V)1.2 channels expressed in Xenopus oocytes and their modulation by the pyrethroid deltamethrin. In the absence of pyrethroid, histograms of channel open times were best-fit by single exponentials. The open time constants at -40 mV for WT (0.53+/-0.05 ms) and M874 (0.65+/-0.08 ms) channels were significantly different and both decreased with depolarisation. At > or =100 nM deltamethrin, WT open time histograms at -40 mV were best-fit by two exponentials (time constants, 0.49+/-0.03 ms (tau(o,fast,WT)) and 5.2+/-0.5 ms (tau(o,slow,WT)). The population of long-duration openings and tau(o,slow,WT) increased when the concentration of deltamethrin was raised, but tau(o,fast,WT) was unaffected. Qualitatively similar results were obtained for the M874 channel, but with > or =10 nM deltamethrin. Deltamethrin also caused a negative shift in the relationships between channel opening probability (P(op)) and membrane potential and first latency and membrane potential, suggesting that the pyrethroid binds to the closed channel. Selectivity for Na was increased by the pyrethroid (10 microM, WT; 1 microM, mutant).

Polyamine amide toxins obtained from venoms of spiders and wasps interact selectively with ionotropic glutamate receptors (GLU-R) of vertebrate central nervous systems. The sites and modes of action of these polyamine amide toxins are reviewed with particular reference to their structure-activity relationships. Qualitatively, their effects on GLU-R are identical to those exerted by polyamines such as spermine, but the polyamine amides are more potent. These compounds (a) potentiate and (b) antagonize GLU-R, the latter arising through open channel block. For the N- methyl - D - aspartate receptor this non-competitive antagonism probably arises through binding of toxin to the Mg2+ site(s) located in the channel gated by this receptor. Similarities and differences between GLU-R in vertebrates and in invertebrates with respect to their interactions with polyamines and polyamine amide toxins are discussed. In both groups the low specificity of these compounds is illustrated by their antagonism at nicotinic acetylcholine receptors in addition to GLU-R. Electrophysiological studies, including those employing Xenopus oocytes, are reviewed and future prospects for the use of polyamine amides in therapy are discussed.

K+ channel activity in cultured locust myofibres was investigated using gigaohm patch-clamp techniques. After 2 months in vitro the myofibres had a mean resting potential of -39±7mV (±S.D., N=42). Five types of K+ channel were identified at this time. The majority of single-channel events recorded from cell-attached patches were due to a small-conductance (type 1) and a large-conductance (type 2), inward rectifier, K+ channel. In cell-attached patches, with 180 mmol 1−1 KC1 in the patch pipette, the type 1 channel had a chord conductance of 43 pS for inward currents and 8pS for outward currents; the type 2 channel had a chord conductance of 115 pS for inward currents and 29 pS for outward currents. The type 2 channel exhibited bursting kinetics, was ATP-sensitive and could be blocked by Ba2+. Two other channels (types 3 and 4) had linear conductances of 130pS and 207pS, respectively. The type 3 channel was Ca2+-sensitive. A further channel (type 5) appeared to be an inward rectifier with a conductance of 5pS. Openings of types 3, 4 and 5 channels occurred less frequently than openings of the other two channels. Types 1, 2, 3 and 4 channels possessed multiple closed and open states with non-linear gating mechanisms.

Gigaohm recordings have been made from glutamate receptor channels in excised, outside-out patches of collagenase-treated locust muscle membrane. The channels in the excised patches exhibit the kinetic state switching first seen in megaohm recordings from intact muscle fibers. Analysis of channel dwell time distributions reveals that the gating mechanism contains at least four open states and at least four closed states. Dwell time autocorrelation function analysis shows that there are at least three gateways linking the open states of the channel with the closed states. A maximum likelihood procedure has been used to fit six different gating models to the single channel data. Of these models, a cooperative model yields the best fit, and accurately predicts most features of the observed channel gating kinetics.

Synthetic analogues of arthropod polyamine-containing toxins have been investigated for antagonism of the quisqualate-sensitive L-glutamate receptor (quis-GluR) of locust muscle. Block of the cation-selective channel gated by quis-GluR was demonstrated for N-(4-hydroxyphenylpropanoyl)-and N-(4-hydroxyphenylacetyl)-spermine at significantly lower concentrations than that for spermine.

Philanthotoxin-343 (PhTX-343), a synthetic analogue of wasp toxin PhTX-433, is a noncompetitive antagonist at ionotropic receptors (e.g., AChR or iGluR). To determine possible effects of variations of the amino acid side chain, a library consisting of seventeen PhTX-343 analogues was prepared. Thus, tyrosine was replaced by either apolar, conformationally constrained, or bulky amino acids, whereas the acyl unit and the polyamine moiety were kept unchanged. Analogues with tertiary amide groups were prepared for the first time. Pentafluorophenyl esters were employed for amide bond formation, establishing general protocols for philanthotoxin solution- and solid-phase synthesis (39-90% and 42-54% overall yields, respectively). The analogues were tested for their ability to antagonize kainate-induced currents of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazoyl)propanoic acid receptors (AMPAR) expressed in Xenopus oocytes from rat brain mRNA. This showed that steric bulk in the amino acid moiety is well tolerated and suggests that binding to AMPAR does not involve the alpha-NHCO group as a donor in hydrogen bonding.

This review summarises current knowledge of polyamine-containing spider toxins and their interactions with ionotropic receptors of invertebrate and vertebrate excitable cells. Their diverse actions on ionotropic glutamate and acetylcholine receptors, which include potentiation, closed channel block and open channel block, are discussed in the context of toxin and target structures. Factors that complicate attempts to identify and pharmacologically characterise the binding sites for these toxins include their ability to permeate channels of some ionotropic receptors and their apparent accumulation in a cellular compartment, possibly the membrane bilayer.

Black widow spider venom (BWSV) kills Caenorhabditis elegans after injection owing to the presence of heat- and detergent-sensitive components, which are high-molecular-mass latrotoxins. A C. elegans homologue of latrophilin/CIRL (calcium-independent receptor for latrotoxin), B0457.1, was identified and shown to have five conserved domains. RNAi (RNA interference) of this gene rendered C. elegans resistant to BWSV, whereas RNAi for CYP37A1 or a neurexin I homologue, and a deletion mutant of the related B0286.2 gene, had no effect on BWSV toxicity. The latrophilin RNAi mutants exhibit changes in defaecation cycle and alterations in drug sensitivity. These results demonstrate that latrophilin mediates the toxicity of BWSV and provide evidence for a physiological function of this receptor.

Whole-cell recordings and outside-out patch recordings from TE671 cells were made to investigate antagonism of human muscle nicotinic acetylcholine receptors (nAChR) by the philanthotoxins, PhTX-343 and PhTX-(12). When coapplied with acetylcholine (ACh), PhTX-343 caused activation-dependent, noncompetitive inhibition (IC50 = 17 microM at -100 mV) of whole-cell currents that was strongly voltage-dependent. However, preapplication of PhTX-343 unveiled a voltage-independent antagonism that also required receptor activation, which is suggestive of desensitization enhancement. In single-channel studies, 10 microM PhTX-343 significantly reduced the mean open time of channel openings evoked by 1 microM ACh from 4.42 +/- 0.44 to 1.58 +/- 0.10 ms with a minor increase (1.26-fold) in mean closed time. These data indicate that PhTX-343 predominantly blocks the open channel gated by ACh. In contrast, PhTX-(12) caused potent (IC50 = 0.77 microM at-100 mV), activation-dependent, noncompetitive inhibition of ACh-induced whole-cell currents that was only weakly voltage-dependent and suggestive of desensitization enhancement. It caused only a small decrease (7.5%) in the mean open time of channel openings induced by 1 microM ACh, whereas the mean closed time was significantly increased from 200 +/- 45 ms to 586 +/- 145 ms. The different voltage-dependencies of the two modes of action of these philanthotoxins suggest two binding sites, one deep in the nAChR pore, the other near the extracellular entrance to the pore.

Philanthotoxin-12 (PhTX-12) is a novel potent and selective, noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). Homologues of PhTX-12 with 7-11 methylene groups between the primary amino group and the aromatic head-group were synthesized using solid-phase methodology. In vitro electrophysiological studies of nAChR demonstrated that decreasing the number of methylene groups from 12 to 11 significantly increased potency. Antagonism by PhTX-11, like that of PhTX-12, was only weakly voltage-dependent. When the methylene spacer was reduced further, antagonism was decreased below that of PhTX-12, and in some cases potentiation of ACh responses by up to 60% was observed.

Biochemical and immunological studies have shown that mice infected with LP-BM5 virus develop antibodies to ionotropic glutamate receptors. Here, IgG isolated from brain of infected mice has been tested electrophysiologically on cultured rat cortical and hippocampal neurons. The IgG elicited glycine-independent currents that reversed at ∼0 mV. Equivalent concentrations of IgG from uninfected mice were inactive. The glycine-independent currents were less influenced by DNQX and GYKI-52466 than currents elicited by AMPA and KA. The IgG also elicited glycine-dependent currents that reversed at −10 mV and were blocked by dl -AP5, 5,7-DCKA, and polyamine amides. Glycine-dependent and -independent currents were unaffected by tetrodotoxin, strychnine, the transmembrane Cl − gradient or d -tubocurare. Although part of the glycine-independent current remains uncharacterized, these results confirm that a virus-induced immunopathology produces IgG clones that activate ionotropic glutamate receptors and that could, thereby, contribute to the excitotoxic neurological syndrome observed in LP-BM5-infected mice.

Autoantibodies to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors may contribute to chronic hyperexcitability syndromes and neurodegeneration, but their origin is unclear. We examined LP-BM5 murine leukemia virus-infected mice, which manifest excitotoxic brain lesions and hypergammaglobulinemia, for the presence of AMPA-receptor Ab's. Endogenous IgG accumulated upon neurons in the neocortex and caudate/putamen of infected mice and interacted with native and recombinant AMPA-receptor subunits with the following relative abundance: GluR3 > or = GluR1 > GluR2 = GluR4, as determined by immunoprecipitation. In a radioligand assay, IgG preparations from infected mice specifically inhibited [(3)H]AMPA binding to receptors in brain homogenates, an activity that was lost after preadsorbing the IgG preparation to immobilized LP-BM5 virus. These IgGs also evoked currents when applied to hippocampal pyramidal neurons or to damaged cerebellar granule neurons. These currents could be blocked using any of several AMPA receptor antagonists. Thus, anti-AMPA-receptor Ab's can be produced as the result of a virus infection, in part through molecular mimicry. These Ab's may alter neuronal signaling and contribute to the neurodegeneration observed in these mice, actions that may be curtailed by the use of AMPA-receptor antagonists.

To map the structure of a ligand-gated ion channel, we used the photolabile polyamine-containing toxin MR44 as photoaffinity label. MR44 binds with high affinity to the nicotinic acetylcholine receptor in its closed channel conformation. The binding stoichiometry was two molecules of MR44 per receptor monomer. Upon UV irradiation of the receptor-ligand complex, (125)I-MR44 was incorporated into the receptor alpha-subunit. From proteolytic mapping studies, we conclude that the site of (125)I-MR44 cross-linking is contained in the sequence alpha His-186 to alpha Leu-199, which is part of the extracellular domain of the receptor. This sequence partially overlaps in its C-terminal region with one of the three loops that form the agonist-binding site. The agonist carbachol and the competitive antagonist alpha-bungarotoxin had only minor influence on the photocross-linking of (125)I-MR44. The site where the hydrophobic head group of (125)I-MR44 binds must therefore be located outside the zone that is sterically influenced by agonist bound at the nicotinic acetylcholine receptor. In binding and photocross-linking experiments, the luminal noncompetitive inhibitors ethidium and triphenylmethylphosphonium were found to compete with (125)I-MR44. We conclude that the polyamine moiety of (125)I-MR44 interacts with the high affinity noncompetitive inhibitor site deep in the channel of the nicotinic acetylcholine receptor, while the aromatic ring of this compound binds in the upper part of the ion channel (i.e. in the vestibule) to a hydrophobic region on the alpha-subunit that is located in close proximity to the agonist binding site. The region of the alpha-subunit labeled by (125)I-MR44 should therefore be accessible from the luminal side of the vestibule.

Philanthotoxin-433 (PhTX-433), a natural polyamine wasp toxin, is a noncompetitive antagonist of certain ionotropic receptors. Six analogues of PhTX-343 (a synthetic analogue of the natural product), in which the secondary amino groups are systematically replaced by oxygen or methylene groups, have been synthesized by coupling of N-(1-oxobutyl)tyrosine with 1,12-dodecanediamine, 4,9-dioxa-1, 12-dodecanediamine, or appropriately protected di- and triamines, the latter being obtained by multistep syntheses. The resulting PhTX-343 analogues were purified and characterized, and their protolytic properties (stepwise macroscopic pK(a) values) were determined by (13)C NMR titrations. All analogues are fully protonated at physiological pH. The effects of these compounds on acetylcholine-induced currents in TE671 cells clamped at various holding potentials were determined. All of the analogues noncompetitively antagonized the nicotinic acetylcholine receptor (nAChR) in a concentration-, time-, and voltage-dependent manner. The amplitudes of acetylcholine-induced currents were compared at their peaks and at the end of a 1 s application in the presence or absence of the analogues. Most of the analogues were equipotent with or more potent than PhTX-343. The dideaza analogue PhTX-12 [IC(50) of 0.3 microM (final current value)] was the most potent, representing the highest potency improvement (about 50-fold) yet achieved by modification of the parent compound (PhTX-343). Thus, the presence of multiple positive charges in the PhTX-343 molecule is not necessary for antagonism of nAChR. In contrast, the compounds were much less potent than PhTX-343 at locust muscle ionotropic glutamate receptors sensitive to quisqualate (qGluR). The results demonstrate that the selectivity for different types of ionotropic receptors can be achieved by manipulating the polyamine moiety of PhTX-343.

Pyrethroid insensitivity in resistant (kdr) insects has been correlated with a leucine to phenylalanine replacement in the S6 transmembrane segment of domain II of the axonal sodium channel alpha(para)-subunit. An alpha-subunit of rat brain type II sodium channel containing this mutation has been expressed and its sensitivity to permethrin compared with that of the wild-type channel. The steady-state activation curve of the mutant was shifted 14 mV in the depolarizing direction, We propose that an equivalent shift of the sodium current activation curve in kdr insects could account for their low sensitivity to permethrin toxicity. (C) 1997 Federation of European Biochemical Societies.

A cDNA encoding a 100-kDa subunit (XenNR1) of the N-methyl-D-aspartate (NMDA) glutamate receptor type has been cloned from Xenopus central nervous system. When XenNR1 is coexpressed in a mammalian cell line with a recently cloned 51-kDa non-NMDA receptor subunit (XenU1), also from Xenopus, it forms a functional unitary receptor exhibiting the pharmacological properties characteristic of both NMDA and non-NMDA receptors. Firstly, XenU1 can replace NR2 subunits, in complementing XenNR1 to introduce the ligand binding properties of a complete NMDA receptor. Second, responses to both NMDA and non-NMDA receptor agonists and antagonists were obtained in patch-clamp recordings from the cotransfected cells, but no significant responses were recorded when the cells were singly transfected. Third, from solubilized cell membranes from the cotransfected cells, an antibody to the NR1 subunit coprecipitated the binding sites of the non-NMDA receptor subunit. The unitary glutamate receptor has a unique set of properties that denote intersubunit interaction, including a glycine requirement for the responses to non-NMDA as well as to NMDA receptor agonists and voltage-dependent block by Mg2+ of the non-NMDA agonist responses.

Primary cultures of rat cerebellar granule cells have been used to assess the potential neuroprotective effects of philanthotoxins and argiotoxin-636 (ArgTX-636). These polyamine amides are potent antagonists of ionotropic l -glutamate ( l -Glu) receptors. In granule cells loaded with fluo-3, ArgTX-636 and philanthotoxin-343 (PhTX-343) antagonised increases of intracellular free calcium concentration ([Ca 2+ ];) that were stimulated by N -methyl- d -aspartate (NMDA). The antagonism was use-dependent. Antagonism by PhTX-343 was fully reversible, but recovery following antagonism by ArgTX-636 was slow and only partial during the time-course of an experiment. Neither compound inhibited K+-induced increases in [Ca 2+];. In excitotoxicity studies with cerebellar granule cells, the release of lactate dehydrogenase (LDH) and morphological observations were used to assess cell death. A 20–30 min exposure to 500 μM NMDA, 100 μM t,-Glu or 500 μM kainate was sufficient to kill > 90% of the cells after 18–20 h. When added 5 min prior to, and during agonist exposure, PhTX-343 and ArgTX-636 provided total neuroprotection. ArgTX-636 was about 20–30 fold more potent than PhTX-343 against NMDA, but was approximately equipotent with PhTX-343 against a kainate challenge. Neither of the toxins showed any inherent toxicity even at 400 μM and 100 μM respectively. Some analogues of PhTX-343 are more potent, both in terms of antagonism of NMDA-stimulated increases of [Ca 2+ ] i and neuroprotection, than PhTX-343 and ArgTX-636.

Background: The nicotinic acetylcholine receptors (nAChRs) and glutamate receptors are ligand-gated cation channels composed of five separate polypeptide chains. A 43 kDa protein of unknown function is noncovalently associated with the cytoplasmic side of nAChR in vivo . The -venoms of many wasps and spiders ccntain toxins that block the activity of these channels. Philanthotoxin-433 (PhTX-433) is a non-competitive channel blocker found in the venom of the wasp Philanthus . We have used a photolabile derivative to investigate how PhTX-433 interacts with nAChRs. Results: A radiolabeled PhTX analog, containing a photolabile group substituted on one of its aromatic rings, photocrosslinked to all five subunits (α, α′, β, γ, δ) of purified nAChR in the absence of the 43 kDa protein. In the presence of the 43 kDa protein, the a subunit was preferentially labeled. Proteolysis of the receptor after crosslinking indicated that the hydrophobic end (head) of the PhTx-433 analog bound to the cytoplasmic loop(s) of the a-subunit. Binding is inhibited by other non-competitive channel blockers such as the related polyamine-amide toxins from spiders and chlorpromazine. Conclusions: These results, coupled with previous structure/ activity studies, lead to a putative model of the binding of PhTx and related polyamine toxins to nAChRs in vitro . The 43 kDa protein appears to influence the orientation of toxin binding. Further binding studies are necessary to confirm the model and to define how toxins enter the receptor and how they are oriented within it. A precise understanding of ligand/receptor interaction is crucial for the design of drugs specific for a particular subtype of receptor.

Physical and biophysical mechanisms of mechano-sensitivity of cell membranes are reviewed. The possible roles of the lipid matrix and of the cytoskeleton in membrane mechanoreception are discussed. Techniques for generation of static strains and dynamic curvatures of membrane patches are considered. A unified model for stress-activated and stress-inactivated ion channels under static strains is described. A review of work on stress-sensitive pores in lipid-peptide model membranes is presented. The possible role of flexoelectricity in mechano-electric transduction, e.g. in auditory receptors is discussed. Studies of flexoelectricity in model lipid membranes, lipid-peptide membranes and natural membranes containing ion channels are reviewed. Finally, possible applications in molecular electronics of mechanosensors employing some of the recognized principles of mechano-electric transduction in natural membranes are discussed.

The effects of high pressure on the channel gating kinetics of the quisqualate-sensitive L-glutamate receptor (qGluR) of locust muscle have been investigated using a megaohm seal patch-clamp technique. Pressure was applied with helium gas and recordings were carried out at 20.5 degrees C with Rb+ as the main charge-carrying cation in the patch pipette. The mean open time of the qGluR channel was unaffected by 10 and 30 MPa, but it was significantly reduced at 50 MPa. A high proportion of brief openings (mean 0.808 ms) was seen at 50 MPa but not at lesser pressures. Also, in contrast to lesser pressures, 50 MPa prolonged the mean closed time and reduced both the frequency and probability of channel opening. 10(-6) M ketamine significantly reduced the mean channel open time, as previously reported. A pressure of 10 MPa which alone had no effect on the qGluR channel, restored the mean open time in the presence of 10(-6) M ketamine to the value obtained in the absence of the anaesthetic. This implies the shortening of qGluR channel open time by ketamine involves a large + delta V and, therefore, probably conformational changes in the channel. However 10 MPa did not restore the distribution of open times to normal.

An experimental study of flexoelectricity in model membranes containing ion pores and native membranes containing ion channels has been undertaken with the objective of determining the relationship, if any, between flexoelectricity and ion transport. Model membrane patches containing ion pores induced by a blue-green algal toxin, microcystin-LR, and locust muscle membrane patches containing potassium channels were studied using patch-clamp techniques. A correspondence was established between the presence of open channels and pores and the amplitude of the 1st harmonic of the total membrane current when the membranes or patches were subjected to pressure oscillations. The 2nd harmonic of the membrane current provided a measure of the amplitude of a membrane curvature induced by pressure, thus making it possible to determine the membrane flexoelectric coefficient. This study shows that flexoelectricity could be an effective driving force for ion transport through membrane pores and channels, thus further highlighting the possible biological significance of this mechano-electric phenomenon.

In the search for new toxins, preferably with new sites of action, the polyamine amides represent a new class of compounds with potential as insecticides and as pharmaceutical agents due to their antagonism of ligand-gated cation channels. In particular, they are potent antagonists of the l -glutamate receptors of insect skeletal muscle. In this paper, we report on synthetic studies to produce hybrid analogues based upon the argiotoxin spider toxins and philanthotoxin-433 which is obtained from a solitary, parasitic wasp. We speculate upon possible modes and sites of action for these antagonists and we discuss their potential as insecticides and in the possible treatment of ischaemic damage. The synthesis and characterization of 4-hydroxyphenylpropanoylspermine is reported and the locust muscle biological assay is described. Using this pharmacological screen, structure-activity relationships have been determined in our laboratories. These are reviewed in the light of the current literature. Voltage clamp studies of the synthetic analogue philanthotoxin-343 and the effects of this polyamine amide on glutamate receptors expressed in Xenopus oocytes are outlined. In conclusion, a description of our current ideas and understanding of the many sites and modes of action of the polyamine amides, based both upon our own studies and also upon those recently reported, is presented.

The interactions of the general anaesthetic ketamine with the quisqualate-sensitive L-glutamate receptor (QUIS-R) of locust muscle have been investigated at the single channel level using a M omega seal patch clamp technique. Low concentrations (10(-10) to 10(-9) M) of ketamine did not significantly alter the kinetics of the QUIS-R channel. Higher concentrations of ketamine decreased the probability of the channel being open, the frequency of channel opening and the channel mean open time, and increased the channel mean closed time. Probability density functions of channel dwell times indicate that during application of greater than 10(-8) M ketamine the distribution of channel openings becomes restricted mainly to brief events. These results are consistent with the view that ketamine blocks the open, and possibly also the closed, channel of locust muscle QUIS-R and that this anaesthetic dissociates only slowly from its blocking site(s).

Piek and co-workers reported that the solitary digger wasp Philanthus triangulum F., a wasp found in the Sahara Desert that preys on honeybees, makes a venom which blocks glutamate receptors (GluR) on locust muscles.1 A toxin, δ-philanthotoxin (δ-PhTX), which is an antagonist of GluR, was isolated from the venom sacs of the female wasp and assigned structure 1 (Fig. 1) from spectroscopic data and the synthesis of its DL-form.2 Using a honeybee worker paralysis assay, we independently isolated the toxin PhTX-433 (numerals denote the number of methylenes in the polyamine moiety) and established its structure as 1, with the S configuration, by synthesis and circular dichroic spectroscopy. Natural PhTX-433 and two other isomers, -343 and -334, were also synthesized.3 PhTX-433 and its analogs are efficient noncompetitive reversible inhibitors of the quisqualate-sensitive glutamate receptor (QUISR) in locust skeletal muscle.3 A number of similar but structurally more complex toxins have been isolated from various spider venoms4-8 (Fig. 1). The isolation and characterization of polyamine toxins from spiders and wasps has stimulated considerable interest in view of the important involvement of GluR in mammalian central nervous systems and in insect neurotransmission.9,10

The effects of intracellularly and extracellularly applied synthetic analogues of delta-philanthotoxin (PhTX-433) and the polyamine spermine on the excitatory postsynaptic current (EPSC) of glutamatergic synapses and single channel currents gated by quisqualate-sensitive glutamate receptors (QUIS-R) on locust leg muscle have been compared. When applied extracellularly all 3 compounds reversibly antagonised the EPSC and the single channel currents. Antagonism was voltage independent, but use (agonist) dependent. Antagonism also occurred when they were injected into muscle fibres, but in this case it was not use dependent. It is proposed that spermine and the two toxins bind to the closed and open channel conformations of QUIS-R at a site near the intracellular opening of the channel gated by this receptor.