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Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 non-selectively inhibits several excitatory ligand-gated ion channels, and we recently showed that its synthetic analogue, PhTX-343, exhibits strong selectivity for neuronal over muscle-type nicotinic acetylcholine receptors (nAChRs). Here we examined the action of seventeen analogues of PhTX-343 against ganglionic (?3?4) and brain (?4?2) nAChRs expressed in Xenopus oocytes by using two-electrode voltage-clamp at -100 mV. IC50 values for PhTX-343 inhibition of ?3?4 and ?4?2 receptors were 7.7 nM and 80 nM, respectively. All of the studied analogues had significantly higher potency at ?3?4 nAChRs with IC50 values as low as 0.16 nM and with up to a 91-fold selectivity for ?3?4 over ?4?2 receptors. We conclude that PhTX-343 analogues displaying both a saturated ring and an aromatic moiety in the hydrophobic headgroup of the molecule demonstrate exceptional potency and selectivity for ?3?4 nAChRs. 2

An array of analogues of the wasp toxin philanthotoxin-433, in which the asymmetric polyamine moiety was exchanged for spermine and the headgroup replaced with a variety of structurally diverse moieties, was prepared using parallel solid-phase synthesis approaches. In three analogues, the spermine moiety was extended with an amino acid tail, six compounds contained an N-acylated cyclohexylalanine, and four analogues were based on a novel diamino acid design with systematically changed spacer length between N-cyclohexylcarbonyl and N-phenylacetyl substituents. The analogues were studied using two-electrode voltage-clamp electrophysiology employing Xenopus laevis oocytes expressing GluA1(i) AMPA or GluN1/2A NMDA receptors. Several of the analogues showed significantly increased inhibition of the GluN1/2A NMDA receptor. Thus, an analogue containing N-(1-naphtyl)acetyl group showed an IC(50) value of 47 nM. For the diamino acid-based analogues, the optimal spacer length between two N-acyl groups was determined, resulting in an analogue with an IC(50) value of 106 nM.

Ca2+-permeable AMPA receptors (AMPARs) are expressed throughout the adult CNS but yet their role in development is poorly understood. In the developing retina, most investigations have focused on Ca2+ influx through NMDARs in promoting synapse maturation and not on AMPARs. However, NMDARs are absent from many retinal cells suggesting that other Ca2+-permeable glutamate receptors may be important to consider. Here we show that inhibitory horizontal and AII amacrine cells lack NMDARs but express Ca2+-permeable AMPARs. Before eye-opening, AMPARs were fully blocked by philanthotoxin (PhTX), a selective antagonist of Ca2+-permeable AMPARs. After eye-opening, however, a subpopulation of Ca2+-permeable AMPARs were unexpectedly PhTX resistant. Furthermore, Joro spider toxin (JSTX) and IEM-1460 also failed to antagonize, demonstrating that this novel pharmacology is shared by several AMPAR channel blockers. Interestingly, PhTX-insensitive AMPARs failed to express in retinae from dark-reared animals demonstrating that light entering the eye triggers their expression. Eye-opening coincides with the consolidation of inhibitory cell connections suggesting that the developmental switch to a Ca2+-permeable AMPAR with novel pharmacology may be critical to synapse maturation in the mammalian retina.

A protocol for parallel solid-phase synthesis of ω-amino acid-elongated philanthotoxins was developed for exploration of structure-activity relationships for unnatural wasp toxin analogues. Several methods for on-resin activation of N-trityl-linked ω-amino acids were investigated, and the influence of chain length upon activation as pentafluorophenyl (Pfp) esters was examined. Comparison of Pfp-ester activation with phosphonium- and aminium/ uronium-promoted coupling without pre-activation of the resin-bound carboxylic acids showed that only benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) exhibited a comparable efficiency. The present Pfp ester protocol gave high yields and purities of the resulting philanthotoxin analogues; however, in the case of γ-aminobutyric acid (GABA), use of PyBOP was required in order to avoid lactamization to 2-pyrrolidone.

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.

Philanthotoxin (PhTX-433), a potent noncompetitive inhibitor of the L-glutamate receptors and the nicotinic acetylcholine receptors of vertebrates and invertebrates, has a butyryl-tyrosyl-thermospermine structure. Several synthetic analogs of PhTX with hydrophobic alkyl branches in the polyamine chain exhibit 6- to 10-fold enhanced activities to various receptors. Because the branched analogs exhibit such unique activities and because of their importance in tertiary structural studies of ligand/receptor binding, methods for preparing branched PhTX analogs, including photolabile analogs, are presented

The synthetic methods for 27 novel and photolabile philanthotoxin analogs are described. Most analogs were synthesized by two general methods with modifications of these methods where necessary.

The synthetic methods for 27 novel and photolabile philanthotoxin analogs are described. Most analogs were synthesized by two general methods with modifications of these methods where necessary.

The wasp venom philanthotoxin-4.3.3 (PhTX-4.3.3) is an antagonist of glutamate transmission in the insect as well as in the mammalian brain. It was recently shown that PhTX-4.3.3 inhibits the N-methyl-D-aspartate (NMDA) transmission in rat hippocampus. In this study we show that dideaza-philanthotoxin-12 (dideaza-PhTX-12), an analogue of PhTX-4.3.3, is a potent antagonist of voltage-dependent Ca2+ currents in rat hippocampal CA1 neurons. At a concentration of 10 microM it reduces the Ca2+ current to 40%. Two voltage-dependent potassium currents, the A current and the delayed rectifier, were hardly affected by dideaza-PhTX-12, indicating selectivity of the drug for Ca2+ currents. As a consequence the philanthotoxins will inhibit the calcium influx via voltage dependent as well as NMDA mediaded calcium channels and thus reduce excitability in the hippocampus.

The effects of spermine and a synthetic analogue (PhTX-343) of the polyamine amide toxin, delta-philanthotoxin, on the responses of Xenopus oocytes to application of amino acids were examined using voltage clamp. The oocytes were injected with either total rat brain RNA or chick cerebrum, poly(A+)RNA. The responses to N-methyl-D-aspartate and L-kainate were potentiated by low concentrations (10(-11)-10(-7) M) of PhTX-343 and by 10(-5)-10(-4) M spermine. There was variability between oocytes in terms of their responsiveness to these compounds and recovery from their effects was slow and often incomplete. Prolonged or repeated applications of PhTX-343 and spermine eventually resulted in inhibition. Higher concentrations of these compounds always inhibited the responses to acidic amino acids. Low concentrations of PhTX-343 and spermine also potentiated the responses to nicotine and gamma-aminobutyric acid. These results are discussed in terms of the postulated polyamine binding site on the N-methyl-D-aspartate receptor.

1. The molecular structure of β-philanthotoxin (β-PTX), a toxin from the insect paralysing venom of the wasp Philanthus triangulum, has been elucidated using NMR and mass spectrometry. β-PTX has a polyamine character: C5H11·NH·CO·(CH2)4·NH·(CH2)3·NH2. This structure has been confirmed by synthesis. 2. In the locust muscle fibre β-PTX blocks iontophoretically evoked glutamate potentials in a non-activation induced manner. β-PTX also blocks the nicotinic transmission in the insect CNS, however, at much higher toxin concentrations. 3. β-PTX reduces the frequency of postsynaptic channel opening and reduces the duration of open times. 4. These results suggest an effect of β-PTX on kinetics of glutamate activated ion channels, different from acting as an open channel blocker.

A general, improved procedure for rapid synthesis of philanthotoxin analogues, a pharmacologically important class of polyamine conjugates, is described. The solution-phase procedure is illustrated by gram-scale synthesis of philanthotoxins PhTX-343 and PhTX-12. Selectively protected polyamines are coupled to N(alpha)-Fmoc-protected amino acid pentafluorophenyl esters. After removal of the N(alpha)-Fmoc group, the amine is coupled with carboxylic acid pentafluorophenyl esters. Deprotection followed by a rapid and efficient purification by vacuum liquid chromatography on octadecylsilyl silica (RP-18 phase) gave the philanthotoxin analogues in 74-78% overall yield.

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.

Three photosensitive, synthetic analogues of δ-philanthotoxin (PhTX-433) have been tested on the locust, excitatory nerve-muscle system. At 10 −9 M they inhibit reversibly the postjunctional currents (EPSCs) recorded from muscle fibres during motor nerve stimulation, mainly by non-competitively antagonizing postjunctional quisqualate-sensitive glutamate receptors (Quis-R), probably through open channel block. This use-dependent antagonism is characteristic of the philanthotoxins. When the preparation was irradiated with 270 nm U.V. during toxin application and nerve stimulation the EPSCs were inhibited irreversibly. Irradiation of the preparation alone or in the presence of philanthotoxins (e.g. PhTX-433) which are not photosensitive did not lead to irreversible inhibition of the EPSC. It is concluded that the three photosensitive toxins bind covalently to Quis-R in its open channel conformation during U.V. irradiation, thereby irreversibly blocking the channel gated by this receptor.

1. The effect of electrophoretic ejection of philanthotoxin (the polyamine toxin, from the Egyptian digger wasp) was tested on responses of brainstem and spinal neurones in the pentobarbitone-anaesthetized rat to excitatory amino acids. 2. Philanthotoxin caused a dose-dependent reduction of responses to quisqualate, alpha-amino-3-hydroxy-5-phenyl-4-isoxazolepropionate (AMPA) and kainate with little effect on those to N-methyl-D-aspartate (NMDA). 3. The time-course of this antagonist action was slow. In particular the rate of recovery was dependent on frequency of ejection of the agonist. This agonist-dependent recovery suggests that philanthotoxin has a channel blocking mode of action on mammalian central neurones.

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.

M. Bruce , R. Bukownik , A. T. Eldefrawi , M. E. Eldefrawi , R. Goodnow , Jr, T. Kallimopoulos , K. Konno , K. Nakanishi , M. Niwa and P. N. R. Usherwood . Structure-activity relationships of analogues of the wasp toxin philanthotoxin: non-competitive antagonists of quisqualate receptors. Toxicon 28, 1333–1346, 1990.—Fifty-two analogues of the wasp toxin, philanthotoxin-433, have been synthesized and tested on a glutamatergic, nerve-muscle preparation from locust leg. Reduction in amplitude of the neurally-evoked muscle twitch was used to construct dose-inhibition relationships from which ic 50 s were estimated. The most active analogues were characterized by one or more of the following: increased hydrophobicity of aromatic and tyrosyl regions; an increased number of protonated groups in the polyamine region; a guanidinium instead of a spermine terminal amino moiety. The incorporation of a butyl side-group in the polyamine also enhanced potency. These results are explained on the basis of the known non-competitive antagonistic blockage by philanthotoxin-433 of the channel gated by postjunctional glutamate receptors when the channel is open.

In this chapter we will briefly review some of the evidence from electrophysiological studies of mammalian neurones in vivo which relates to the subtypes of glutamate receptors. In particular we will concentrate on recent experiments elucidating the mechanism of action of HA-966 as an N-methyl-D-aspartate (NMDA) antagonist and of a new tricyclic quinoxalinedione (NBQX) and a wasp venom, philanthotoxin, as quisqualate antagonists. These studies were designed to test the hypothesis that the NMDA subtype and one other non-NMDA subtype of glutamate receptor mediate the excitation of rat spinal and brainstem neurones produced by acidic amino acids.

Ca2+-permeable AMPA receptors (AMPARs) are expressed throughout the adult CNS but yet their role in development is poorly understood. In the developing retina, most investigations have focused on Ca2+ influx through NMDARs in promoting synapse maturation and not on AMPARs. However, NMDARs are absent from many retinal cells suggesting that other Ca2+-permeable glutamate receptors may be important to consider. Here we show that inhibitory horizontal and AII amacrine cells lack NMDARs but express Ca2+-permeable AMPARs. Before eye-opening, AMPARs were fully blocked by philanthotoxin (PhTX), a selective antagonist of Ca2+-permeable AMPARs. After eye-opening, however, a subpopulation of Ca2+-permeable AMPARs were unexpectedly PhTX resistant. Furthermore, Joro spider toxin (JSTX) and IEM-1460 also failed to antagonize, demonstrating that this novel pharmacology is shared by several AMPAR channel blockers. Interestingly, PhTX-insensitive AMPARs failed to express in retinae from dark-reared animals demonstrating that light entering the eye triggers their expression. Eye-opening coincides with the consolidation of inhibitory cell connections suggesting that the developmental switch to a Ca2+-permeable AMPAR with novel pharmacology may be critical to synapse maturation in the mammalian retina.

1. The digger wasp, Philanthus triangulum, which preys on honeybees, produces a paralysing venom possessing a wide variety of activities. 2. In insects, the venom has a central as well as a peripheral effect; the latter effect consists of a presynaptic as well as a postsynaptic block of the skeletal neuromuscular transmission. 3. The presynaptic block is probably caused by an inhibition of the re-uptake of the transmitter. The postsynaptic effect probably consists of a block of open ion channels. 4. The venom contains at least four active toxins called alpha-, beta-, gamma- and delta-philanthotoxin (PTX). alpha-PTX blocks transmission in the cockroach CNS. The other three toxins block neuromuscular transmission. delta-PTX being the most active toxin in blocking glutamate evoked postsynaptic depolarizations. 5. In the junctional, as well as in the extrajunctional, muscle fibre membrane delta-PTX blocks ion channels in a use-dependent manner. Once the channel has been blocked, unblocking seems to be channels in a use-dependent manner. Once the channel has been blocked, unblocking seems to be semi-irreversible when agonist activation is low (spontaneous release of transmitter and/or leak of glutamate from the pipette). 6. The time constant of blocking is roughly estimated to be in the order of 10 msec, that of unblocking seems to be several hundreds of msec.

This paper provides answers to the questions which of the toxins present in the venom of the wasp Philanthus triangulum may be responsible for the previously reported blockage of transmission through the sixth abdominal ganglion of the cockroach, and whether this may occur by block of synaptic transmission or by affecting axonal exitability. In current clamp experiments the crude venom induces a slight depolarization of the membrane of the giant axon from the sixth abdominal ganglion of the cockroach and a small and irreversible decrease in the amplitude of the action potential. These marginal effects are not seen with relatively high concentrations of the philanthotoxins β-PTX and δ-PTX. It appears that neither the crude venom nor the toxins significantly affect the excitability of the cockroach giant axon. At a concentration of 20 μg ml−1 δ-PTX causes a slowly reversible block of synaptic transmission from the cercal nerve XI to a giant interneuron without any change in resting membrane potential, whereas β-PTX is inactive. Iontophoretically evoked acetylcholine potentials of the giant neuron are more sensitive to δ-PTX than excitatory postsynaptic potentials. This suggests that the toxin acts on the postsynaptic membrane.

The effects of varying the structure of philanthotoxin (PhTX) were investigated on binding of the channel blockers: [3H]perhydrohistrionicotoxin (H12-HTX) to the nicotinic acetylcholine receptor (nACh-R) of Torpedo electric organ and [3H]MK-801 [( 3H]-5-methyl-10,11-dihydro-5H-dibenzocyclo-hepten-5,10-imine maleate) to the N-methyl-D-aspartate receptor (NMDA-R) of rat brain cortex. The four moieties of PhTX (tyrosine, butyrate, spermine and the terminal amino group) were modified or conjugated resulting in 36 compounds. Although the potencies of the PhTX analogs on both receptors were higher with increasing lipophilicity and the polyamine chain length, there was considerable divergence between the two receptors' channels in the structural activity requirements for blockade by PhTX analogs. A major difference was the more critical role of the amine terminal for inhibition of the nACh-R than the NMDA-R, whereas the reverse might be true for the tyrosine moiety. The potency range of PhTX analogs on [3H]H12-HTX binding was 1070, but only 21 on [3H]MK-801 binding. Adding a lysine or arginine onto the spermine moiety increased the compound's potency on the nACh-R with little effect on the NMDA-R. Because spermine is a component of PhTX, the effects of five polyamines were also studied. Spermine and spermidine potentiated [3H]MK-801 binding, whereas putrescine, cadeverine and agmatine inhibited it. In presence of glutamate, higher concentrations of all polyamines inhibited [3H]MK-801 binding. On the nACh-R, spermine, spermidine and agmatine inhibited [125I]alpha-bungarotoxin and also [3H]H12-HTX binding in presence of carbamylcholine. The complex nature of PhTX interactions with the two receptors suggests that PhTX may bind to two sites: an external polyamine binding site and a channel binding site.

One component (delta-philanthotoxin (delta-PTX) of the venom from the wasp Philanthus triangulum blocks transmission postsynaptically at excitatory synapses on locust muscle. delta-PTX depresses both the iontophoretic glutamate potential and the excitatory junctional current (e.j.c.) in a glutamate receptor activation-dependent manner. The rate of recovery from the effects of the toxin is reduced following either prolonged application of L-glutamate or repetitive iontophoretic application of this amino acid or high frequency neural stimulation of the muscle in the presence of delta-PTX. The decay phase of the e.j.c. is shortened by delta-PTX. The effects of delta-PTX on the e.j.c. are not voltage dependent. The open-close kinetics of glutamate channels in extrajunctional muscle membrane are modified by delta-PTX as shown by patch clamp analysis. The mean life time of the glutamate channel is reduced, whilst the mean interval between single opening events is increased with the events often occurring in bursts. These data are consistent with glutamate channel blocking by this toxin. It is proposed that the toxin blocks open channels gated by both junctional and extrajunctional glutamate receptors on locust muscle. It is further proposed that delta-PTX enters a compartment of the muscle through the glutamate open channels and that it can also block the open channels from this site.

The beta-fraction of the venom of the solitary wasp Philanthus triangulum inhibited glutamate-induced potentials in muscle fibres of the locust in a dose-dependent manner (50% block at 2 micrograms/ml). Single channel behaviour and miniature excitatory postsynaptic currents were not affected. The blocking effect disappeared after treatment of the muscle with the lectin concanavalin A which prevents desensitization of glutamate receptors. Block of potentials induced by iontophoretic application of glutamate was strongly dependent on the frequency of stimulation: no block occurred at stimulus intervals of 30 sec or more. The nature of this inhibition is discussed and proposed to be a delay of recovery from desensitization.

Alterations in the polyamine and amino acid (tyrosine) moieties of philanthotoxin-343 (PhTX-343) were investigated for their effects on the antagonism of nicotinic acetylcholine receptors (nAChRs) isolated from the locust (Schistocerca gregaria) mushroom body. Through whole-cell patch-clamp recordings, the philanthotoxin analogues in this study were shown to cause inhibition of the inward current when co-applied with acetylcholine (ACh). PhTX-343 (IC50 = 0.80 μM at −75 mV) antagonised locust nAChRs in a use-dependent manner, suggesting that it acts as an open-channel blocker. The analogue in which both the secondary amine functionalities were replaced with methylene groups (i.e., PhTX-12) was ~6-fold more potent (IC50 (half-maximal inhibitory concentration) = 0.13 μM at −75 mV) than PhTX-343. The analogue containing cyclohexylalanine as a substitute for the tyrosine moiety of PhTX-343 (i.e., Cha-PhTX-343) was also more potent (IC50 = 0.44 μM at −75 mV). A combination of both alterations to PhTX-343 generated the most potent analogue, i.e., Cha-PhTX-12 (IC50 = 1.71 nM at −75 mV). Modulation by PhTX-343 and Cha-PhTX-343 fell into two distinct groups, indicating the presence of two pharmacologically distinct nAChR groups in the locust mushroom body. In the first group, all concentrations of PhTX-343 and Cha-PhTX-343 inhibited responses to ACh. In the second group, application of PhTX-343 or Cha-PhTX-343 at concentrations ≤100 nM caused potentiation, while concentrations ≥ 1 μM inhibited responses to ACh. Cha-PhTX-12 may have potential to be developed into insecticidal compounds with a novel mode of action.

Glutamate uptake in insect neuromuscular junctions is inhibited by delta-philanthotoxin. Two other components of the philanthus venom (beta- and gamma-philanthotoxin) do not affect this glutamate uptake. No synergism between the active delta-philanthotoxin and the two inactive components had to be assumed to explain the inhibition by the Philanthus venom.

The actions of philanthotoxin (PhTX) were studied on the function of glutamate receptors expressed in Xenopus oocytes injected with rat brain mRNA and on binding of radioligands to rat brain glutamate receptors. PhTX reversibly inhibited the oocyte responses to quisqualate, N-methyl-D-aspartate (NMDA) and kainate in a dose-dependent manner. The NMDA receptor was the most sensitive to PhTX action (10-fold more than the kainate receptor) and the least sensitive was the smooth current component of the quisqualate response. Recovery from PhTX block differed among the three amino acids. NMDA responses recovered completely within a few minutes whereas responses to kainate and quisqualate recovered more slowly. PhTX had no effect on equilibrium binding of [3H]glutamate to rat brain cortical membranes studied in buffer treated to eliminate microorganisms. Based on the drug specificity of this [3H]glutamate binding, it is suggested to be mostly to the NMDA receptor. Low concentrations of PhTX (1-10 microM) potentiated binding of [3H] MK-801, a specific noncompetitive inhibitor of the NMDA receptor. However, higher PhTX concentrations inhibited this binding with an IC50 of 20 microM, similar to its inhibition of the oocyte-expressed NMDA receptor. Inhibition of [3H]MK-801 binding by PhTX was noncompetitive. It is suggested that PhTX, like the more potent MK-801, binds to an allosteric site on the NMDA receptor and inhibits its function but its binding site is not identical with the MK-801 binding site.

The effects of pure philanthotoxin (PhTX), a component of the venom of the wasp Philanthus triangulum, were studied on nicotinic acetylcholine receptors (nAChRs) of vertebrates and insects so as to compare their sensitivities and the mechanism of action of PhTX. Electrophysiological techniques were used on frog muscles and cockroach thoracic ganglia and biochemical techniques were applied to membranes from Torpedo electric organ and honeybee brain. PhTX (1-20 microM) inhibited reversibly the indirectly elicited muscle twitch and reduced the endplate current peak amplitude and its decay time constant in a concentration-dependent manner. In patch clamp studies, PhTX (1-5 microM) when combined with acetylcholine, induced a concentration-dependent decrease in frequency of channel openings and in channel open and burst times. The cockroach fast coxal depressor neuron was inhibited by PhTX in a time- and voltage-dependent manner. The initial rate of binding of [3H]perhydrohistrionicotoxin to Torpedo nAChR in the presence of carbamylcholine was inhibited competitively by PhTX. Binding of alpha-[125I] bungarotoxin to electric organ and honeybee brain membranes was inhibited by PhTX. Binding of [3H]acetylcholine to the electric organ receptor was potentiated by low concentrations of PhTX but inhibited by high concentrations. PhTX, therefore, inhibits both vertebrate and insect nAChRs, which may be important molecular targets for its toxicity. It is suggested that PhTX at high concentration may have some competitive action on nAChR, but it acts mainly as a blocker of the ion channel of the nAChR in its open conformation.

Alterations in the polyamine and amino acid (tyrosine) moieties of philanthotoxin-343 (PhTX-343) were investigated for their effects on the antagonism of nicotinic acetylcholine receptors (nAChRs) isolated from the locust (Schistocerca gregaria) mushroom body. Through whole-cell patch-clamp recordings, the philanthotoxin analogues in this study were shown to cause inhibition of the inward current when co-applied with acetylcholine (ACh). PhTX-343 (IC50 = 0.80 μM at −75 mV) antagonised locust nAChRs in a use-dependent manner, suggesting that it acts as an open-channel blocker. The analogue in which both the secondary amine functionalities were replaced with methylene groups (i.e., PhTX-12) was ~6-fold more potent (IC50 (half-maximal inhibitory concentration) = 0.13 μM at −75 mV) than PhTX-343. The analogue containing cyclohexylalanine as a substitute for the tyrosine moiety of PhTX-343 (i.e., Cha-PhTX-343) was also more potent (IC50 = 0.44 μM at −75 mV). A combination of both alterations to PhTX-343 generated the most potent analogue, i.e., Cha-PhTX-12 (IC50 = 1.71 nM at −75 mV). Modulation by PhTX-343 and Cha-PhTX-343 fell into two distinct groups, indicating the presence of two pharmacologically distinct nAChR groups in the locust mushroom body. In the first group, all concentrations of PhTX-343 and Cha-PhTX-343 inhibited responses to ACh. In the second group, application of PhTX-343 or Cha-PhTX-343 at concentrations ≤100 nM caused potentiation, while concentrations ≥ 1 μM inhibited responses to ACh. Cha-PhTX-12 may have potential to be developed into insecticidal compounds with a novel mode of action.

Synaptic transmission relies on presynaptic neurotransmitter (NT) release from synaptic vesicles (SVs) and on NT detection by postsynaptic receptors. Transmission exists in two principal modes: action-potential (AP) evoked and AP-independent, “spontaneous” transmission. AP-evoked neurotransmission is considered the primary mode of inter-neuronal communication, whereas spontaneous transmission is required for neuronal development, homeostasis, and plasticity. While some synapses appear dedicated to spontaneous transmission only, all AP-responsive synapses also engage spontaneously, but whether this encodes functional information regarding their excitability is unknown. Here we report on functional interdependence of both transmission modes at individual synaptic contacts of Drosophila larval neuromuscular junctions (NMJs) which were identified by the presynaptic scaffolding protein Bruchpilot (BRP) and whose activities were quantified using the genetically encoded Ca2+ indicator GCaMP. Consistent with the role of BRP in organizing the AP-dependent release machinery (voltage-dependent Ca2+ channels and SV fusion machinery), most active BRP-positive synapses (>85%) responded to APs. At these synapses, the level of spontaneous activity was a predictor for their responsiveness to AP-stimulation. AP-stimulation resulted in cross-depletion of spontaneous activity and both transmission modes were affected by the non-specific Ca2+ channel blocker cadmium and engaged overlapping postsynaptic receptors. Thus, by using overlapping machinery, spontaneous transmission is a continuous, stimulus independent predictor for the AP-responsiveness of individual synapses.

Earlier findings had suggested that spontaneous and evoked glutamate release activates non-overlapping populations of NMDA receptors. Here, we evaluated whether AMPA receptor populations activated by spontaneous and evoked release show a similar segregation. To track the receptors involved in spontaneous or evoked neurotransmission, we used a polyamine agent, philanthotoxin, that selectively blocks AMPA receptors lacking GluR2 subunits in a use-dependent manner. In hippocampal neurons obtained from GluR2-deficient mice, philanthotoxin application decreased AMPA-receptor-mediated spontaneous miniature EPSCs (AMPA-mEPSCs) down to 20% of their initial level within 5 min. In contrast, the same philanthotoxin application at rest decreased the subsequent AMPA-receptor-mediated evoked EPSCs (eEPSCs) only down to 80% of their initial value. A 10-min-long perfusion of philanthotoxin further decreased AMPA-eEPSC amplitudes to 60% of their initial magnitude, which remained substantially higher than the level of AMPA-mEPSC block achieved within 5 min. Finally, stimulation after removal of philanthotoxin resulted in reversal of AMPA-eEPSC block, verifying strict use dependence of philanthotoxin. These results support the notion that spontaneous and evoked neurotransmission activate distinct sets of AMPA receptors and bolster the hypothesis that synapses harbor separate microdomains of evoked and spontaneous signaling.

BackgroundSpontaneous "miniature" transmitter release takes place at low rates at all synapses. Long thought of as an unavoidable leak, spontaneous release has recently been suggested to be mediated by distinct pre- and postsynaptic molecular machineries and to have a specialized role in setting up and adjusting neuronal circuits. It remains unclear how spontaneous and evoked transmission are related at individual synapses, how they are distributed spatially when an axon makes multiple contacts with a target, and whether they are commonly regulated.ResultsElectrophysiological recordings in the Drosophila larval neuromuscular junction, in the presence of the use-dependent glutamate receptor (GluR) blocker philanthotoxin, indicated that spontaneous and evoked transmission employ distinct sets of GluRs. Invivo imaging of transmission using synaptically targeted GCaMP3 to detect Ca(2+) influx through the GluRs revealed little spatial overlap between synapses participating in spontaneous and evoked transmission. Spontaneous and evoked transmission were oppositely correlated with presynaptic levels of the protein Brp: synapses with high Brp favored evoked transmission, whereas synapses with low Brp were more active spontaneously. High-frequency stimulation did not increase the overlap between evoked and spontaneous transmission, and instead decreased the rate of spontaneous release from synapses that were highly active in evoked transmission.ConclusionsAlthough individual synapses can participate in both evoked and spontaneous transmission, highly active synapses show a preference for one mode of transmission. The presynaptic protein Brp promotes evoked transmission and suppresses spontaneous release. These findings suggestthe existence of presynaptic mechanisms that promote synaptic specialization to either evoked or spontaneous transmission.

We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor (KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 μM) produced an increase in 4-aminopyridine (4-AP)-evoked glutamate release. In thalamocortical slices, KA (1 μM) produced an increase in the amplitude of evoked excitatory post-synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione, and persisted after pre-treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G-protein-independent regulation further in thalamocortical slices, the KAR-mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca(2+) permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca(2+) stores by thapsigargin, or inhibition of Ca(2+) -induced Ca(2+) -release by ryanodine. Thus, the KA-mediated modulation was contingent on both Ca(2+) entry through Ca(2+) -permeable KARs and liberation of intracellular Ca(2+) stores. Finally, sensitivity to W-7 indicated that the increased cytosolic [Ca(2+) ] underpinning KAR-mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre-synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca(2+) -calmodulin dependent activation of an adenylyl cyclase/cAMP/protein kinase A signalling cascade, independent of G-protein involvement.

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)

There is accumulating evidence that sleep contributes to memory formation and learning, but the underlying cellular mechanisms are incompletely understood. To investigate the impact of sleep on excitatory synaptic transmission, we obtained whole-cell patch-clamp recordings from layer V pyramidal neurons in acute slices of somatosensory cortex of juvenile rats (postnatal days 21–25). In animals after the dark period, philanthotoxin 74 (PhTx)-sensitive calcium-permeable AMPA receptors (CP-AMPARs) accounted for ∼25% of total EPSP size, and current–voltage (I–V) relationships of the underlying EPSCs showed inward rectification. In contrast, in similar experiments after the light period, EPSPs were PhTx insensitive with linearI–Vcharacteristics, indicating that CP-AMPARs were less abundant. Combined EEG and EMG recordings confirmed that slow-wave sleep-associated delta wave power peaked at the onset of the more quiescent, lights-on phase of the cycle. Subsequently, we show that burst firing, a characteristic action potential discharge mode of layer V pyramidal neurons during slow-wave sleep has a dual impact on synaptic AMPA receptor composition: repetitive burst firing without synaptic stimulation eliminated CP-AMPARs by activating serine/threonine phosphatases. Additionally, repetitive burst-firing paired with EPSPs led to input-specific long-term depression (LTD), affecting Ca2+impermeable AMPARs via protein kinase C signaling. In agreement with two parallel mechanisms, simple bursts were ineffective after the light period but paired bursts induced robust LTD. In contrast, incremental LTD was generated by both conditioning protocols after the dark cycle. Together, our results demonstrate qualitative changes at neocortical glutamatergic synapses that can be induced by discharge patterns characteristic of non-rapid eye movement sleep.

The effects of the arylalkyamine arthropod toxins argiotoxin 636 and philanthotoxin 343 were studied on N-methyl-D-aspartate receptor currents in cultured rat hippocampal neurons using whole-cell recording techniques. Argiotoxin 636 and philanthotoxin 343 blocked 10 microM N-methyl-D-aspartate (+10 microM glycine) currents in a concentration-dependent fashion (steady-state IC50 values, 0.9 and 56 microM, respectively). The onset and recovery from argiotoxin 636 block occurred slowly (forward and reverse rate constants, 7.5 x 10(3) s-1 M-1 and 6.9 x 10(-3) s-1, respectively) whereas the philanthotoxin 343 block was more rapid (forward and reverse rate constants, 1.1 x 10(5) s-1 M-1 and 0.1 s-1). A portion, but not all, of the block by the two toxins could be reversed by depolarization to positive holding potentials, indicating that there are voltage-dependent and non-voltage-dependent components of the block. The long-lasting argiotoxin 636 block at -60 mV occurred in a use-dependent fashion and could be substantially reduced by co-application with 10 mM Mg2+, providing evidence that the toxin has a channel blocking action. In contrast to the use dependence of the voltage-dependent argiotoxin 636 block, the non-voltage-dependent component of block (at +60 mV) did not require agonist gating of the channel. The non-voltage-dependent block by argiotoxin 636 was unaffected by increasing the glycine concentration, but was reversed by increasing the N-methyl-D-aspartate concentration, suggesting that the toxin may act as a competitive antagonist at the N-methyl-D-aspartate recognition site. This mechanism was further supported by the near identity of the time constant for argiotoxin 636 block with the time constant for agonist dissociation, irrespective of whether the rapidly dissociating agonist N-methyl-D-aspartate or the more slowly dissociating agonist glutamate was used. With high concentrations of N-methyl-D-aspartate (> or = 100 microM), argiotoxin 636 produced a potentiation of the peak N-methyl-D-aspartate response (at +60 mV) that was accompanied by a slowing in the rate of current desensitization and an increase in the affinity for glycine. We conclude that the arylalkylamine toxins antagonize N-methyl-D-aspartate receptor currents by two distinct mechanisms: use-dependent and voltage-dependent open channel block and competitive antagonism at the N-methyl-D-aspartate recognition site. In addition, argiotoxin 636 exerts a polyamine-like facilitation of N-methyl-D-aspartate receptor currents. This facilitation occurs via both the glycine-dependent and glycine-independent mechanisms, whereby spermine is known to potentiate N-methyl-D-aspartate receptor responses.

Recent studies in Jurkat T cells and in rat basophilic leukemia cells revealed an Mg(2+)-inhibited cation (MIC) channel that has electrophysiological properties similar to TRPM7 Eyring rate model expressed exogenously in mammalian cells. Here we compare the characteristics of several polyvalent cations and Mg(2+) to block monovalent MIC current from the outside. Putrescine, spermidine, spermine, PhTX-343 (a derivative of the naturally occurring polyamine toxin philanthotoxin), and Mg(2+) each blocked in a dose- and voltage-dependent manner, indicating a blocking site within the electric field of the ion channel. Spermine and the relatively bulky PhTX-343 exhibited voltage dependence steeper than that expected for the number of charges on the molecule. Polyamines and Mg(2+) are permeant blockers, as judged by relief of block at strongly negative membrane potentials. Intracellular dialysis with spermine (300 microM) had no effect, indicating an asymmetrical pore. At the single-channel level, spermine and Mg(2+) induced flickery block of 40-pS single channels. I/V characteristics and polyamine block are similar in expressed TRPM7 and in native MIC currents, consistent with the conclusion that native MIC channels are composed of TRPM7 subunits. An Eyring rate model is developed to account for I/V characteristics and block of MIC channels by polyvalent cations from the outside.

Although the N-methyl-D-aspartate (NMDA) subtype of L-glutamate receptor is well characterized, the significance of non-NMDA glutamate-sensitive binding sites is not well documented. In this study, a new tricyclic quinoxalinedione (NBQX) and an arthropod toxin (philanthotoxin) were shown to block responses of spinal neurones in vivo to kainate, quisqualate, and AMPA in parallel but had little effect on responses to NMDA. Philanthotoxin appeared to be a use-dependent antagonist consistent with a channel-blocking mode of action. On cortical wedges in vitro, however, NBQX proved to be a more potent antagonist of AMPA and quisqualate than of kainate (pA2 values of 7.1, 7.0, and 5.6, respectively) with no effect at 10 μM on responses to NMDA. These studies provide evidence that on cortical neurones, but not on spinal neurones, AMPA and kainate depolarize by pharmacologically different mechanisms.Key words: glutamate receptors, quinoxalinediones, philanthotoxin, AMPA, kainate.

The α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are glutamate-gated cation channels that mediate fast excitatory synaptic transmission in the central nervous system. AMPARs are tetramers formed by homo- or heteromeric assembly of GluA1-4 subunits to produce multiple subtypes with varying biophysical properties. Polyamine toxins such as joro spider toxins, philanthotoxins (PhTXs), and argiotoxins are use-dependent ion channel blockers of AMPARs widely employed as highly potent antagonists of GluA2-lacking receptor subtypes. In addition to this use, recent findings have indicated that a philanthotoxin analog, PhTX-74, can distinguish among GluA2-containing AMPAR subtypes in the presence of the prototypical transmembrane AMPAR regulatory protein γ-2 (or stargazin). Thus, PhTX-74 may be of potential use in studies of the neurobiological role of GluA2-containing subtypes. We have evaluated the pharmacological profile of PhTX-74 and related polyamine toxins at homo- and heteromeric AMPARs in the presence and absence of γ-2. Determination of IC(50) values for inhibition of glutamate-evoked currents from Xenopus oocytes expressing recombinant homo- or heteromeric combinations of GluA1, GluA2, and GluA3 in the presence of γ-2 shows that PhTX-74 inhibits homomeric GluA1 and GluA3 receptors nonselectively, with IC(50) values in the nanomolar range (252-356 nM), and heteromeric GluA1/A2 and GluA2/A3 receptors nonselectively, with IC(50) values in the micromolar range (22 μM). Thus, in contrast to earlier findings, we find that PhTX-74 cannot pharmacologically discriminate between GluA2-containing AMPAR subtypes.

Background Ca2+-permeable non-N-methyl-D-aspartate receptors are found in the spinal dorsal horn and represent a presumptive target for glutamatergic transmission in nociceptive processing. This study characterized the analgesic profile associated with the blockade of these spinal receptors by intrathecally delivered agents known to act at these receptors, the spider venom Joro toxin (JST) and philanthotoxin. Methods Philanthotoxin (0.5, 2.5, or 5 microg) or JST (5 microg) was given spinally before thermal injury to the paw. JST (5 microg) was also given 10 min before subcutaneous formalin injection, after intraplantar administration of carrageenan, and to rats that were allodynic due to tight ligation of spinal nerves. Lower doses of JST (0.25 and 1.0 microg) were given before formalin injection and testing of thermal latencies. Thermal latencies were measured using a Hargreaves box, mechanical thresholds using von Frey hairs, and formalin response by means of counting flinches. Results Both agents blocked thermal injury-induced mechanical allodynia. JST (5 microg) given 1 h after carrageenan blocked induction of thermal hyperalgesia and mechanical allodynia. JST (5 microg) had no effect in the formalin test, on allodynia after spinal nerve ligation, or when given 3 h after carrageenan. The lowest dose (0.25 microg JST) at pretreatment intervals of 60-120 min resulted in modest hypoalgesia during phase 1 formalin and thermal testing. Conclusions The behavioral effect of intrathecal Ca2+-permeable non-N-methyl-D-aspartate antagonists indicates an important role for this spinal receptor in regulating hyperalgesic states induced by tissue injury and inflammation and reveals an action that is distinct from those observed with other glutamate receptor antagonists.

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.

1. The blocking action of δ-philanthotoxin (PhTX-4.3.3), a polyamine amide wasp toxin, and 33 structural analogues was studied at the nicotinic acetylcholine receptor of isolated neuronal somata from locust thoracic ganglia and at the cockroach cercal nerve-giant interneuron nicotinic cholinergic synapse. 2. A comparison of the structure-activity relationships reported here for the locust somal and cockroach synaptic nicotinic receptors and for the glutamatergic neuromuscular synapses of housefly larvae reported previously suggests a generally similar pharmacology for the channel-blocking action at ligand-activated ion channels, but with differences that might be due to variation in accessibility between synaptic receptors and those on the neuronal somata or to genuine divergence in ligand-activated ion channel pharmacology.

Voltage-dependent, non-competitive inhibition by philanthotoxin-343 (PhTX-343) analogues, with reduced charge or length, of nicotinic acetylcholine receptors (nAChR) of TE671 cells and ionotropic glutamate receptors (N-methyl-D-aspartate receptors (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR)) expressed in Xenopus oocytes from rat brain RNA was investigated. At nAChR, analogues with single amine-to-methylene or amine-to-ether substitutions had similar potencies to PhTX-343 (IC(50)=16.6 microM at -100 mV) whereas PhTX-(12), in which both secondary amino groups of PhTX-343 were replaced by methylenes, was more potent than PhTX-343 (IC(50)=0.93 microM at -100 mV). Truncated analogues of PhTX-343 were less potent. Inhibition by all analogues was voltage-dependent. PhTX-343 (IC(50)=2.01 microM at -80 mV) was the most potent inhibitor of NMDAR. At AMPAR, most analogues were equipotent with PhTX-343 (IC(50)=0.46 microM at -80 mV), apart from PhTX-83, which was more potent (IC(50)=0.032 microM at -80 mV), and PhTX-(12) and 4,9-dioxa-PhTX-(12), which were less potent (IC(50)s>300 microM at -80 mV). These studies show that PhTX-(12) is a selective nAChR inhibitor and PhTX-83 is a selective AMPAR antagonist.