Search

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.

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.

Calcium ions play an important role in many signalling pathways involved in normal cell metabolism. Pertrebations of normal Ca++ signalling may also play a pivotal role in the initiation of cell death. In these studies I have examined the influx of 45Ca++ into the extensor tibiae muscle of the locust (Schistocerca gregaria ). 45Ca++ entry could be stimulated by the addition of glutamate receptor-agonists or by activation of voltage activated calcium channels. L-glutamate, L-quisqualate and NMDA stimulated the influx of 45Ca++ while L-aspartate had only a small effect. DL-ibotenate, kainate, AMPA and glycine had no effect on 45Ca++ uptake (all agonists were tested at concentrations up to (100μM). Glycine (1μM) enhanced the 45Ca++ entry induced by NMDA and L-glutamate. Only the glycine potentiation of L-glu-stimulated responses was abolished in the presence of Mg++ (2mM) or AP5 (10μM) whereas the NMDA-stimulated response was completely abolished by these agents. These finding suggests that in the presence of glycine, L-glutamate may activate NMDA receptors and that in the absence of glycine L-glu-stimulated 45Ca++ entry occurs via activation of the qGluR. Depolarisation of the extensor tibiae muscles (50mM KCl) stimulated 45Ca++ influx by activation of voltage-sensitive calcium channels. Philanthotoxin-343 (0.1μM) had no effect on depolarisation activated calcium entry, however, nifedipine (1μM) an L-type calcium channel antagonist inhibited this Ca++ influx. Nifedipine did not inhibit L-glu-stimulated Ca++ entry suggesting that in these muscles L-type Ca++ channels are not involved in the Ca++ influx pathway following G1uR activation. Philanthotoxin-433 (PhTX-433) and many of its synthetic analogues are potent inhibitors of locust GluR. In the future these analogues may prove as useful potential neuroprotective agents or as novel pesticides. Over 100 analogues of PhTX-433 have been synthesized with changes made in the four regions of the structure, the thermospermine moiety, the tyrosyl moiety, the butyryl moiety and the terminal amino moiety. The effects of different concentrations (10-4M to 10-14M) of synthetic analogues of PhTX-433 (PhTX-343, PhTX-343-Arg, PhTX-4) were investigated in the 45Ca++ influx assay using locust extensor tibiae muscle. PhTX-343-Arg was more potent (IC50= - 7x10-9) than PhTX- 343 (IC50= - 10-8M) or PhTX-4 in blocking 45Ca H uptake. These findings were further supported by electrophysiological studies. The interaction of these synthetic analogues of philanthotoxin with GluR of locust muscle were further investigated by examining the effect of these compounds on evoked excitatory post synaptic potentials. In recent years control of ticks have been very important issue because of the social and economical damage they cause. Neuromuscular transmission is a main target site for the chemical control of many pests. Philanthotoxin and its analogues block the glutamate receptors which are involved in arthropod neuromuscular transmission and thus may prove useful as novel pesticides. The action of synthetic analogues of philanthotoxin (C7PhTX-343, DNP12-, PhTX-343 and PhTX-343) were examined on evoked excitatory postsynaptic potential in tick coxal muscle. These compounds all antagonized the evoked EPSP. C7PhTX-343 and DNP12-PhTX-343 exhibited same potency (IC50 = 10-8M) and both were more potent than PhTX-343 (IC50 ='2X10-5M). In recent years Xenopus oocyte has taken over a new role as a test tube for the study of the biogenesis, functional architecture and modulation of plasma membrane protein. Attempts were made to express mRNA from embryonic tissue of tick and locust leg muscle in to Xenopus oocyte for pharmacological studies. Xenopus oocytes failed to translate RNA faithfully and efficiently from these sources. Rat brain RNA injected oocytes used as control, expressed routinely.

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.

Philanthotoxin-433 (PhTX-433) is an active component of the Egyptian solitary digger wasp, Philanthus triangulum, venom which non-selectively inhibits several excitatory ion channels. With the aim of improving potency and selectivity, several synthetic analogues were developed based on the single or multiple modifications in the hydrophilic polyamine tail and hydrophobic aromatic head group of the PhTX. \ud In the first part of this study, we investigated the pharmacological actions of two synthetic analogues, Philanthotoxin-343 (PhTX-343) and Philanthotoxin-12 (PhTX-12), on mammalian hetero- and homooligomeric nicotinic acetylcholine receptor (nAChR) subunit combinations expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX-analogue were studied electrophysiologically using two-electrode voltage-clamp at three different membrane holding potentials (VH = -60 mV, -80 mV and -100 mV). Concentration-inhibition curves were constructed and IC50 values estimated for each holding potential. The IC50 (95% CI, n=oocytes) values for PhTX-343 inhibition of α3β4, α3β2, α4β2, α4β4, α7, α3β4(F255V), α4β2(V253F) and α1β1δγ peak currents at -100 mV were 0.07 µМ (0.05-0.10 µМ, n=9), 3.20 µМ (1.69-6.26 µМ, n=8), 0.14 µМ (0.09-0.21 µМ, n=7), 0.28 µМ (0.17-0.46 µМ, n=6), 8.7 µМ (6.9-11.0 µМ, n =9), 0.01 µМ (0.01-0.02 µМ, n=8), 0.10 µМ (0.06-0.16 µМ, n=8) and 3.2 µМ (2.5-4.1 µМ, n=9) respectively; for PhTX-12 they were 2.03 µМ (1.22-3.26 µМ, n=8), 74.0 µМ (21-259 µМ, n=10), 1.6 µМ (0.7-3.6 µМ, n=11), 1.82 µМ (0.81-4.11 µМ, n=9), 12.1 µМ (9.0-16.0 µМ, n =10), 1.0 µМ (0.5-2.0 µМ, n=5), 3.4 µМ (1.3-8.7 µМ, n=9) and 5.0 µМ (2.8-8.8 µМ, n=8) respectively; i.e. in contrast to M-nAChR, PhTX-343 was more potent than PhTX-12 in all cases. All IC50s were lower when inhibition was measured after 1 minute of application indicating use dependence. For inhibition of heteromeric nAChRs, the peak potency of PhTX-343 was strongly augmented by holding the cell at more negative VH while this was not the case for PhTX-12 where only weak voltage-dependence was observed. The inhibition of homomeric α7 receptors by PhTX-343 was voltage-independent, whereas the block by PhTX-12 was voltage-dependent. In addition, these two synthetic analogues were different in their recovery rates (PhTX-12 was generally faster than PhTX-343) but they were similar in their mechanism of action, non-competitive inhibition.\ud In the second part of the project, I explored the structure-activity relations of twenty one synthetic analogues, with a view to improving their activity and selectivity on rat neuronal α4β2 and α3β4 nAChRs at VH = -100 mV. We showed that the presence of positive charge in the polyamine tail of PhTX compounds is essential for nAChR subtype selectivity and its removal made the molecule lose its selectivity. Also, it appears that adding a bulky group to the terminal ammonium drastically reduced activity whereas a similar addition to the head region it increased their potency. In addition, we identified the key regions and substitutions responsible for increasing PhTX activity, cyclohexylalanine in place of tyrosine, and selectivity, phenyl group. Analogues having cyclohexylalanine and a phenolic group in the head region showed IC50 values in the low nano-molar and pico-molar (160-400 pM) range. These data suggest that PhTXs could serve as lead compounds for highly potent and selective inhibitors of N-nAChRs.

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.

We elucidated the mechanisms underlying the kainate receptor (KAR)-mediated facilitatory modulation of synaptic transmission in the cerebellum. In cerebellar slices, KA (3 μM) increased the amplitude of evoked excitatory postsynaptic currents (eEPSCs) at synapses between axon terminals of parallel fibers (PF) and Purkinje neurons. KA-mediated facilitation was antagonized by NBQX under condition where AMPA receptors were previously antagonized. Inhibition of protein kinase A (PKA) suppressed the effect of KA on glutamate release, which was also obviated by the prior stimulation of adenylyl cyclase (AC). KAR-mediated facilitation of synaptic transmission was prevented by blocking Ca2+ permeant KARs using philanthotoxin. Furthermore, depletion of intracellular Ca2+ stores by thapsigargin, or inhibition of Ca2+-induced Ca2+-release by ryanodine, abrogated the synaptic facilitation by KA. Thus, the KA-mediated modulation was conditional on extracellular Ca2+ entry through Ca2+-permeable KARs, as well as and mobilization of Ca2+ from intracellular stores. Finally, KAR-mediated facilitation was sensitive to calmodulin inhibitors, W-7 and calmidazolium, indicating that the increased cytosolic [Ca2+] sustaining KAR-mediated facilitation of synaptic transmission operates through a downstream Ca2+/calmodulin coupling. We conclude that, at cerebellar parallel fiber-Purkinje cell synapses, presynaptic KARs mediate glutamate release facilitation, and thereby enhance synaptic transmission through Ca2+-calmodulin dependent activation of adenylyl cyclase/cAMP/protein kinase A signaling.

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.