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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.

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.

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.

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 IC50S 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.