Abstract
Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 inhibits several excitatory ligand-gated ion channels, and to improve selectivity two synthetic analogues, PhTX-343 and PhTX-12, were developed. Previous work showed a 22-fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3β4 nAChRs were most sensitive to PhTX-343 (IC50 = 12 nM at −80 mV) with α4β4, α4β2, α3β2, α7 and α1β1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1β1γδ was most sensitive to PhTX-12 along with α3β4 (IC50 values of 100 nM) with α4β4, α4β2, α3β2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage dependence was observed. We conclude that PhTX-343 mainly acts as an open-channel blocker of nAChRs with strong subtype selectivity.
Highlights
Philanthotoxin-433 (PhTX-433; Fig. 1) is a polyamine-based toxin found in the venom of the Egyptian digger wasp, Philanthus triangulum, used to paralyse insect prey by inhibiting nicotinic acetylcholine receptors and ionotropic glutamate receptors[1,2]
Based on the observation that in both ionotropic receptor types the inhibition by PhTX-343 is use- and voltage-dependent, it has been proposed that the dominant mode of action involves an open-channel blocking mechanism whereby the polyamine tail penetrates deep into the channel pore where the environment is hydrophilic, while the head group interacts with the more hydrophobic outer parts of the pore[9,10,11]
The results presented in this investigation show that the mechanism of inhibition and recovery of nicotinic acetylcholine receptors (nAChRs) in response to PhTX-343 is strongly influenced by their subunit combination
Summary
Philanthotoxin-433 (PhTX-433; Fig. 1) is a polyamine-based toxin found in the venom of the Egyptian digger wasp, Philanthus triangulum, used to paralyse insect prey by inhibiting nicotinic acetylcholine receptors (nAChRs) and ionotropic glutamate receptors (iGluRs)[1,2]. Apart from its inhibitory action on insect nAChRs and iGluRs, PhTX-433 and its closely related synthetic analogue, PhTX-343 (Fig. 1), exhibit potent activity at vertebrate ionotropic receptors, and their receptor interactions have been quite extensively characterized for mammalian iGluRs, including the AMPA, kainate and NMDA receptor subtypes[3,4,5,6], as well as for vertebrate muscle-type nAChRs7,8 These investigations have inferred that philanthotoxins (PhTXs) display some selectivity towards iGluRs over nAChRs. Based on the observation that in both ionotropic receptor types the inhibition by PhTX-343 is use- and voltage-dependent (i.e. more potent inhibition is observed with increasing negative membrane potentials), it has been proposed that the dominant mode of action involves an open-channel blocking mechanism whereby the polyamine tail penetrates deep into the channel pore where the environment is hydrophilic, while the head group interacts with the more hydrophobic outer parts of the pore[9,10,11]. A single study has investigated the effects of PhTX-343 at nAChRs expressed by PC12 cells, showing that it potently antagonised responses to ACh in a voltage-dependent manner[14]
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