Subunit-Dependent Inhibition of Neuronal Nicotinic Acetylcholine Receptors by Philanthotoxins

Abstract

Philanthotoxin-433 (PhTX-433) is the active component of the Egyptian solitary digger wasp, Philanthus triangulum, venom which non-selectively inhibits several excitatory ion channels. To improve selectivity two synthetic analogues, Philanthotoxin-343 (PhTX-343) and Philanthotoxin-12 (PhTX-12), were developed. Previous work showed a 22-fold selectivity for PhTX-12 on embryonic muscle-type nicotinic acetylcholine receptors (M-nAChR) naturally expressed in the TE671 cell line in comparison to PhTX-343. In this study, we investigated the pharmacological action of both analogues on different mammalian hetero-oligomeric neuronal nicotinic acetylcholine receptor (N-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 values for PhTX-343 inhibition of α3β4, α3β2, α4β2 and α4β4 peak currents at −100 mV were 0.077 µM (n=9), 3.20 µM (n=8), 0.170 µM (n=7) and 0.28 µM (n=6) respectively; for PhTX-12 they were 2.03 µM (n=8), 36.0 µM (n=10), 0.430 µM (n=7) and 1.82 µM (n=9) respectively; i.e. in contrast to M-nAChR, PhTX-343 was more potent than PhTX-12 in all cases. The 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 weak voltage-dependence was observed. The variation in potency is most likely due to a single amino acid change in the β2/4 subunit pore lining region. Therefore, we conclude that PhTX-343 works as a potent open channel blocker of the mammalian heteromeric N-nAChR and has binding site deep in the channel while the PhTX-12 site is near to the outside of channel.