ABSTRACT: Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated ?-bungarotoxin ([125I]-?Bgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-?Bgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 ?M) than d-TC (IC50 = 0.39 ?M), while with ?7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 ?M and 7.77 ?M, respectively), but BBIQA2 was similar (IC50 = 5.52 ?M). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult ?1?1?? nAChR or human ?7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 ?M). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle ?1?1?? nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal ?3?2, ?4?2, and ?9?10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With ?3?2?2 GABAAR currents were almost completely inhibited by d-TC at a high (100 ?M) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-?-cobratoxin for binding to ?1?3?2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.
