Fig 1.
Chemical structures of d-tubocurarine (d-TC) and bisbenzyltetrahydroisoquinoline alkaloids (BBIQAs).
Fig 2.
Inhibition of [125I]-labeled α-bungarotoxin binding.
(a) to A. californica AChBP, (b) to nAChR from Torpedo californica electric organ membranes, and (c) to human α7 nAChR in GH4C1 cells with BBIQA1 (open blue triangles), BBIQA2 (open red squares), BBIQA3 (open green rhombuses), and d-TC (open black circles). Data are mean ± SEM of two biological replicates with duplicates for each point (the number of technical replicates is 2), i.e. n = 4. IC50 values derived from these data are shown in Table 1.
Table 1.
Inhibitory effects of the alkaloids from Matis Dart Poison and d-TC on [125I]-αBgt binding, agonist-induced calcium responses, and/or currents mediated by A. californica AChBP, T. californica nAChR, mouse adult α1β1εδ nAChR, human α7 nAChR, and mouse 5-HT3AR.
Fig 3.
Dose-response curves of inhibitory activity of d-TC (open black circles), BBIQA1 (open blue triangles), and BBIQA2 (open red squares).
(a) on the 30 μM (approx. EC50) acetylcholine-evoked intracellular calcium ion concentration ([Ca2+]i) rise in Neuro2a cells expressing mouse adult α1β1εδ nAChRs; and (b) on the 10 μM (approx. EC50) acetylcholine-evoked [Ca2+]i rise in Neuro2a cells expressing human α7 nAChRs in the presence of 10 μM PNU120596. Data are presented as mean ± SEM, n = 3. The respective IC50 values are shown in the Table 1. (c) Dose-response curves of acetylcholine (ACh)-evoked [Ca2+]i rise in the absence (grey circles, EC50 = 8.4 ± 0.6 μM) and presence of d-TC (black circles) or its analogs, BBIQA1 (blue triangles), and BBIQA2 (red squares) at different concentrations in Neuro2a cells expressing mouse adult α1β1εδ nAChRs. Data are presented as mean ± SEM, n = 3. EC50 values are shown in the Table 2.
Table 2.
Inhibitory effects of the alkaloids from Matis Dart Poison and d-TC at different concentrations (IC50 and 3x IC50) on the acetylcholine dose–response curve at mouse adult α1β1εδ nAChR expressed in Neuro2a cells.
Fig 4.
Activity of d-TC, BBIQAs1 and 2 against heteromeric neuronal nAChRs, GABAAR, and 5-HT3AR.
(a) Representative current traces of human α3β2 nAChR, showing inhibition of nicotine (50 μM)-induced current by 10 μM d-TC, BBIQA1, or BBIQA2. (b) Bar graph for d-TC and BBIQAs (1 and 10 μM) inhibition of agonist-evoked currents mediated by human α3β2 (50 μM Nicotine), rat α4β2 (10 μM Nicotine), and human α9α10 (25 μM Acetylcholine) nAChRs. (c) Bar graph for d-TC and BBIQAs (100 μM) inhibition of agonist-evoked currents mediated by mouse α3β2γ2 GABAAR (100 μM GABA). (d) Inhibition of Alexa Fluor 546 α-cobratoxin (αCtx, 50 nM) binding to α1β3γ2 GABAAR expressed in Neuro2a cells by 50 μM d-TC, BBIQAs 1 and 2. The bar graph represents the remaining fluorescence of Alexa Fluor 546 αCtx (50 nM). In (a, b, c, d) data are presented as mean ± SEM, n = 3–6. One-way ANOVA with Tukey’s HSD test, (black asterisks, p<0.05, normalized current evoked by agonist in the presence of d-TC, BBIQA1, or BBIQA2 vs normalized current induced by agonist in the absence of antagonists). (e) Dose-response curves of d-TC (open black circles), BBIQA1 (open blue triangles), or BBIQA2 (open red squares) inhibitory action on 1 μM 5-HT-evoked ion currents mediated by mouse 5-HT3AR. Data are presented as mean ± SEM, n = 3–5. IC50 values determined from these data are shown in Table 1.