Article Figures & Data
Figures
- Fig. 1.
Paralyzing effects of bephenium on C. elegans. Chemical structures of bephenium and levamisole are shown at the top of the figure. Assays were carried out on agar plates with synchronized populations of C. elegans at 20–22°C. (A) Dose-response curve for the paralyzing effect of bephenium (1-hour exposure) on wild-type C. elegans (N2 strain). The percentage of paralyzed animals was related to the control condition in the absence of bephenium and in the presence of the corresponding amount of dimethyl sulfoxide. Body paralysis was defined as the lack of complete body movement in response to prodding. Values are mean percentages ± S.D. (B) Comparison of the percentage of paralyzed animals containing (wild-type) or lacking L-AChRs (unc38(e264)I) after exposure of 1 or 2 hours to 3 mM bephenium. Values are mean percentages ± S.D. derived from seven dishes per condition. *** Indicates statistically significant differences with respect to the wild-type condition (P < 0.001). (C) Percentage of paralyzed worms exposed for 1 or 2 hours to 0.03 mM levamisole, 1 mM bephenium, or the two drugs combined. Values are mean percentages ± S.D. derived from three simultaneous assays for all conditions and two complete experiments performed on separate days. Statistical comparisons between conditions with drug combination and individual drugs (***P < 0.001; **P < 0.01; *P < 0.05).
- Fig. 2.
Single-channel currents elicited by bephenium from cultured L1 muscle cells. (A) Traces of single-channel activity in the presence of 50 μM ACh or bephenium from wild-type L1 muscle cells are shown at two different temporal scales. At the right, representative open- and closed-duration histograms are shown for each condition. Channel openings are shown as upward deflections. Pipette potential: 100 mV. (B) Traces of single-channel activity in the presence of 30 μM bephenium at the indicated holding potential are shown filtered at 9 kHz with channel openings as upward deflections. Middle: Corresponding amplitude histograms. Right: Current-voltage relationship. The conductance was obtained by linear regression analysis. Each point corresponds to the mean ± S.D. of three different recordings at each condition. (C) Continuous recordings from muscle cells derived from the lev-8(x15) mutant strain at 50 μM ACh and 30 μM bephenium. Pipette potential: 100 mV. Filter: 9 kHz. Single-channel activity of L-AChRs lacking LEV-8 decreases markedly with time and occurs in clusters.
- Fig. 3.
Activation of L-AChR as a function of bephenium concentration. (A) L-AChR channels were recorded from cultured muscle cells of PD4251 worms at different bephenium concentrations and are shown at different temporal scales. Filter: 9 kHz. Holding potential: 100 mV. Representative open duration histograms are shown. Channel openings are shown as upward deflections. (B) Relationship between the reciprocal of the mean open duration (s−1) as a function of bephenium concentration. The slope of the curves fitted by linear regression estimates the value of the forward blocking constant. Each point corresponds to the mean ± S.D. of three different patches for each condition.
- Fig. 4.
Activation of mammalian nAChR by bephenium. Single-channel recordings from BOSC cells transfected with mammalian α1, β, ε, and δ subunit cDNAs. (A) Mammalian nAChR channels activated by 30 μM ACh or bephenium are shown at two different time scales. In the presence of ACh, clusters of channel activity can be clearly distinguished. A representative cluster is shown at the lower amplitude resolution. No clusters are distinguished in the presence of bephenium. At the right, representative open-time histograms are shown for each condition. Holding potential: 70 mV. Channels are shown as upward deflections. (B) Mammalian nAChR channels activated by bephenium at different concentrations. No clusters are distinguished at any concentration. At the right, representative open-time histograms are shown. Holding potential: 70 mV. Channels are shown as upward deflections. (C) Single channel currents in the presence of 30 μM ACh alone or together with 30 μM bephenium were recorded at a pipette potential of +70 mV (top) or −70 mV (bottom). Channel openings are shown as upward deflections at +70 mV and as downward deflections at −70 mV.
- Fig. 5.
Molecular docking of bephenium into homology-modeled binding-site interfaces. Homology models of the extracellular domain of two adjacent subunits were created using the structure of PDB 5KXI [(A) and (C), loop C closed] and PDB 5AFM [(B) and (D), loop C partially closed]. (A) and (B) correspond to the LEV-8/UNC-38 interface and (C) and (D) to α1/ε interface. Loops from the principal face are shown in green and from the complementary face in blue. Bephenium is shown in yellow. Cation-π interactions are shown in orange, π-π in pink, H-bonds in light blue, π-sulfur in red, and π-alkyl in purple.
