|
Figure 1. Inhibition of acetylcholine-elicited currents (IAChs) by peimine (Pm). (A) Molecular structure of Pm showing the charged nitrogen. (B) Superimposed IAChs evoked by 10 µM ACh either alone (Ctr) or co-applied with different Pm concentrations, as stated on the right. Note that Pm accelerates IACh decay when applied at concentrations of 0.1 μM or above. Hereafter, unless otherwise stated, the holding potential was −60 mV, downward deflections represent inward currents and the bars above recordings indicate the timing of drug application. (C) Pm concentration-IACh inhibition relationship. IACh amplitudes at their peak (Ip; filled symbols) and at their steady state (Iss, measured 20 s after the peak; open symbols) were normalized to the IACh evoked by ACh alone and plotted against the logarithm of Pm concentration. Solid and dashed lines are sigmoid curves fitted to Ip and Iss data, respectively. Error bars indicate SEM. Each point is the average of 5–16 oocytes from 3–8 frogs.
|
|
Figure 2. Pharmacological profile of nAChR blockade by Pm. (A) Representative IAChs evoked by ACh at 10 µM (left), 100 µM (middle), or 1 mM (right), either alone (black recordings) or together with 3 µM Pm (red recordings). (B) ACh concentration-IACh amplitude relationship when the cell was bathed with either just ACh (black color; filled symbols denote Ip values whereas open circles correspond to Iss) or ACh plus 3 µM Pm (red symbols). The EC50 values of the sigmoid curves fitting the experimental data were 31 µM (range 11–52 µM) and 47 µM (range 46–49 µM) for control and 3 µM Pm, respectively. (C) Percentage of Ip (solid symbols) and Iss (open symbols) inhibition elicited by 3 µM Pm when co-applied with the indicated ACh concentrations. (*) indicates significant differences between Ip and Iss inhibition, for each ACh concentration (p < 0.05, paired t-test). (#) indicates significant differences among Iss inhibition at 10 µM ACh and other concentrations (p < 0.05, ANOVA followed by Bonferroni t-test). Each point of panels B and C is the average of 4–14 cells from 1–2 donors.
|
|
Figure 3. Pm accelerates IACh decay and shortens the time to reach Ip. (A1,A2) Superimposed IAChs elicited by 10 µM ACh either alone (black and grey recordings) or together with different Pm concentrations (shown at right). IAChs were scaled to the same Ip amplitude to better compare the differences in time to reach Ip (aTtP; A1) and kinetics of IACh decay after Ip (A2). (B1,B2) Column graphs displaying Pm effects on aTtP (B1) and τ-values of IACh-decay (B2). (*) indicates significant differences among IAChs in presence of Pm (colored columns; same color code as in (A1,A2)) and their control values (Ctr, black column; p < 0.05, ANOVA and Bonferroni t-test). Note that post-control values (after Pm applications; grey column) were similar to control ones. Each point is the average of 4–24 cells (N = 3–12).
|
|
Figure 4. IACh decay and deactivation kinetics depend on Pm concentration. (A1,A2,A3) Representative IAChs elicited by 100 µM ACh either alone (black recording) or together with 1 (orange) or 5 (purple) µM Pm (A1). Pm superfusion remained 12 s after ACh washout (as indicated by the application bars). These recordings were normalized to either the same Ip, to better compare their IACh decay (A2), or the same Iss, to facilitate comparisons of deactivation kinetics (A3). (B1,B2) Column bar plots displaying the effect of 1 (orange) or 5 µM (purple) Pm on the IACh decay time-constant (τDesensitization; (B1)) and the deactivation kinetics (τDeactivation; (B2)), as compared to control IAChs (in the presence of ACh alone; black). (*) indicates significant differences with the control group (p < 0.05, paired t-test) and (#) indicates differences between 1 and 5 µM Pm groups (n = 9, N = 3; the same cells for all comparisons; p < 0.05, paired t-test). Notice that Pm accelerated the desensitization rate and slowed down the deactivation kinetics.
|
|
Figure 5. nAChR blockade by Pm is voltage-dependent. (A) IAChs evoked by 10 µM ACh alone (black recording) or in the presence of either 1 (orange) or 5 µM (purple) Pm when applying voltage pulses from −120 to +60 mV, as illustrated underneath. (B) Net i/v relationship of IAChs elicited by the protocol shown in (A). Black symbols are for control IAChs, whereas those evoked in the presence of Pm are drawn in either orange (+1 µM Pm) or purple (+5 µM Pm). Net IAChs were normalized as the percentage of their control IACh at −60 mV (n = 5–11; N = 2–3). (C) Plot displaying the IACh remnant after co-application of either 1 (orange) or 5 µM (purple) Pm (IACh+Pm), normalized to their control (IACh), versus the membrane potential (same cells as in (B)). Notice that 1 µM Pm, in contrast to 5 µM, did not significantly decrease IACh at +60 mV.
|
|
Figure 6. Blockade of open nAChRs by Pm. (A,B) Superimposed IAChs elicited by 50 s pulses of 10 µM ACh either alone (black recordings) or together with 1 (A) or 5 µM (B) Pm, applied at the IACh plateau. Ips were normalized to the same amplitude to facilitate the kinetics comparisons. The kinetics of IACh inhibition and its recovery from blockade followed exponential functions (green traces (A,B)). (C) Column graph of the τ values found for IACh blockade onset (“On” columns) when 1 (orange) or 5 µM (purple) Pm was co-applied with 10 µM ACh. The “Off” columns correspond to the kinetics of recovery (τ) from blockade, following Pm removal (same color code). (*) indicates significant differences of τ values between Pm concentrations for either “On” or “Off” data (p < 0.05, ANOVA and Bonferroni t-test). (#) denotes differences between “On” and “Off” values for either 1 or 5 µM Pm (p < 0.05, paired t-test). Data are for 10 and 8 oocytes (N = 5) for 1 µM and 5 µM Pm, respectively.
|
|
Figure 7. Kinetics of the voltage-dependent blockade of nAChR by Pm. (A1,B1) IAChs elicited by 10 µM ACh either alone (black recordings) or in the presence of 1 (orange; (A1)) or 5 µM (purple; (B1)) Pm, at −60 mV. A 2 s voltage jump to +40 mV was given at the IACh plateau to unplug the channel pore of the positively-charged Pm. Membrane leak-currents (recorded in the absence of ACh) have been subtracted. (A2,B2) Zooming in to the area indicated by the arrows in panels (A1,B1) (just after the voltage jump). The τ of the voltage-dependent blockade of nAChRs by Pm was determined by fitting an exponential function (green curve over the recording) to the net IACh decay. Before fitting, the smaller and slower IAChs evoked by ACh alone (black recordings of panels (A1,B1)) were subtracted from the IAChs in the presence of Pm. (C) Column-graph of τ values of the voltage-dependent blockade of nAChR by 1 and 5 µM Pm (same color code as in panels (A,B)). (*) indicates significant differences of τ values between both Pm concentrations (p < 0.05, t-test). Data are for 10 (N = 3) and 7 (N = 2) oocytes for 1 µM and 5 µM Pm, respectively.
|
|
Figure 8. Effect of Pm-application timing and holding potential on nAChR blockade. (A1–A3) IAChs elicited at −60 mV (downward deflections) and at +40 mV (upward deflections) by co-application of 10 µM ACh and 1 µM Pm (A1), solely Pm pre-application before superfusing the agonist (A2) or Pm pre-application followed by its co-application with ACh (A3). (B1–B3) As in panels (A1–A3), but in the presence of 5 µM Pm instead of 1 µM. (C) Column graph shows the percentages of Ip inhibition by Pm when applied as indicated in panels (A1–A3,B1–B3), at −60 mV (on the left) and +40 mV (on the right). (*) indicates significant differences between Ip inhibition elicited by 1 and 5 µM Pm (p < 0.05, t-test). (#) denotes significant differences, for each Pm concentration, among the percentages of Ip inhibition elicited by ACh and Pm co-application and other Pm-application protocols, at the same holding potential (p < 0.05, ANOVA and Bonferroni t-test). Each column is the average of 5–11 and 5–17 oocytes, for −60 mV and +40 mV, respectively.
|
|
Figure 9. Predicted binding sites for Pm-nAChR complexes. (A,B) Lateral view of the nAChR displaying the main Pm (labelled in cyan) clusters bound to the open (A) and closed (B) conformations. The predicted loci are numbered consecutively, beginning in the transmembrane (TMD) and later in the extracellular (ECD) domain. (C, D) Top view of the nAChR (from the synaptic cleft) displaying representative Pm clusters binding to residues located within the channel pore, TMD, and ECD in the open (C) and closed (D) conformations. The inset, in the upper right corner, displays the nAChR subunits with their color code.
|
|
Figure 10. Analysis of MD simulations for Pm bound to the ECD and TMD of nAChR in open and closed conformations. Panels (A) (open conformation) and (B) (closed conformation) display the trajectory through a 100 ns simulation of a Pm molecule sited in each main cluster at the TMD and the ECD, as numbered in Figure 9A,B. Panels (C) (TMD, open), (D) (ECD, open), (E) (TMD, closed), and (F) (ECD, closed) display the free energy analysis (MM|PBSA) of the Pm-nAChR complexes at two MD trajectory intervals (40 to 100 ns and the last 30 ns). YASARA-calculated binding energy provides positive values when the predicted binding is strong and stable, whereas negative values indicate unstable or no binding.
|
|
Figure 11. Empty volume (left panels) and number of water molecules (right panels) within the hydrophobic gate of the nAChR pore region (between Val255 and Glu262 of the alpha subunit; see inset in the upper right corner), through the period of 40 to 100 ns of MD simulations. Top panels show the empty volume (A) and the number of water molecules (B) at the hydrophobic gate region in control nAChR (in the absence of Pm), both in the open (yellow) and the closed (blue) conformations; also, it displays the effect of ACh on the closed conformation (green). Middle panels (C,D) display the effect of Pm on these parameters when located at representative sites of the nAChR in the open conformation: within the channel pore (Pm 1), at the TMD (Pm 6), and the ECD (Pm 14). Lower panels (E,F) demonstrate the effect of Pm at representative loci of the nAChR in the closed conformation: inside the channel (Pm 1), at TMD (Pm 2), and at ECD (Pm 13).
|
