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FIGURE 1. 2,6-Dimethylaniline (DMA) inhibits ACh-induced currents (IAChs). (A) Molecular structures of lidocaine and DMA, showing the resemblance of DMA to the phenolic ring of lidocaine. (B) Superimposed IAChs, recorded in the same nAChR-bearing oocyte, by application of 10 μM ACh either alone (Control) or together with DMA, at the indicated concentrations. In this and following figures, unless otherwise stated, the holding potential was -60 mV, downward deflections denote inward currents and the horizontal bar above records corresponds to the timing of drug application. (C) DMA concentration-IACh inhibition relationship. Amplitude of the IAChs evoked in presence of DMA was normalized to the IACh elicited by ACh alone (Control) and plotted as a function of the logarithm of the DMA concentration. Solid line is a sigmoid curve fitted to the data and error bars are SEM. Each point is the average of 4–28 oocytes from 3 to 13 frogs.
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FIGURE 2. Slow recovery from nAChR blockade by DMA. (A) Superimposed IAChs evoked sequentially, in the same oocyte, by superfusing the cell with 10 μM ACh alone [(1), black bar and recording], co-applied with 2 mM DMA [(2), green bar and recording] or when changing from ACh plus DMA to ACh alone at the time indicated by the bars [(3), green followed by black bars and blue recording]. Note the incomplete recovery of IACh amplitude after washing DMA for 20 s. (B) Superimposed currents obtained by superfusing one oocyte with 10 μM ACh alone (Control, black recording) or plus DMA (+ 2 mM DMA, green recording). Seven min after DMA withdrawal (Postcontrol, gray recording), IACh did not fully reach the control amplitude. (C) Column graph showing the percentages of IACh recovery after 20 s or 7 min from DMA washout. Asterisks indicate significant differences respect to the control response.
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FIGURE 3. IACh blockade by DMA lacks of voltage dependence. (A) Whole membrane currents (upper traces) evoked by applying to an oocyte the voltage protocol shown on bottom, during the current plateau elicited by 10 μM ACh, either alone (black) or with 2 mM DMA (green). (B) Net i/v relationships for IACh, obtained by applying the voltage protocol shown in (A) while superfusing the cells with 10 μM ACh either alone (black filled circles) or co-applied with 2 mM DMA (green open circles). Values represent the percentage of current referred to their control IACh at -60 mV; each point is the average of 5 cells (N = 3). (C) Plot showing the fraction of plateau IACh left by 2 mM DMA (IACh+DMA), normalized to its control (IACh), versus the membrane potential. Same cells than in (B). Note the lack of a clear voltage dependence of IACh blockade by DMA. The dashed red line shows the best linear fit to the data; the fitted line has a correlation coefficient of -0.21, giving a p of 0.58 (the probability for the t-test of the slope = 0).
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FIGURE 4. IACh rebound elicited by DMA washout. When an oocyte was challenged with a high ACh concentration (1 mM), while holding its membrane potential at -60 mV (Vh = -60 mV), the IACh showed a marked desensitization and a noticeable rebound-current (A2, black recording and arrow) when the agonist was rinsed. By contrast, both when applying the same ACh concentration to the cell at a membrane potential of +40 mV (A1, black recording), or when decreasing the ACh concentration to 10 μM (B, black recording), the IACh rebound was not evoked. However, when ACh was co-applied with 2 mM DMA the IACh rebound was evident at any potential or ACh concentration tested (A1,A2,B, green recordings and arrows).
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FIGURE 5. 2,6-Dimethylaniline effects on IACh decay and time-to-peak. (A) Superimposed IACh recordings evoked by application of 100 μM ACh either alone (black recoding) or plus 2 mM DMA (green recording) and by re-applying 100 μM ACh alone 7 min after DMA washout (Postcontrol, gray trace overlapping the control one). Note that all IACh amplitudes have been scaled to the same size to better showing differences on IACh desensitization. Inset shows, at an expanded temporal scale, the IACh peaks elicited by ACh either alone or co-applied with DMA. (B) Plots showing the percentage of IACh decay obtained at different times (2, 10, and 20 s) after IACh peak. Data were measured from recordings as those shown in (A), by applying 100 μM ACh either alone (Control, filled circles and continuous black line; Postcontrol, filled triangles and dashed black line) or plus 2 mM DMA (open circles and continuous green line). (C) Column graph showing the IACh time-to-peak values when applying 100 μM ACh either alone (Control and Postcontrol, empty columns) or together with 2 mM DMA (filled green column). Values of n and N, given in each column, are common to (B,C); in both panels, asterisks indicate significant differences among groups (p < 0.05, ANOVA and Bonferroni t-test). (D) Plot displays the DMA dose-dependence of IACh decay hastening. Desensitization values (Dtis) at 2 (orange), 10 (pink) and 20 s (violet) from IACh peaks, elicited by co-applying 100 μM ACh with 100, 200, 500, or 2000 μM DMA, were expressed as percentage respect to their control Dtis and plotted against the log of DMA concentration. Each point is the average of 4–12 oocytes from three frogs. Asterisks of different colors indicate significant differences respect to the control values for the color-coded time (p < 0.05, one sample t-test). Inset shows superimposed recordings evoked by 100 μM ACh either alone or together with 200 μM DMA; recording colors are as in (A) and IACh amplitudes have also been scaled to the same size.
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FIGURE 6. IACh decay hastening elicited by DMA is dependent on ACh concentration. (A) Superimposed recordings of IAChs elicited by 10 μM (A1) or 1 mM (A2) ACh either alone (black recordings) or co-applied with 2 mM DMA (green recordings) in oocytes with the membrane potential held at +40 mV. IACh decays were fitted to exponential curves (red discontinuous lines) and the time constant (τ) values for each group were determined. (B). Column graph of τ values for IACh decays. Data of each column are mean ± SEM from 4 to 6 oocytes (N = 3). When co-applying DMA and 1 mM ACh, the IACh decay was best fitted to double exponential curves and the τ value shown in B corresponds to the fast component. Note that both control IACh amplitude and desensitization rate increased with ACh concentration (see black recordings in A1,A2; B, open columns) and mind the presence of rebound-currents when ACh and DMA were co-applied (green records), independently of the ACh dose used. Observe that DMA co-application caused a stronger blocking effect at low (A1) than at high (A2) ACh concentrations and that DMA enhancement of the rate of IACh decay was greater for higher ACh doses (compare recordings of A1, A2; B). The asterisk indicates significant differences between both DMA groups (p < 0,05, t-test), and the pound sign denotes that this column is truncated, because IAChs elicited by 10 μM ACh showed almost no desensitization.
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FIGURE 7. 2,6-Dimethylaniline effects on ACh concentration-IACh amplitude relationship. (A)
IACh recordings evoked by applying, successively, ACh at increasing concentrations (10, 100 μM, and 1 mM) either alone (black traces) or co-applied with 2 mM DMA either directly (A1, green recordings) or after being pre-applied for 12 s (A2, red recordings). (B) Averaged ACh concentration-IACh amplitude curves obtained following the experimental protocol shown in (A). Black filled circles are for ACh alone (n = 10–23, N = 4–5), green open circles for co-application of ACh plus 2 mM DMA (n = 4–5, N = 3) and red open circles when ACh and DMA co-application was preceded by 12 s of 2 mM DMA pre-application (n = 4–7, N = 2–4). All data were normalized to the maximal IACh elicited by ACh alone and fitted to the Hill equation (continuous lines). (C) Plot showing the percentage of IACh inhibition at different ACh concentrations when ACh was directly co-applied with 2 mM DMA (open green circles and solid line; n = 4–28, N = 4–10), or when ACh and 2 mM DMA co-application was preceded by 12 s DMA pre-application (open red circles; n = 5–15, N = 4–9). Asterisks indicate significant differences (p < 0.05, ANOVA and Bonferroni t-test) respect to the IACh blockade caused by solely co-applying 10 μM ACh and 2 mM DMA; pound signs indicate significant differences (t-test), for each ACh dose, between the IACh blockade caused by direct co-application of ACh with DMA and when it was preceded by a 12 s DMA application. The dashed line indicates 50% inhibition. Note the reduction of IACh inhibition when DMA was co-applied with high ACh concentrations (1 mM) and the strong IACh blockade when DMA was pre-applied before its co-application with high ACh concentrations.
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FIGURE 8. Additive inhibitory effects of DMA and DEA on IACh. (A1–A3) Representative IACh recordings obtained when superfusing the oocyte with 10 μM ACh either alone (A1–A3; Control, black) or co-applied with 70 μM DEA (A1; + 70 μM DEA, orange), 2 mM DMA (A2; + 2 mM DMA, green) or 70 μM DEA plus 2 mM DMA (A3; + 70 μM DEA + 2 mM DMA, red). (B) Column graph showing the average IACh inhibition elicited by co-application of 10 μM ACh with the different combinations showed in (A) as indicated below each column. The two right most columns show the values predicted by the allotopic and syntopic models of interaction (see text for details). The asterisks above the bars indicate significant differences between groups (p < 0.05, t-test; comparisons of DEA + DMA values with those estimated by each model of inhibition were carried out with one-sample t-test).
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FIGURE 9. Similar pharmacological profile of nAChRs in the presence of either combined DEA and DMA or lidocaine. (A)
IACh recordings elicited by applying, sequentially, ACh at increasing concentrations (10, 100 μM, and 1 mM) either alone (black traces) or co-applied with 1 mM DMA and 30 μM DEA (red traces). (B) Averaged ACh concentration-IACh amplitude curves attained following the experimental protocol shown in (A). Black filled circles are for ACh alone (n = 4–7, N = 1) and red open circles when ACh was co-applied with DMA and DEA (same cells than the control curve). All data were normalized to the maximal IACh elicited by ACh alone and fitted to Eq. (3). Continuous black and red lines are the fitted curves, labeled as ControlDEA+DMA and DEA+DMA, respectively. Added to this plot are the values we reported for the dose-response curves of nAChRs activated by ACh either alone (gray symbols and discontinuous line; ControlLid) or in the presence of 70 μM lidocaine (orange circles and discontinuous line; Lid; data from Alberola-Die et al., 2011). Notice the similarities among both control curves and between DEA+DMA and lidocaine curves.
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FIGURE 10. Modeling of DMA binding to nAChR EC- and TM-domains in the open and closed states. (A) Lateral view, in the membrane plane (top corresponding to the EC side) of nAChR, in the closed state, with bound DMA molecules. Subunits are colored for this and following panels as follows: α1 (blue), α2 (cyan), β (magenta), γ (orange), and δ (green). DMA molecules are colored brown and represented as van der Waals spheres. Notice that DMA binds both at the EC and TM domains. The red arrow indicates the orthosteric binding site at the α1-γ interface. (B1,C1) Top view (from the synaptic cleft) of nAChR structures in the closed (B1) and open (C1) states with bound DMA molecules. Note that, when closed, at the EC domain, DMA binds to intrasubunit loci (arrows in B1), mainly located on α1, α2 and β subunits, whereas at the TM domain DMA preferentially interact with residues located at intersubunit crevices. Also note that DMA binds within the channel pore only on nAChRs in the open state (red circle in C1). (B2,C2) Expanded top view of nAChR TM domains in the closed (B2) and open (C2) states with bound DMA. Note that whereas in the closed state DMA binds at all intersubunit assemblies (arrows in B2), in the open state these binding sites were less favored.
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