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Muscle-Type Nicotinic Receptor Modulation by 2,6-Dimethylaniline, a Molecule Resembling the Hydrophobic Moiety of Lidocaine

View Article: PubMed Central - PubMed

ABSTRACT

To identify the molecular determinants responsible for lidocaine blockade of muscle-type nAChRs, we have studied the effects on this receptor of 2,6-dimethylaniline (DMA), which resembles lidocaine’s hydrophobic moiety. Torpedo marmorata nAChRs were microtransplanted to Xenopus oocytes and currents elicited by ACh (IACh), either alone or co-applied with DMA, were recorded. DMA reversibly blocked IACh and, similarly to lidocaine, exerted a closed-channel blockade, as evidenced by the enhancement of IACh blockade when DMA was pre-applied before its co-application with ACh, and hastened IACh decay. However, there were marked differences among its mechanisms of nAChR inhibition and those mediated by either the entire lidocaine molecule or diethylamine (DEA), a small amine resembling lidocaine’s hydrophilic moiety. Thereby, the IC50 for DMA, estimated from the dose-inhibition curve, was in the millimolar range, which is one order of magnitude higher than that for either DEA or lidocaine. Besides, nAChR blockade by DMA was voltage-independent in contrast to the increase of IACh inhibition at negative potentials caused by the more polar lidocaine or DEA molecules. Accordingly, virtual docking assays of DMA on nAChRs showed that this molecule binds predominantly at intersubunit crevices of the transmembrane-spanning domain, but also at the extracellular domain. Furthermore, DMA interacted with residues inside the channel pore, although only in the open-channel conformation. Interestingly, co-application of ACh with DEA and DMA, at their IC50s, had additive inhibitory effects on IACh and the extent of blockade was similar to that predicted by the allotopic model of interaction, suggesting that DEA and DMA bind to nAChRs at different loci. These results indicate that DMA mainly mimics the low potency and non-competitive actions of lidocaine on nAChRs, as opposed to the high potency and voltage-dependent block by lidocaine, which is emulated by the hydrophilic DEA. Furthermore, it is pointed out that the hydrophobic (DMA) and hydrophilic (DEA) moieties of the lidocaine molecule act differently on nAChRs and that their separate actions taken together account for most of the inhibitory effects of the whole lidocaine molecule on nAChRs.

No MeSH data available.


Related in: MedlinePlus

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 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.

Mentions: When either 10 or 100 μM ACh were co-applied with DMA, at roughly its IC50, IACh decays were significantly accelerated with respect to those evoked by ACh alone, suggesting an enhancement of nAChR desensitization by DMA. Dti values at 2 and 20 s (see Eq. (2) in Materials and Methods) were: 36 ± 5% and 92 ± 1%, for 100 μM ACh alone versus 50 ± 5% and 99 ± 1% for 100 μM ACh plus 2 mM DMA, respectively (same cells in both groups; n = 18, N = 14; p < 0.05, ANOVA; see Figures 5A,B). This effect was fully reverted 7 min after DMA rinsing with ANR (40 ± 5% and 93 ± 2%; p > 0.05, ANOVA, Figure 5B). Additionally, DMA diminished the apparent time-to-peak, i.e., the time elapsed from IACh onset to IACh peak, from 1.6 ± 0.2 s for 100 μM ACh alone to 1.1 ± 0.2 s for 100 μM ACh plus 2 mM DMA (same cells that IACh decay measurements; p < 0.05, ANOVA; Figures 5A,C). Noteworthy, the time-to-peak reverted to control values 7 min after DMA washout (1.6 ± 0.3 s; see Postcontrol of Figures 5A,C), as the IACh decay rate did. Interestingly, DMA hastening of IACh decay was dose-dependent, starting the increase of desensitization at concentrations as low as 100 μM DMA (Figure 5D; p < 0.05, one sample t-test).


Muscle-Type Nicotinic Receptor Modulation by 2,6-Dimethylaniline, a Molecule Resembling the Hydrophobic Moiety of Lidocaine
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 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.
Mentions: When either 10 or 100 μM ACh were co-applied with DMA, at roughly its IC50, IACh decays were significantly accelerated with respect to those evoked by ACh alone, suggesting an enhancement of nAChR desensitization by DMA. Dti values at 2 and 20 s (see Eq. (2) in Materials and Methods) were: 36 ± 5% and 92 ± 1%, for 100 μM ACh alone versus 50 ± 5% and 99 ± 1% for 100 μM ACh plus 2 mM DMA, respectively (same cells in both groups; n = 18, N = 14; p < 0.05, ANOVA; see Figures 5A,B). This effect was fully reverted 7 min after DMA rinsing with ANR (40 ± 5% and 93 ± 2%; p > 0.05, ANOVA, Figure 5B). Additionally, DMA diminished the apparent time-to-peak, i.e., the time elapsed from IACh onset to IACh peak, from 1.6 ± 0.2 s for 100 μM ACh alone to 1.1 ± 0.2 s for 100 μM ACh plus 2 mM DMA (same cells that IACh decay measurements; p < 0.05, ANOVA; Figures 5A,C). Noteworthy, the time-to-peak reverted to control values 7 min after DMA washout (1.6 ± 0.3 s; see Postcontrol of Figures 5A,C), as the IACh decay rate did. Interestingly, DMA hastening of IACh decay was dose-dependent, starting the increase of desensitization at concentrations as low as 100 μM DMA (Figure 5D; p < 0.05, one sample t-test).

View Article: PubMed Central - PubMed

ABSTRACT

To identify the molecular determinants responsible for lidocaine blockade of muscle-type nAChRs, we have studied the effects on this receptor of 2,6-dimethylaniline (DMA), which resembles lidocaine&rsquo;s hydrophobic moiety. Torpedo marmorata nAChRs were microtransplanted to Xenopus oocytes and currents elicited by ACh (IACh), either alone or co-applied with DMA, were recorded. DMA reversibly blocked IACh and, similarly to lidocaine, exerted a closed-channel blockade, as evidenced by the enhancement of IACh blockade when DMA was pre-applied before its co-application with ACh, and hastened IACh decay. However, there were marked differences among its mechanisms of nAChR inhibition and those mediated by either the entire lidocaine molecule or diethylamine (DEA), a small amine resembling lidocaine&rsquo;s hydrophilic moiety. Thereby, the IC50 for DMA, estimated from the dose-inhibition curve, was in the millimolar range, which is one order of magnitude higher than that for either DEA or lidocaine. Besides, nAChR blockade by DMA was voltage-independent in contrast to the increase of IACh inhibition at negative potentials caused by the more polar lidocaine or DEA molecules. Accordingly, virtual docking assays of DMA on nAChRs showed that this molecule binds predominantly at intersubunit crevices of the transmembrane-spanning domain, but also at the extracellular domain. Furthermore, DMA interacted with residues inside the channel pore, although only in the open-channel conformation. Interestingly, co-application of ACh with DEA and DMA, at their IC50s, had additive inhibitory effects on IACh and the extent of blockade was similar to that predicted by the allotopic model of interaction, suggesting that DEA and DMA bind to nAChRs at different loci. These results indicate that DMA mainly mimics the low potency and non-competitive actions of lidocaine on nAChRs, as opposed to the high potency and voltage-dependent block by lidocaine, which is emulated by the hydrophilic DEA. Furthermore, it is pointed out that the hydrophobic (DMA) and hydrophilic (DEA) moieties of the lidocaine molecule act differently on nAChRs and that their separate actions taken together account for most of the inhibitory effects of the whole lidocaine molecule on nAChRs.

No MeSH data available.


Related in: MedlinePlus