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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.


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 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.

Mentions: In a previous work we have found that DEA, a structural analogous of lidocaine’s hydrophilic moiety, mimics some, but not all, of the modulating effects of the entire lidocaine molecule on muscle-type nAChRs (Alberola-Die et al., 2016). Consequently, the present study is aimed, first, to unravel the effects of DMA, which resembles lidocaine’s hydrophobic ring (see molecular structures in Figure 1A), on this receptor and to decipher the nAChR loci at which DMA binds. The second goal is to correlate the mechanisms of action of DMA on nAChRs with those reported for either the entire lidocaine molecule (Alberola-Die et al., 2011) or the hydrophilic moiety of lidocaine, DEA (Alberola-Die et al., 2016). Our results indicate that although both DEA and DMA block nAChRs, their mechanisms of action and binding sites on this receptor are markedly different.


Muscle-Type Nicotinic Receptor Modulation by 2,6-Dimethylaniline, a Molecule Resembling the Hydrophobic Moiety of Lidocaine
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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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121239&req=5

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.
Mentions: In a previous work we have found that DEA, a structural analogous of lidocaine’s hydrophilic moiety, mimics some, but not all, of the modulating effects of the entire lidocaine molecule on muscle-type nAChRs (Alberola-Die et al., 2016). Consequently, the present study is aimed, first, to unravel the effects of DMA, which resembles lidocaine’s hydrophobic ring (see molecular structures in Figure 1A), on this receptor and to decipher the nAChR loci at which DMA binds. The second goal is to correlate the mechanisms of action of DMA on nAChRs with those reported for either the entire lidocaine molecule (Alberola-Die et al., 2011) or the hydrophilic moiety of lidocaine, DEA (Alberola-Die et al., 2016). Our results indicate that although both DEA and DMA block nAChRs, their mechanisms of action and binding sites on this receptor are markedly different.

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.