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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

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 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).

Mentions: We measured IAChs at different membrane potentials by applying voltage jumps (from -120 to +60 mV, in 20 mV steps) in absence (not shown) or presence of 10 μM ACh applied either alone or together with 2 mM DMA (Figure 3A) to determine if IACh inhibition by DMA has any voltage-dependence, which would suggest its binding into the channel pore.


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

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

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).
Mentions: We measured IAChs at different membrane potentials by applying voltage jumps (from -120 to +60 mV, in 20 mV steps) in absence (not shown) or presence of 10 μM ACh applied either alone or together with 2 mM DMA (Figure 3A) to determine if IACh inhibition by DMA has any voltage-dependence, which would suggest its binding into the channel pore.

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