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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.


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 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).

Mentions: Since DMA is a non-charged molecule, the lack of voltage-dependence of nAChRs blockade by DMA does not fully exclude that this molecule can bind into the channel pore. Therefore, to ascertain if DMA actually binds into the channel pore we analyzed the “rebound” currents elicited by ACh either alone or in the presence of 2 mM DMA (Figure 4). It is well-established that high doses of ACh elicit open-channel blockade of nAChRs, evidenced by an IACh rebound just when rinsing out the agonist. This current arises during the agonist washout because then the ACh leaves the channel, unplugging the pore, when it can be still bound to the high affinity orthosteric sites (Legendre et al., 2000; Liu et al., 2008). This open-channel blockade of nAChRs by high ACh concentrations is only found at negative membrane potentials, because at positive voltages the positively charged ACh is electrostatically repelled from the channel pore (compare control IAChs, black recordings, of Figures 4A1,A2). However, when 1 mM ACh was co-applied with 2 mM DMA, rebound currents were elicited both at positive and negative potentials (Figures 4A1,A2, green traces), indicating that the uncharged DMA is binding into the channel pore with low affinity, and thereby eliciting an open-channel blockade of nAChRs. Furthermore, this IACh rebound was also elicited when 2 mM DMA was co-applied with a low ACh concentration (10 μM) at negative potentials, in spite of the fact that ACh, at this concentration, cannot block by its own the channel pore (Figure 4B). Noticeably, when DMA concentration decreased below 500 μM, this rebound currents were not elicited (see in the recordings of Figure 1B that the rebound current appears at 2 mM DMA) and they were of larger amplitude when the cell was challenged with a relatively high ACh concentration (100 μM or higher; compare recordings of panels A2,B of Figure 4).


Muscle-Type Nicotinic Receptor Modulation by 2,6-Dimethylaniline, a Molecule Resembling the Hydrophobic Moiety of Lidocaine
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 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).
Mentions: Since DMA is a non-charged molecule, the lack of voltage-dependence of nAChRs blockade by DMA does not fully exclude that this molecule can bind into the channel pore. Therefore, to ascertain if DMA actually binds into the channel pore we analyzed the “rebound” currents elicited by ACh either alone or in the presence of 2 mM DMA (Figure 4). It is well-established that high doses of ACh elicit open-channel blockade of nAChRs, evidenced by an IACh rebound just when rinsing out the agonist. This current arises during the agonist washout because then the ACh leaves the channel, unplugging the pore, when it can be still bound to the high affinity orthosteric sites (Legendre et al., 2000; Liu et al., 2008). This open-channel blockade of nAChRs by high ACh concentrations is only found at negative membrane potentials, because at positive voltages the positively charged ACh is electrostatically repelled from the channel pore (compare control IAChs, black recordings, of Figures 4A1,A2). However, when 1 mM ACh was co-applied with 2 mM DMA, rebound currents were elicited both at positive and negative potentials (Figures 4A1,A2, green traces), indicating that the uncharged DMA is binding into the channel pore with low affinity, and thereby eliciting an open-channel blockade of nAChRs. Furthermore, this IACh rebound was also elicited when 2 mM DMA was co-applied with a low ACh concentration (10 μM) at negative potentials, in spite of the fact that ACh, at this concentration, cannot block by its own the channel pore (Figure 4B). Noticeably, when DMA concentration decreased below 500 μM, this rebound currents were not elicited (see in the recordings of Figure 1B that the rebound current appears at 2 mM DMA) and they were of larger amplitude when the cell was challenged with a relatively high ACh concentration (100 μM or higher; compare recordings of panels A2,B of Figure 4).

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.