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

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.

Mechanisms Underlying the Strong Inhibition of Muscle-Type Nicotinic Receptors by Tetracaine

“…Membrane current recordings were performed at 21–25°C, 16–72 h after injection of proteoliposomes, using a high-compliance two-microelectrode voltage-clamp system (TurboTEC-10CD, npi Tamm, Germany). The recording methodology has been previously described (Morales et al, 1995 ; Alberola-Die et al, 2016b ). Briefly, oocytes were placed in a 150-μL recording chamber and continuously superfused with normal frog Ringer's solution (NR: 115 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 5 mM HEPES, pH 7.0) supplemented with 0.5 μM atropine sulfate (normal Ringer with atropine, ANR) to block any muscarinic response (Kusano et al, 1982 ).…”

“…Experimental procedures were similar to those used to study the effects of lidocaine (Alberola-Die et al, 2011 ) and other modulators (Alberola-Die et al, 2016a , b ) on nAChRs. Briefly, the Ttc concentration- I ACh inhibition relationship was determined by measuring I ACh s evoked by 10 μM ACh alone, or together with different concentrations of Ttc.…”

“…Notably, we have found that lidocaine exerts multiple inhibitory actions on muscle- and neuronal-type nAChRs (Alberola-Die et al, 2011 , 2013 ). Furthermore, most actions of lidocaine on the muscle-type nAChR can be ascertained by using structural analogs of either its hydrophilic (diethylamine; DEA) or hydrophobic (dimethylaniline; DMA) moieties (Alberola-Die et al, 2016a , b ). The polar, charged DEA is responsible for the voltage-dependent blockade of nAChRs.…”

“…In contrast, nAChR blockade by the uncharged, hydrophobic DMA is voltage-independent (although it can bind to the open-channel pore), and it mainly occurs through interactions outside the pore both at the ECD and, preferentially, at inter-subunit crevices on the transmembrane-spanning domain (TMD) to elicit closed-channel blockade. Moreover, DMA enhances nAChR desensitization (Alberola-Die et al, 2016b ).…”

Makaluvamine G from the Marine Sponge Zyzzia fuliginosa Inhibits Muscle nAChR by Binding at the Orthosteric and Allosteric Sites

“…In terms of peak current amplitude, the inhibition extent ranged from 27% at 10 μM acetylcholine to 81% at 1000 μM ( Figure 2 B). Un-competitive (not to be confused with non-competitive, which does not correlate positively with agonist concentration) inhibition could arise from the direct open-channel blocking, as it is known for memantine blocking of the N -methyl- d -aspartate (NMDA) receptor [ 15 ], as well as from the receptor modulation, as described for muscle nAChR interaction with 2,6-dimethylaniline [ 16 ]. In this case, the antagonist can bind to some allosteric site and change the kinetic properties of the ion channel.…”