Muscle-Type Nicotinic Receptor Blockade by Diethylamine, the Hydrophilic Moiety of Lidocaine

Abstract

Lidocaine bears in its structure both an aromatic ring and a terminal amine, which can be protonated at physiological pH, linked by an amide group. Since lidocaine causes multiple inhibitory actions on nicotinic acetylcholine receptors (nAChRs), this work was aimed to determine the inhibitory effects of diethylamine (DEA), a small molecule resembling the hydrophilic moiety of lidocaine, on Torpedo marmorata nAChRs microtransplanted to Xenopus oocytes. Similarly to lidocaine, DEA reversibly blocked acetylcholine-elicited currents (IACh) in a dose-dependent manner (IC50 close to 70 μM), but unlike lidocaine, DEA did not affect IACh desensitization. IACh inhibition by DEA was more pronounced at negative potentials, suggesting an open-channel blockade of nAChRs, although roughly 30% inhibition persisted at positive potentials, indicating additional binding sites outside the pore. DEA block of nAChRs in the resting state (closed channel) was confirmed by the enhanced IACh inhibition when pre-applying DEA before its co-application with ACh, as compared with solely DEA and ACh co-application. Virtual docking assays provide a plausible explanation to the experimental observations in terms of the involvement of different sets of drug binding sites. So, at the nAChR transmembrane (TM) domain, DEA and lidocaine shared binding sites within the channel pore, giving support to their open-channel blockade; besides, lidocaine, but not DEA, interacted with residues at cavities among the M1, M2, M3, and M4 segments of each subunit and also at intersubunit crevices. At the extracellular (EC) domain, DEA and lidocaine binding sites were broadly distributed, which aids to explain the closed channel blockade observed. Interestingly, some DEA clusters were located at the α-γ interphase of the EC domain, in a cavity near the orthosteric binding site pocket; by contrast, lidocaine contacted with all α-subunit loops conforming the ACh binding site, both in α-γ and α-δ and interphases, likely because of its larger size. Together, these results indicate that DEA mimics some, but not all, inhibitory actions of lidocaine on nAChRs and that even this small polar molecule acts by different mechanisms on this receptor. The presented results contribute to a better understanding of the structural determinants of nAChR modulation.

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

“…The recording methodology has been described previously (Morales et al, 1995; Alberola-Die et al, 2016). 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 (ANR) to block any muscarinic response (Kusano et al, 1982).…”

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

“…Nicotinic acetylcholine receptor function can be modulated by a broad number of molecules, some of them containing tertiary-amino or quaternary-ammonium groups in their structure, including: (i) local anesthetics (LAs) like lidocaine (Alberola-Die et al, 2011, 2013) or its structural analogs, QX-314 and QX-222 (Neher and Steinbach, 1978; Pascual and Karlin, 1998); (ii) cholinesterase inhibitors as BW284c51, edrophonium or decamethonium (Olivera-Bravo et al, 2007) and (iii) small molecules such as choline (Grosman and Auerbach, 2000; Lape et al, 2009), TMA and TEA (Akk and Steinbach, 2003) or DEA (Alberola-Die et al, 2016). All these molecules are totally or partially protonated at physiological pH and, thus, their quaternary-ammonium group might be responsible for nAChR inhibition by open-channel blockade, acting within the channel pore (Arias, 2006).…”

“…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.…”

“…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.…”