Mechanisms of Blockade of the Muscle -Type Nicotinic Receptor by Benzocaine, a Permanently Uncharged Local Anesthetic

Most local anesthetics (LAs) are amine compounds bearing one or several phenolic rings. Many of them are protonated at physiological pH, but benzocaine (Bzc) is permanently uncharged, which is relevant because the effects of LAs on nicotinic acetylcholine (ACh) receptors (nAChRs) depend on their presence as uncharged or protonated spe- cies. The aims of this study were to assess the effects of Bzc on nAChRs and to correlate them with its binding to puta- tive interacting sites on this receptor. nAChRs from Torpedo electroplaques were microtransplanted to Xenopus oocytes and currents elicited by ACh ( I ACh s), either alone or together with Bzc, were recorded at different potentials. Co -application of ACh with increasing concentrations of Bzc showed that Bzc reversibly blocked nAChRs. I ACh inhibi- tion by Bzc was voltage -independent, but the I ACh rebound elicited when rinsing Bzc suggests an open -channel block- ade. Besides, ACh and Bzc co -application enhanced nAChR desensitization. When Bzc was just pre -applied it also inhibited I ACh , by blocking closed (resting) nAChRs. This blockade slowed down the kinetics of both the I ACh activation and the recovery from blockade. The electrophysiological results indicate that Bzc effects on nAChRs are similar to those of 2,6-dimethylaniline, an analogue of the hydrophobic moiety of lidocaine. Furthermore, docking assays on models of the nAChR revealed that Bzc and DMA binding sites on nAChRs overlap fairly well. These results demon- strate that Bzc inhibits nAChRs by multiple mechanisms and contribute to better understanding both the modulation of nAChRs and how LAs elicit some of their clinical side effects. This article is part of a Special Issue entitled: Honoring Ricardo Miledi -outstanding neuroscientist of XX-XXI centuries. (c) 2019 IBRO. Published by Elsevier Ltd. All rights reserved.