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

Abstract

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 species. The aims of this study were to assess the effects of Bzc on nAChRs and to correlate them with its binding to putative 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 inhibition by Bzc was voltageindependent, but the I ACh rebound elicited when rinsing Bzc suggests an open-channel blockade. 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 demonstrate 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.

Peimine, an Anti-Inflammatory Compound from Chinese Herbal Extracts, Modulates Muscle-Type Nicotinic Receptors

“…These τ values were rather slow, as compared to those previously reported, under similar experimental conditions, for I ss inhibition elicited by different local anesthetics (LAs), as tetracaine or benzocaine, at their IC 50 . In fact, for these latter compounds, τ values were faster than the solution exchange kinetics (roughly 1.5 s [ 26 , 28 ]). Furthermore, the voltage-dependent blockade of nAChRs by Pm allowed us to determine more accurately the rate of open-channel blockade, by jumping the membrane potential during the I ACh elicited in the presence of Pm.…”

“…This interaction between neighboring nAChRs might account for the differences in the δ-subunit between both structural models since dimers were reduced to monomeric receptors in the Rahman’s model. In fact, we chose for our structural studies Unwin’s models because of: (i) the structures for the open and closed conformations are available, (ii) the nAChR is present in their original membrane, and (iii) we have significant experience correlating structural and functional results using these commonly accepted models; actually, Unwin’s models have so far provided a coherent correlation with our functional results [ 21 , 25 , 26 , 28 ].…”

“…Experimental procedures were similar to those previously used to study the effects of either acetylcholinesterase inhibitors [ 44 , 45 ] or LAs on nAChRs [ 24 , 26 , 28 ]. Briefly, Pm concentration- I ACh inhibition relationship was determined by measuring I ACh s evoked by 10 μM ACh alone or together with different Pm concentrations.…”

Xenopus Oocytes as a Powerful Cellular Model to Study Foreign Fully-Processed Membrane Proteins

“…After confirming that purified and reconstituted nAChRs microtransplanted to the oocyte membrane preserve their native biophysical properties [ 67 ], this technique has been used to study the modulation of this complex protein by different molecules of therapeutic relevance. Thus, this experimental approach has been used to assess the allosteric modulation of muscle-type nAChR function by several cholinesterase inhibitors [ 105 , 106 ], local anesthetics such as lidocaine [ 107 ] or akin molecules [ 59 , 60 ], tetracaine [ 108 ], and benzocaine [ 109 ] or natural anti-inflammatory agents such as peimine [ 110 ]. By combining electrophysiological and virtual docking assays, it has been possible to identify specific nAChR residues interacting with different drugs, which support the open-channel blockade elicited by either lidocaine or diethylamine, a molecule resembling the hydrophilic moiety of lidocaine [ 59 , 107 , 110 ] or tetracaine [ 108 ].…”