Abstract
Nicotinic acetylcholine (ACh) receptors (nAChRs) are included among the targets of a variety of local anesthetics, although the molecular mechanisms of blockade are still poorly understood. Some local anesthetics, such as lidocaine, act on nAChRs by different means through their ability to present as both charged and uncharged molecules. Thus, we explored the mechanisms of nAChR blockade by tetracaine, which at physiological pH is almost exclusively present as a positively charged local anesthetic. The nAChRs from Torpedo electroplaques were transplanted to Xenopus oocytes and the currents elicited by ACh (IAChs), either alone or co-applied with tetracaine, were recorded. Tetracaine reversibly blocked IACh, with an IC50 (i.e., the concentration required to inhibit half the maximum IACh) in the submicromolar range. Notably, at very low concentrations (0.1 μM), tetracaine reduced IACh in a voltage-dependent manner, the more negative potentials produced greater inhibition, indicating open-channel blockade. When the tetracaine concentration was increased to 0.7 μM or above, voltage-independent inhibition was also observed, indicating closed-channel blockade. The IACh inhibition by pre-application of just 0.7 μM tetracaine before superfusion of ACh also corroborated the notion of tetracaine blockade of resting nAChRs. Furthermore, tetracaine markedly increased nAChR desensitization, mainly at concentrations equal or higher than 0.5 μM. Interestingly, tetracaine did not modify desensitization when its binding within the channel pore was prevented by holding the membrane at positive potentials. Tetracaine-nAChR interactions were assessed by virtual docking assays, using nAChR models in the closed and open states. These assays revealed that tetracaine binds at different sites of the nAChR located at the extracellular and transmembrane domains, in both open and closed conformations. Extracellular binding sites seem to be associated with closed-channel blockade; whereas two sites within the pore, with different affinities for tetracaine, contribute to open-channel blockade and the enhancement of desensitization, respectively. These results demonstrate a concentration-dependent heterogeneity of tetracaine actions on nAChRs, and contribute to a better understanding of the complex modulation of muscle-type nAChRs by local anesthetics. Furthermore, the combination of functional and virtual assays to decipher nAChR-tetracaine interactions has allowed us to tentatively assign the main nAChR residues involved in these modulating actions.
Highlights
The muscle-type nicotinic acetylcholine (ACh) receptor is the prototypical member of the Cys-loop family of ligand-gated ion channels
The nicotinic acetylcholine (ACh) receptor (nAChR) from Torpedo marmorata electroplaques were purified by bromoacetylcholine-affinity chromatography in the presence of asolectin lipids, using cholate as a detergent
The superfusion of Ttc on either uninjected oocytes or those bearing nAChRs, with the membrane potential clamped at −60 mV, did not modify their cell membrane conductance, even at concentrations as high as 1 mM
Summary
The muscle-type nicotinic acetylcholine (ACh) receptor (nAChR) is the prototypical member of the Cys-loop family of ligand-gated ion channels. All nAChRs share many structural properties, neuronal nAChRs differ from their muscle-type counterparts in the large diversity of their subunit compositions, which in some cases are tissue specific, and in the associated heterogeneity of their physiological and pharmacological properties (Albuquerque et al, 2009; Taly et al, 2009) These receptors constitute a key therapeutic target, given the high prevalence and relevance of disorders related to nAChR dysfunction, including some myasthenias, addictive behaviors, some types of epilepsy, schizophrenia, Parkinson’s and Alzheimer’s diseases, inflammation, pain, and even cancer (Hurst et al, 2013; Wu et al, 2015; Parikh et al, 2016; Schulte et al, 2016). Over the last few decades, much effort has been devoted to understand the mechanisms underlying nAChR modulation, as a large number of highly different molecules affect their functional properties, enabling these receptors to act as allosteric proteins (Changeux, 2014)
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