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Front Mol Neurosci
2016 Feb 15;9:12. doi: 10.3389/fnmol.2016.00012.
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Muscle-Type Nicotinic Receptor Blockade by Diethylamine, the Hydrophilic Moiety of Lidocaine.
Alberola-Die A
,
Fernández-Ballester G
,
González-Ros JM
,
Ivorra I
,
Morales A
.
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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 (I ACh ) in a dose-dependent manner (IC 50 close to 70 μM), but unlike lidocaine, DEA did not affect I ACh desensitization. I ACh 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 I ACh 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.
Figure 1. DEA effects on ACh-induced currents (IAChs). (A) Molecular structures of protonated lidocaine and diethylamine (DEA), showing the resemblance of DEA to the hydrophilic moiety of lidocaine. Note that DEA is positively charged at physiological pH, as most lidocaine molecules. (B) Superimposed IAChs recorded in the same nAChR-bearing oocyte by successive applications of 10 μM ACh either alone (Control, black) or together with DEA (orange) at the indicated concentrations. In this and subsequent figures, unless otherwise stated, the holding potential was â60 mV, downward deflections denote inward currents and the horizontal bar above records corresponds to the timing of drug application. (C) DEA concentration-IACh inhibition relationship. Amplitude of the IAChs evoked in presence of DEA was normalized to the IACh elicited by ACh alone (Control) and plotted vs. the logarithm of DEA concentration. Data are the average of four oocytes from different donors and error bars, in this and following figures, are SEM; solid line is a sigmoid curve fitted to the data. (D) Superimposed IAChs recorded sequentially in the same oocyte by superfusing the cell with 10 μM ACh alone [(1), bar of solid circles and black recording], co-applied with DEA [100 μM; (2), bar of open circles and orange recording] or when changing from ACh plus DEA to ACh alone at the time indicated by the corresponding bar [(3), bar of open circles followed by solid circles and blue recording].
Figure 2. DEA neither affects IACh desensitization nor apparent time-to-peak. (A) Superimposed IAChs elicited by application of 100 μM ACh either alone (Control, black recoding) or together with DEA (+200 μM DEA, orange recording), and by re-applying 100 μM ACh alone 7 min after DEA washout (Postcontrol, gray recording). Note that IACh amplitudes have been scaled to the same size to better showing IACh desensitization. (B) Plots showing the percentage of IACh desensitization at different times (2, 10, and 20 s) after the IACh peak. Data were computed from recordings as shown in (A), by applying 100 μM ACh either alone (Control, filled circles and continuous black line; Postcontrol, filled triangles and dashed line) or plus 200 μM DEA (orange circles and line). (C) Column graph of the IACh apparent time-to-peak when applying 100 μM ACh alone (Control and Postcontrol) or with DEA (+200 μM DEA). The number of oocytes (n) and donors (N) given in each column is common to (B,C).
Figure 3. Voltage-dependent blockade of IACh by DEA. (A)
IAchs (upper traces) recorded in an oocyte when applying the voltage protocol shown on bottom, during the current plateau elicited by 10 μM ACh either alone (Control, black) or together with DEA (+100 μM DEA, orange). (B) Net i/v relationships for IACh evoked by ACh alone (10 μM ACh, black filled circles and line) or co-applied with 100 μM DEA (+100 μM DEA, orange open circles and line), obtained when applying the voltage protocol shown in (A). Normalized values represent the percentage of each IACh referred to its control at â60 mV and each point is the average of eight cells (N = 5). (C) Plot showing the fraction of the IACh left by 100 μM DEA (IACh+DEA), normalized to its control (IACh), vs. the membrane potential. Note the linear voltage dependence of IACh blockade by DEA in the range between â70 and â20 mV; the discontinuous red line shows the best linear fit to these points and the continuous line indicates the fraction of IACh remaining at positive potentials in presence of DEA. Inset shows the maximum longitudinal and transversal dimensions of the DEA molecule.
Figure 4. DEA effects on ACh concentration-IACh amplitude relationship. (A) Recordings obtained by applying sequentially to the same oocyte, at intervals of 5â30 min, increasing ACh concentrations (3â1000 μM) either alone (black) or co-applied with 100 μM DEA (orange). (B) Averaged ACh concentration-IACh amplitude curves for IAChs elicited by ACh either alone (black filled circles; n = 4, N = 3) or plus 100 μM DEA (orange open circles; same oocytes). Data were normalized to the maximal IACh elicited by ACh alone and fitted to the Hill equation (continuous lines).
Figure 5. DEA pre-application increases IACh inhibition and changes the pharmacological profile of nAChR inhibition. (A)
IAChs elicited by pre-application of 100 μM DEA for 12 s followed by its co-application with ACh at the indicated concentrations (red recordings) or by just ACh at the same concentrations (black recordings). (B) Averaged ACh concentration-IACh amplitude curves for IAChs evoked by ACh either alone (black filled circles; n = 4â8, N = 1â3) or co-applied with 100 μM DEA, after 12 s pre-application of DEA at the same concentration (red open circles; same oocytes). Data were normalized to the maximal IACh elicited by ACh alone and fitted to the Hill equation (continuous lines). (C) Plot showing the percentage of IACh inhibition when ACh (at different concentrations) was directly co-applied with 100 μM DEA (orange circles; n = 5â29, N = 4â10), or when ACh and DEA co-applications were preceded by 100 μM DEA pre-application for 12 s (red circles; n = 7â11, N = 1â3). Asterisks indicate significant differences (p < 0.05, Kruskal Wallis ANOVA on ranks) respect to the IACh blockade caused by solely co-applying 10 μM ACh and 100 μM DEA; the dashed line indicates 70% inhibition. Note that the percentage of IACh inhibition decreased markedly when DEA was just co-applied with high ACh concentrations. By contrast, when DEA was pre-applied before its co-application with ACh, the percentage of IACh blockade was similar at the different ACh concentrations (see text). The slight decrease in IACh blockade by DEA observed at 3 μM ACh might not be reliable because of the inaccuracies own to the small size of IACh at this agonist concentration.
Figure 6. Modeling of DEA and lidocaine binding to the EC and TM domains of the nAChR. (A1) Lateral view, in the membrane plane (top corresponding to the synaptic cleft), of nAChR with bound DEA molecules. For this and following panels, the color code for nAChR subunit is: α (blue and cyan), β (magenta), γ (orange), and δ (green). Ligand molecules are colored brown and represented in van der Waals spheres. Note that very few DEA-nAChR binding solutions (clusters) were located at the TM domain, except two inside the channel pore (red circle), but there were several clusters distributed at the EC domain. (A2) Upper view, from the synaptic cleft, of the nAChR with bound DEA. Note that at the EC domain DEA mainly interacts at intersubunit interphases, although some DEA clusters involved single subunits. The red circle indicates the cluster sited within the channel pore. (A3) An expanded view of the EC domain at the α-γ interphase, corresponding to the ACh-binding site (loops A, B, and C of the α subunit are indicated as reference). Note that one DEA cluster is near, but not inside, the pocket of the ACh-binding site (see text for details). (B1) nAChR with lidocaine binding solutions is shown in a similar view as in (A1). Note that lidocaine clusters were more numerous at the TM domain than those observed for DEA. Several TM clusters were located in intra- and intersubunit cavities and others inside the channel pore (red circle). At the EC domain there were also several lidocaine clusters, with a distribution roughly similar to that found for DEA. (B2) Upper view, from the synaptic cleft, of the nAChR with bound lidocaine. Note that both at the EC and TM domains lidocaine interacted at intra and intersubunit interphases and that several clusters were grouped inside the channel pore (red circle). (B3) The same nAChR area as in (A3), showing a lidocaine cluster into the ACh-binding site.
Alberola-Die,
Lidocaine effects on acetylcholine-elicited currents from mouse superior cervical ganglion neurons.
2013, Pubmed
Alberola-Die,
Lidocaine effects on acetylcholine-elicited currents from mouse superior cervical ganglion neurons.
2013,
Pubmed
Alberola-Die,
Multiple inhibitory actions of lidocaine on Torpedo nicotinic acetylcholine receptors transplanted to Xenopus oocytes.
2011,
Pubmed
,
Xenbase
Albuquerque,
Mammalian nicotinic acetylcholine receptors: from structure to function.
2009,
Pubmed
Arias,
Positive and negative modulation of nicotinic receptors.
2010,
Pubmed
Cecchini,
The nicotinic acetylcholine receptor and its prokaryotic homologues: Structure, conformational transitions & allosteric modulation.
2015,
Pubmed
Chatzidaki,
Allosteric modulation of nicotinic acetylcholine receptors.
2015,
Pubmed
Cohen,
Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations.
1992,
Pubmed
,
Xenbase
Corringer,
Nicotinic receptors at the amino acid level.
2000,
Pubmed
Dani,
Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system.
2007,
Pubmed
Duan,
A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations.
2003,
Pubmed
Gallagher,
Identification of amino acids of the torpedo nicotinic acetylcholine receptor contributing to the binding site for the noncompetitive antagonist [(3)H]tetracaine.
1999,
Pubmed
Gentry,
Local anesthetics noncompetitively inhibit function of four distinct nicotinic acetylcholine receptor subtypes.
2001,
Pubmed
Gonzalez-Gutierrez,
The atypical cation-conduction and gating properties of ELIC underscore the marked functional versatility of the pentameric ligand-gated ion-channel fold.
2015,
Pubmed
Gotti,
Neuronal nicotinic receptors: from structure to pathology.
2004,
Pubmed
Guex,
SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling.
1997,
Pubmed
Hara,
The effects of the local anesthetics lidocaine and procaine on glycine and gamma-aminobutyric acid receptors expressed in Xenopus oocytes.
2007,
Pubmed
,
Xenbase
Hilf,
Structural basis of open channel block in a prokaryotic pentameric ligand-gated ion channel.
2010,
Pubmed
,
Xenbase
Hille,
Common mode of action of three agents that decrease the transient change in sodium permeability in nerves.
1966,
Pubmed
Hurst,
Nicotinic acetylcholine receptors: from basic science to therapeutics.
2013,
Pubmed
Ivorra,
Protein orientation affects the efficiency of functional protein transplantation into the xenopus oocyte membrane.
2002,
Pubmed
,
Xenbase
Krieger,
Making optimal use of empirical energy functions: force-field parameterization in crystal space.
2004,
Pubmed
Krieger,
Increasing the precision of comparative models with YASARA NOVA--a self-parameterizing force field.
2002,
Pubmed
Kusano,
Cholinergic and catecholaminergic receptors in the Xenopus oocyte membrane.
1982,
Pubmed
,
Xenbase
Liu,
Common molecular determinants of flecainide and lidocaine block of heart Na+ channels: evidence from experiments with neutral and quaternary flecainide analogues.
2003,
Pubmed
Middleton,
Photoaffinity labeling the torpedo nicotinic acetylcholine receptor with [(3)H]tetracaine, a nondesensitizing noncompetitive antagonist.
1999,
Pubmed
Morales,
Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane.
1995,
Pubmed
,
Xenbase
Morris,
Using AutoDock for ligand-receptor docking.
2008,
Pubmed
Narahashi,
Cationic forms of local anaesthetics block action potentials from inside the nerve membrane.
1969,
Pubmed
Olivera-Bravo,
Diverse inhibitory actions of quaternary ammonium cholinesterase inhibitors on Torpedo nicotinic ACh receptors transplanted to Xenopus oocytes.
2007,
Pubmed
,
Xenbase
Olivera-Bravo,
The acetylcholinesterase inhibitor BW284c51 is a potent blocker of Torpedo nicotinic AchRs incorporated into the Xenopus oocyte membrane.
2005,
Pubmed
,
Xenbase
Pan,
Structure of the pentameric ligand-gated ion channel GLIC bound with anesthetic ketamine.
2012,
Pubmed
,
Xenbase
Papke,
Mechanisms of noncompetitive inhibition of acetylcholine-induced single-channel currents.
1989,
Pubmed
Pascual,
Delimiting the binding site for quaternary ammonium lidocaine derivatives in the acetylcholine receptor channel.
1998,
Pubmed
,
Xenbase
Prince,
Mechanism of tacrine block at adult human muscle nicotinic acetylcholine receptors.
2002,
Pubmed
Sanchez,
Slow permeation of organic cations in acetylcholine receptor channels.
1986,
Pubmed
Scheller,
Ketamine blocks currents through mammalian nicotinic acetylcholine receptor channels by interaction with both the open and the closed state.
1996,
Pubmed
Sine,
Agonists block currents through acetylcholine receptor channels.
1984,
Pubmed
Steinbach,
Alteration by xylocaine (lidocaine) and its derivatives of the time course of the end plate potential.
1968,
Pubmed
Taly,
Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system.
2009,
Pubmed
Trellakis,
Differential lidocaine sensitivity of human voltage-gated potassium channels relevant to the auditory system.
2006,
Pubmed
Ueta,
Local anesthetics have different mechanisms and sites of action at recombinant 5-HT3 receptors.
2007,
Pubmed
,
Xenbase
Unwin,
Refined structure of the nicotinic acetylcholine receptor at 4A resolution.
2005,
Pubmed
Unwin,
Acetylcholine receptor channel imaged in the open state.
1995,
Pubmed
Woodhull,
Ionic blockage of sodium channels in nerve.
1973,
Pubmed
Xiong,
Inhibition by local anesthetics of Ca2+ channels in rat anterior pituitary cells.
1998,
Pubmed
Yamakura,
Subunit-dependent inhibition of human neuronal nicotinic acetylcholine receptors and other ligand-gated ion channels by dissociative anesthetics ketamine and dizocilpine.
2000,
Pubmed
,
Xenbase
