Isabel Ivorra

The acetylcholinesterase inhibitor BW284c51 is a potent blocker of Torpedo nicotinic AchRs incorporated into the Xenopus oocyte membrane

1 This work was aimed to determine if 1,5‐bis(4‐allyldimethylammoniumphenyl)pentan‐3‐one dibromide (BW284c51), the most selective acetylcholinesterase inhibitor (AchEI), affects the nicotinic acetylcholine (Ach) receptor (AchR) function. 2 Purified Torpedo nicotinic AchRs were injected into Xenopus laevis oocytes and BW284c51 effects on Ach‐ and carbamylcholine (Cch)‐elicited currents were assessed using the voltage‐clamp technique. 3 BW284c51 (up to 1 mM) did not evoke any change in the oocyte membrane conductance. When BW284c51 (10 pM–100 μM) and Ach were coapplied, Ach‐evoked currents (IAch) were reversibly inhibited in a concentration‐dependent manner (Hill coefficient, 1; IC50, 0.2–0.5 μM for 0.1–1000 μM Ach). Cch‐elicited currents showed a similar inhibition by BW284c51. 4 IAch blockade by BW284c51 showed a strong voltage dependence, being only apparent at hyperpolarising potentials. BW284c51 also enhanced IAch desensitisation. 5 BW284c51 changed the Ach concentration‐dependence curve of Torpedo AchR response from two‐site to single‐site kinetics, without noticeably affecting the EC50 value. 6 The BW284c51 blocking effect was highly selective for nicotinic over muscarinic receptors. BW284c51 inhibition potency was stronger than that of tacrine, and similar to that of d‐tubocurarine (d‐TC). Coapplication of BW284c51 with either tacrine or d‐TC revealed synergistic inhibitory effects. 7 Our results indicate that BW284c51 antagonises nicotinic AchRs in a noncompetitive way by blocking the receptor channel, and possibly by other, yet unknown, mechanisms. 8 Therefore, besides acting as a selective AchEI, BW284c51 constitutes a powerful and reversible blocker of nicotinic AchRs that might be used as a valuable tool for understanding their function.

Multiple inhibitory actions of lidocaine on Torpedo nicotinic acetylcholine receptors transplanted to Xenopus oocytes

J. Neurochem. (2011) 117, 1009–1019.

Membrane currents in immature oocytes of the Rana perezi frog

Abstract Immature oocytes of the Rana perezi frog were studied electrophysiologically to see if some of the unusual ionic channels found in Xenopus oocytes were also expressed in these cells. Growing oocytes showed a fairly linear current/voltage relationship (from –200 to +60 mV), whereas fully grown cells had several voltage-dependent conductances. Depolarizing pulses elicited a potassium current blocked by tetraethylammonium (TEA) and two kinetically different Ca2+-dependent Cl–currents (ICl(Ca)), both sensitive to niflumic acid. ICl(Ca), which have not been previously observed in Rana immature oocytes, were also found in response to acetylcholine or rabbit serum superfusion or intracellular injection of Ca2+. In addition, three different Cl–currents were activated in these cells by hyperpolarization: (1) a transient inward current dependent on a critical intracellular Ca2+ concentration; (2) an inward rectifier Cl–current, which was Ca2+ independent; and (3) a high threshold (over –140 mV), slow Cl–current, blocked by several divalent cations, 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanatostilbene-2,2′-disulphonic acid (SITS). The presence of most of these infrequent currents in immature oocytes of several frogs and toads suggests that they are not merely the result of random genomic expression but a programmed decision, probably related to a definite functional role.

Diverse inhibitory actions of quaternary ammonium cholinesterase inhibitors on Torpedo nicotinic ACh receptors transplanted to Xenopus oocytes

This work was aimed at comparing and analysing the effects and mechanisms of action of the quaternary ammonium cholinesterase inhibitors (QChEIs) BW284c51, decamethonium and edrophonium, on nicotinic ACh receptor (nAChR) function.

Structural and functional changes induced in the nicotinic acetylcholine receptor by membrane phospholipids

Ligand-gated ion channels (LGICs) constitute an important family of complex membrane proteins acting as receptors for neurotransmitters (Barnard, 1992; Ortells and Lunt, 1995). The nicotinic acetylcholine receptor (nAChR) from Torpedo is the most extensively studied member of the LGIC family and consists of a pentameric transmembrane glycoprotein composed of four different polypeptide subunits (alpha, beta, gamma, and delta) in a 2:1:1:1 stoichiometry (Galzi and Changeux, 1995; Hucho et al., 1996) that are arranged pseudosymmetrically around a central cation-selective ion channel. Conformational transitions, from the closed (nonconducting), to agonist-induced open (ion-conducting), to desensitized (nonconducting) states, are critical for functioning of the nAChR (Karlin, 2002). The ability of the nAChR to undergo these transitions is profoundly influenced by the lipid composition of the bilayer (Barrantes, 2004). Despite existing information on lipid dependence of AChR function, no satisfactory explanation has been given on the molecular events by which specific lipids exert such effects on the activity of an integral membrane protein. To date, several hypotheses have been entertained, including (1) indirect effects of lipids through the alteration of properties of the bilayer, such as fluidity (an optimal fluidity hypothesis [Fong and McNamee, 1986]) or membrane curvature and lateral pressure (Cantor, 1997; de Kruijff, 1997), or (2) direct effects through binding of lipids to defined sites on the transmembrane portion of the protein (Jones and McNamee, 1988; Blanton and Wang, 1990; Fernández et al., 1993; Fernández-Ballester et al., 1994), which has led to the postulation of a possible role of certain lipids as peculiar allosteric ligands of the protein. In this paper we have reconstituted purified AChRs from Torpedo into complex multicomponent lipid vesicles in which the phospholipid composition has been systematically altered. Stopped-flow rapid kinetics of cation translocation and Fourier transform-infrared (FT-IR) spectroscopy studies have been used to illustrate the lipid dependence of both AChR function and AChR secondary structure, respectively.

Quaternary ammonium anticholinesterases have different effects on nicotinic receptors

Copious current research is devoted to finding new modulators of nicotine acetylcholine receptors (nAChRs), in view of their high relevance for nervous system physiology and because alterations in their number and/or function are related to several neurological disorders and neurodegenerative diseases (Pereira et al., 2002). Recently, we have shown that 1,5-bis(4-allyldimethyl ammonium phenyl) pentan-3-one dibromide (BW284c51), a quaternary ammonium cholinesterase inhibitor (QChEI), selectively inhibits nicotinic currents (IACh) by blocking the nAChR channel (Olivera et al., 2005). Interestingly, we found different effects of BW284c51 on nAChRs from those described previously for other QChEIs (Bertrand et al., 1992; Yost and Maestrone, 1994), such as decamethonium and edrophonium. Therefore, this work was aimed at characterizing, comparing, and elucidating the mechanisms underlying the action of different QChEIs on nAChRs.

Nicotinic acetylcholine receptor properties are modulated by surrounding lipids: an in vivo study.

In vitro studies carried out on liposomes of defined composition showed that nicotinic acetylcholine receptors (nAChRs) are fully functional when they are reconstituted in a heterogeneous lipid matrix, such as that provided by crude soybean (asolectin [R-Aso]) lipids. However, when they are reconstituted in plain phosphatidylcholine (R-PC) lipids, their functional activity is completely lost (Fong and McNamee, 1986). This kind of study also pointed out that phosphatidic acid (PA) and cholesterol (Chol) play an important role in preserving the ability of this protein to exhibit an optimal channel activity (Fong and McNamee, 1986). Furthermore, it has been shown recently that nAChR, itself, induces the formation of specific PA-rich lipid domains (Poveda et al., 2002). Because Xenopus oocytes incorporate functionally into their plasma membrane nAChRs after intracellular injection of liposomes bearing this protein (Morales et al., 1995), the aim of this work was to determine the effect of the reconstitution lipid matrix on the functional properties of the transplanted nAChRs.

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

To identify the molecular determinants responsible for lidocaine blockade of muscle-type nAChRs, we have studied the effects on this receptor of 2,6-dimethylaniline (DMA), which resembles lidocaine’s hydrophobic moiety. Torpedo marmorata nAChRs were microtransplanted to Xenopus oocytes and currents elicited by ACh (IACh), either alone or co-applied with DMA, were recorded. DMA reversibly blocked IACh and, similarly to lidocaine, exerted a closed-channel blockade, as evidenced by the enhancement of IACh blockade when DMA was pre-applied before its co-application with ACh, and hastened IACh decay. However, there were marked differences among its mechanisms of nAChR inhibition and those mediated by either the entire lidocaine molecule or diethylamine (DEA), a small amine resembling lidocaine’s hydrophilic moiety. Thereby, the IC50 for DMA, estimated from the dose-inhibition curve, was in the millimolar range, which is one order of magnitude higher than that for either DEA or lidocaine. Besides, nAChR blockade by DMA was voltage-independent in contrast to the increase of IACh inhibition at negative potentials caused by the more polar lidocaine or DEA molecules. Accordingly, virtual docking assays of DMA on nAChRs showed that this molecule binds predominantly at intersubunit crevices of the transmembrane-spanning domain, but also at the extracellular domain. Furthermore, DMA interacted with residues inside the channel pore, although only in the open-channel conformation. Interestingly, co-application of ACh with DEA and DMA, at their IC50s, had additive inhibitory effects on IACh and the extent of blockade was similar to that predicted by the allotopic model of interaction, suggesting that DEA and DMA bind to nAChRs at different loci. These results indicate that DMA mainly mimics the low potency and non-competitive actions of lidocaine on nAChRs, as opposed to the high potency and voltage-dependent block by lidocaine, which is emulated by the hydrophilic DEA. Furthermore, it is pointed out that the hydrophobic (DMA) and hydrophilic (DEA) moieties of the lidocaine molecule act differently on nAChRs and that their separate actions taken together account for most of the inhibitory effects of the whole lidocaine molecule on nAChRs.

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

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.

Mechanisms Underlying the Strong Inhibition of Muscle-Type Nicotinic Receptors by Tetracaine

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.