Lidocaine effects on acetylcholine-elicited currents from mouse superior cervical ganglion neurons

Lidocaine is a commonly used local anaesthetic that, besides blocking voltage-dependent Na+ channels, has multiple inhibitory effects on muscle-type nicotinic acetylcholine (ACh) receptors (nAChRs). In the present study, we have investigated the effects of lidocaine on ACh-elicited currents (IAChs) from cultured mouse superior cervical ganglion (SCG) neurons, which mainly express heteromeric α3β4 nAChRs. Neurons were voltage-clamped by using the perforated-patch method and IAChs were elicited by fast application of ACh (100-300 μM), either alone or in presence of lidocaine at different concentrations. IAChs were reversibly blocked by lidocaine in a concentration-dependent way (IC50 = 41 μM; nH close to 1) and the inhibition was, at least partially, voltage-dependent, indicating an open-channel blockade. Besides, lidocaine blocked resting (closed) nAChRs, as evidenced by the increased inhibition caused by a 12 s lidocaine application just before its co-application with the agonist, and also enhanced IAChs desensitisation, at concentrations close to the IC50. These results indicate that lidocaine has diverse inhibitory actions on neuronal heteromeric nAChRs resembling those previously reported for Torpedo (muscle-type) nAChRs ( Alberola-Die et al., 2011). The similarity of lidocaine actions on different subtypes of heteromeric nAChRs differs with the specific effects of other compounds, restricted to particular subtypes of nAChRs.This work was supported by the following MICINN (Spanish government) grants: CONSOLIDER-INGENIO 2010 (CSD2008-00005), BFU2011-25371 and BFU2012-31359

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

The use of Xenopus oocytes in electrophysiological and biophysical research constitutes a long and successful story, providing major advances to the knowledge of the function and modulation of membrane proteins, mostly receptors, ion channels, and transporters. Earlier reports showed that these cells are capable of correctly expressing heterologous proteins after injecting the corresponding mRNA or cDNA. More recently, the Xenopus oocyte has become an outstanding host–cell model to carry out detailed studies on the function of fully-processed foreign membrane proteins after their microtransplantation to the oocyte. This review focused on the latter overall process of transplanting foreign membrane proteins to the oocyte after injecting plasma membranes or purified and reconstituted proteins. This experimental approach allows for the study of both the function of mature proteins, with their native stoichiometry and post-translational modifications, and their putative modulation by surrounding lipids, mostly when the protein is purified and reconstituted in lipid matrices of defined composition. Remarkably, this methodology enables functional microtransplantation to the oocyte of membrane receptors, ion channels, and transporters from different sources including human post-mortem tissue banks. Despite the large progress achieved over the last decades on the structure, function, and modulation of neuroreceptors and ion channels in healthy and pathological tissues, many unanswered questions remain and, most likely, Xenopus oocytes will continue to help provide valuable responses.The work in the authors’ laboratories has been supported by grants SAF2017-82977-P (AEI/FEDER, UE) and PGC2018-093505-B-I00 from MINECO and GRE17-01 from Universidad de Alicante (Spain)

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.This work was supported by grants BFU2012-31359, BFU2012-39092-C02-01, BFU2011-25920, and CSD2008-00005 from the MINECO and PROMETEO/2014/11 from GVA (Spain)

Red Scliped: Evaluación del uso de tableros de selección de contenidos online en la docencia de Fisiología Animal I

Durante el curso 2013-2014 se implementó en la asignatura de Fisiología Animal I, del Grado en Biología de la Universidad de Alicante, un entorno personal de aprendizaje (EPA), a partir de la herramienta Scliped. Esta herramienta permitió desarrollar una red social de contenidos seleccionados por el profesorado, como estrategia docente para poner a disposición del alumnado recursos de excelencia que faciliten su aprendizaje. La inversión de tiempo y dinero requerida fue sensiblemente inferior a la necesaria para el desarrollo de materiales específicos equivalentes. El alumnado tuvo a su disposición un tablero de 130 contenidos seleccionados, distribuidos en 34 colecciones. Al finalizar el curso escolar 2013-2014, el EPA había recibido un total de 60.794 visitas. La evaluación del uso y conformidad del alumnado con esta herramienta mostró que, si bien opinaban que su uso era innovador, no podría sustituir la labor del profesorado. El porcentaje de adhesión a la herramienta fue bajo, tan sólo el 16% del alumnado encuestado, sin embargo, este grupo declaró que el uso de Scliped era sencillo y atractivo, que los contenidos eran pertinentes y estaban bien organizados, y que les habían facilitado el estudio y la comprensión de la materia

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 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 (IAChs), 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. IACh inhibition by Bzc was voltage-independent, but the IACh 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 IACh, by blocking closed (resting) nAChRs. This blockade slowed down the kinetics of both the IACh 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.This work was supported by grants BFU2012-31359, BFU2015-66612-P, SAF2015-66275-C2-1-R and SAF2017-82977-P (AEI/FEDER, UE) from MINECO, PROMETEO/2014/11 from Generalitat Valenciana (Spain) and GRE17-01 from Universidad de Alicante. R.C. held a predoctoral fellowship from Universidad de Alicante (FPUUA36) and M.N. a predoctoral industrial fellowship from Ministerio de Economía, Industria y Competitividad (DI-16-08303)

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.This work was supported by grants BFU2012-31359, SAF2015-66275-C2-1-R, BFU2011-25920, BFU2015-66612-P, and CSD2008-00005 from the MINECO and PROMETEO/2014/11 from GVA (Spain)

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.This work was supported by grants BFU2012-31359, SAF2015-66275-C2-1-R, and SAF2017-82977-P (AEI/FEDER, UE) from MINECO and PROMETEO/2014/11 from Generalitat Valenciana (Spain). RC held a predoctoral fellowship from Universidad de Alicante (FPUUA36)

Evaluación del trabajo colaborativo en Iniciación a la Investigación en Biología

Tras el trabajo previo de diseño de la asignatura Iniciación a la Investigación en Biología, el equipo multidisciplinar de profesores y profesoras de la Red Docente INVES ha desarrollado una metodología propia de trabajo en equipo, no sólo entre el diferente profesorado que la compone, sino también con el profesorado de la asignatura Estadística, con la que se comparten objetivos de aprendizaje comunes. Se ha optimizado el sistema de evaluación del trabajo colaborativo del alumnado, mediante el uso de rubricas y auto-evaluación. Dicho trabajo consiste en el diseño y desarrollo de un proyecto de investigación bibliométrico de temática biológica realizado por los estudiantes, propiciando la adquisición de competencias transversales mediante una dinámica de trabajo en grupo que culmina en la edición de unas Jornadas Científicas. Por otra parte, se han consensuado criterios comunes de evaluación continua, mejorando en la eficiencia de la evaluación, y determinado un incremento de la capacidad de aprendizaje del alumnado a lo largo de los cursos 2010-11 al 2013-14. La oferta formativa se completa mediante la formación de un grupo de Alto Rendimiento Académico con docencia en lengua inglesa. Esto permite al alumnado implementar el objetivo general de compresión de lengua extranjera inglés en lo relativo al ámbito científico

Avances en el trabajo colaborativo en Iniciación a la Investigación en Biología

Un equipo multidisciplinar de profesores y profesoras que componen la Red Docente INVES e imparten docencia en la asignatura Iniciación a la Investigación en Biología, ha desarrollado una metodología propia de trabajo en equipo, en coordinación con el profesorado de la asignatura Estadística, con la que se comparten objetivos de aprendizaje comunes. El sistema de evaluación del trabajo colaborativo del alumnado se ha optimizado mediante el uso de rúbricas y auto-evaluación. Se ha propiciado la adquisición de competencias transversales mediante una dinámica de trabajo en grupo. El diseño y desarrollo de un proyecto de investigación bibliométrico, de temática biológica, es realizado por los y las estudiantes, y culmina con la edición de unas Jornadas Científicas. Con el fin de mejorar la eficiencia de la evaluación, se han consensuado criterios comunes de evaluación continua entre el profesorado. Ello ha determinado un incremento de la capacidad de aprendizaje del alumnado a lo largo de los cursos 2010-11 al 2013-14. La lectura y compresión de textos científicos en inglés junto a la formación de un grupo de Alto Rendimiento Académico con docencia en lengua inglesa completa la oferta formativa, permitiendo al alumnado implementar el objetivo general de compresión de lengua extranjera inglés en lo relativo al ámbito científico