Search

Y55W mutants of non-selective NaK and partly K+-selective NaK2K channels have been used to explore the conformational dynamics at the pore region of these channels as they interact with either Na+ or K+. A major conclusion is that these channels exhibit a remarkable pore conformational flexibility. Homo-FRET measurements reveal a large change in W55–W55 intersubunit distances, enabling the selectivity filter (SF) to admit different species, thus, favoring poor or no selectivity. Depending on the cation, these channels exhibit wide-open conformations of the SF in Na+, or tight induced-fit conformations in K+, most favored in the four binding sites containing NaK2K channels. Such conformational flexibility seems to arise from an altered pattern of restricting interactions between the SF and the protein scaffold behind it. Additionally, binding experiments provide clues to explain such poor selectivity. Compared to the K+-selective KcsA channel, these channels lack a high affinity K+ binding component and do not collapse in Na+. Thus, they cannot properly select K+ over competing cations, nor reject Na+ by collapsing, as K+-selective channels do. Finally, these channels do not show C-type inactivation, likely because their submillimolar K+ binding affinities prevent an efficient K+ loss from their SF, thus favoring permanently open channel states. This work was partly supported by grants PGC2018-093505-B-I00 from the Spanish “Ministerio de Ciencia e Innovación”/FEDER, UE, and FCT-Fundação para a Ciência e a Tecnologia, I.P., under the scope of the project UIDB/04565/2020 and UIDP/04565/2020 of the Research Unit Institute for Bioengineering and Biosciences—iBB and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB.

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).

Fritillaria bulbs are used in Traditional Chinese Medicine to treat several illnesses. Peimine (Pm), an anti-inflammatory compound from Fritillaria, is known to inhibit some voltage-dependent ion channels and muscarinic receptors, but its interaction with ligand-gated ion channels remains unexplored. We have studied if Pm affects nicotinic acetylcholine receptors (nAChRs), since they play broad functional roles, both in the nervous system and non-neuronal tissues. Muscle-type nAChRs were incorporated to Xenopus oocytes and the action of Pm on the membrane currents elicited by ACh (IAChs) was assessed. Functional studies were combined with virtual docking and molecular dynamics assays. Co-application of ACh and Pm reversibly blocked IACh, with an IC50 in the low micromolar range. Pm inhibited nAChR by: (i) open-channel blockade, evidenced by the voltage-dependent inhibition of IAch, (ii) enhancement of nAChR desensitization, revealed by both an accelerated IACh decay and a decelerated IACh deactivation, and (iii) resting-nAChR blockade, deduced from the IACh inhibition elicited by Pm when applied before ACh superfusion. In good concordance, virtual docking and molecular dynamics assays demonstrated that Pm binds to different sites at the nAChR, mostly at the transmembrane domain. Thus, Pm from Fritillaria bulbs, considered therapeutic herbs, targets nAChRs with high affinity, which might account for its anti-inflammatory actions. This research was funded by grants SAF2017-82977-P (AEI/FEDER, UE), RTI2018-096724-B-C21, RTI2018-097189-B-C21, and PGC2018-093505-B-I00 from MINECO, PROMETEO/2016/006 from Generalitat Valenciana and GRE17-01 from Universidad de Alicante. R.C. held a predoctoral fellowship from Universidad de Alicante (FPUUA36).

The voltage-gated potassium channel Kv1.3 plays an apparent dual physiological role by participating in activation and proliferation of leukocytes as well as promoting apoptosis in several types of tumor cells. Therefore, Kv1.3 is considered a potential pharmacological target for immunodeficiency and cancer. Different cellular locations of Kv1.3, at the plasma membrane or the mitochondria, could be responsible for such duality. While plasma membrane Kv1.3 facilitates proliferation, the mitochondrial channel modulates apoptotic signaling. Several molecular determinants of Kv1.3 drive the channel to the cell surface, but no information is available about its mitochondrial targeting. Caveolins, which are able to modulate cell survival, participate in the plasma membrane targeting of Kv1.3. The channel, via a caveolin-binding domain (CDB), associates with caveolin 1 (Cav1), which localizes Kv1.3 to lipid raft membrane microdomains. The aim of our study was to understand the role of such interactions not only for channel targeting but also for cell survival in mammalian cells. By using a caveolin association-deficient channel (Kv1.3 CDBless), we demonstrate here that while the Kv1.3–Cav1 interaction is responsible for the channel localization in the plasma membrane, a lack of such interaction accumulates Kv1.3 in the mitochondria. Kv1.3 CDBless severely affects mitochondrial physiology and cell survival, indicating that a functional link of Kv1.3 with Cav1 within the mitochondria modulates the pro-apoptotic effects of the channel. Therefore, the balance exerted by these two complementary mechanisms fine-tune the physiological role of Kv1.3 during cell survival or apoptosis. Our data highlight an unexpected role for the mitochondrial caveolin–Kv1.3 axis during cell survival and apoptosis. Supported by the Ministerio de Ciencia e Innovación (MICINN), Spain (BFU2017-87104-R and PID2020-112647RB-I00 to AF; CSD2008-00005 to AM), the Italian Association for Cancer Research (AIRC IG grant 20286 to IS), the Italian Ministry of University and Education (PRIN 20174TB8KW_004 to IS), the Italian Association for Multiple Sclerosis (to IS), and the European Regional Development Fund.

La asignatura de Inmunología General, impartida en tercero de Grado de Biología presenta algunas dificultades, como la novedad de conceptos y procesos que, hasta ese momento, no han sido estudiados. Por este motivo, hemos decidido mejorar el aprendizaje de esta asignatura y asentar los conocimientos impartidos en clase mediante la utilización de las redes sociales, en concreto, de Instagram. Para ello, hemos publicado en la herramienta de Stories de Instagram, una pregunta por cada tema de teoría, haciendo un total de 17 preguntas binarias, de respuesta múltiple o de desarrollo. Los estudiantes tenían 24 horas para responder a las preguntas y, para aquellos que no tenían acceso a esta red social, se publicó la pregunta en la sección de “Anuncios” de UA Cloud. Una vez transcurridas las 24 horas, se publicaron las respuestas en la aplicación de Stories. Las respuestas puntuaron con 1 punto si eran correctas o con 0, si eran erróneas. Los resultados de esta experiencia educativa mostraron una elevada participación (87,1%) y puntuación (0,9 puntos). Además, el estudiantado se mostró ampliamente satisfecho con la utilidad de esta actividad (90%), así como consideraron que debería implementarse en otras asignaturas (86%).

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).

Cation binding under equilibrium conditions has been used as a tool to explore the accessibility of permeant and nonpermeant cations to the selectivity filter in three different inactivated models of the potassium channel KcsA. The results show that the stack of ion binding sites (S1 to S4) in the inactivated filter models remain accessible to cations as they are in the resting channel state. The inactivated state of the selectivity filter is therefore &ldquo, resting-like&rdquo, under such equilibrium conditions. Nonetheless, quantitative differences in the apparent KD&rsquo, s of the binding processes reveal that the affinity for the binding of permeant cations to the inactivated channel models, mainly K+, decreases considerably with respect to the resting channel. This is likely to cause a loss of K+ from the inactivated filter and consequently, to promote nonconductive conformations. The most affected site by the affinity loss seems to be S4, which is interesting because S4 is the first site to accommodate K+ coming from the channel vestibule when K+ exits the cell. Moreover, binding of the nonpermeant species, Na+, is not substantially affected by inactivation, meaning that the inactivated channels are also less selective for permeant versus nonpermeant cations under equilibrium conditions.

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites. Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines. This work was partly supported by grants PGC2018-093505-B-100, BFU2012-31359 and BFU2015-66612-P from the Spanish MINECO/FEDER (UE), and by BBSRC and Pfizer (BB/L015269/1) through a studentship to V. Oakes.

KcsA, a prokaryote tetrameric potassium channel, was the first ion channel ever to be structurally solved at high resolution. This, along with the ease of its expression and purification, made KcsA an experimental system of choice to study structure–function relationships in ion channels. In fact, much of our current understanding on how the different channel families operate arises from earlier KcsA information. Being an integral membrane protein, KcsA is also an excellent model to study how lipid–protein and protein–protein interactions within membranes, modulate its activity and structure. In regard to the later, a variety of equilibrium and non-equilibrium methods have been used in a truly multidisciplinary effort to study the effects of lipids on the KcsA channel. Remarkably, both experimental and “in silico” data point to the relevance of specific lipid binding to two key arginine residues. These residues are at non-annular lipid binding sites on the protein and act as a common element to trigger many of the lipid effects on this channel. Thus, processes as different as the inactivation of channel currents or the assembly of clusters from individual KcsA channels, depend upon such lipid binding. This work was partly supported by the grant PGC2018-093505-B-I00 from the Spanish “Ministerio de Ciencia, Innovación y Universidades”/FEDER, UE. CD-G acknowledges support from Medical Biochemistry and Biophysics Doctoral Programme (M2B-PhD) and FCT Portugal (SFRH/PD/BD/135154/2017).

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).

The selectivity filter in potassium channels, a main component of the ion permeation pathway, configures a stack of binding sites (sites S1–S4) to which K+ and other cations may bind. Specific ion binding to such sites induces changes in the filter conformation, which play a key role in defining both selectivity and permeation. Here, using the potassium channel KcsA as a model, we contribute new evidence to reinforce this assertion. First, ion binding to KcsA blocked by tetrabutylammonium at the most cytoplasmic site in the selectivity filter (S4) suggests that such a site, when in the nonconductive filter conformation, has a higher affinity for cation binding than the most extracellular S1 site. This filter asymmetry, along with differences in intracellular and extracellular concentrations of K+ versus Na+ under physiological conditions, should strengthen selection of the permeant K+ by the channel. Second, we used different K+ concentrations to shift the equilibrium between nonconductive and conductive states of the selectivity filter in which to test competitive binding of Na+. These experiments disclosed a marked decrease in the affinity of Na+ to bind the channel when the conformational equilibrium shifts toward the conductive state. This finding suggested that in addition to the selective binding of K+ and other permeant species over Na+, there is a selective exclusion of nonpermeant species from binding the channel filter, once it reaches a fully conductive conformation. We conclude that selective binding and selective exclusion of permeant and nonpermeant cations, respectively, are important determinants of ion channel selectivity. This work was funded in part by Grants BFU2012-31359 and BFU2015-66612-P from the Spanish MINECO/FEDER.

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).

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).

Comparative performance of suture and cyanoacrylate adhesives of different alkyl chain length for wound healing were compared in-vivo in New Zealand White rabbits. The alkyl chain length of the cyanoacrylate adhesive determines its effectiveness in tissue repair. The n-butyl cyanoacrylate (BCN) adhesive is very aggressive on the rabbit skin due to high exothermal reaction whereas wound closures with ethyl cyanoacrylate (ECN) and n-octyl cyanoacrylate (OCN) are adequate and similar. No significant alterations were found in the standard biochemical and haematological parameters test. When ECN and OCN adhesives are used, the wounds close with little inflammation, the edges are not separated and the tissues throughout the joined areas and nearby are normal. However, due to BCN stiffness, closed wounds show opened edges and intense inflammation. ECN and OCN adhesives present advantages vs. suture, i.e. less time for application, good confrontation of both sides of the incision, immediate haemostasis, less inflammation and absence of infection. Financial support of the Spanish Research Funding Agency (MICYNN) - PET2008-0264 and MAT2009-10234 projects - is acknowledged.

Results Using insulin and glucagon secretion protocols with fresh islets isolated from C57BL6 mice, we observed that CORT reduced the glucose-stimulated insulin secretion (GSIS) in a similar magnitude from that of SST (p

There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp67–Glu71–Asp80 inactivation triad within the channel structure and its bearing on the selectivity filter. This work was supported in part by grants from the Spanish Ministerio de Ciencia e Innovación Grants BFU2011-25920 and BFU2012-31359 and Consolider-Ingenio 2010 Grant CSD2-2008-00005.

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.

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.

Ivermectin is a veterinary pharmaceutical generally used to control the ecto- and endoparasites of livestock, but its use has resulted in adverse effects on coprophilous insects, causing population decline and biodiversity loss. There is currently no information regarding the direct effects of ivermectin on dung beetle physiology and behaviour. Here, based on electroantennography and spontaneous muscle force tests, we show sub-lethal disorders caused by ivermectin in sensory and locomotor systems of Scarabaeus cicatricosus, a key dung beetle species in Mediterranean ecosystems. Our findings show that ivermectin decreases the olfactory and locomotor capacity of dung beetles, preventing them from performing basic biological activities. These effects are observed at concentrations lower than those usually measured in the dung of treated livestock. Taking into account that ivermectin acts on both glutamate-gated and GABA-gated chloride ion channels of nerve and muscle cells, we predict that ivermectin € s effects at the physiological level could influence many members of the dung pat community. The results indicate that the decline of dung beetle populations could be related to the harmful effects of chemical contamination in the dung., Financial support was provided by the Project OAPN 762/2012 of the Organismo Autónomo de Parques Nacionales-Ministerio de Agricultura, Alimentación y Medio Ambiente.

TRPC5 is an ion channel permeable to monovalent and divalent cations that is widely expressed in different tissues. Although implicated in the control of neurite extension and in the growth cone morphology of hippocampal neurons, as well as in fear-related behaviour, the mechanisms by which TRPC5 is activated remain poorly understood. TRPC5 is known to be activated downstream of Gq-coupled receptors and by membrane stretch, and since there is evidence that mechanical stress may directly activate Gq-coupled receptors, we examined the relationship between the activation of TRPC5 by the type 1 histamine receptor and osmotic stress. Using calcium imaging and patch clamp recordings, we found that a higher proportion of cells expressing TRPC5 respond to hypoosmotic solution when they co-express H1R. This response is associated with a phospholipase C-dependent increase in the cells internal calcium concentration, which is abolished on depletion of calcium stores. We also found that the hypoosmotic stimulus that provokes mechanical stress drives the translocation of TRPC5 to the plasma membrane by a mechanism dependent on PI3K. This increase in TRPC5 at the plasma membrane augments the proportion of cells that respond to hypoosmotic stimulation. Together, these results suggest that hypoosmotic cell-swelling activates Gq-coupled receptors, which in turn enhance the activation of TRPC5 by regulating this channel membrane trafficking. Gq-coupled receptors and TPRC5 are co-expressed in several tissues such as those of the vascular system and in somatosensory neurons, suggesting that this mechanism of TRPC5 activation may have interesting and important implications in arterial pressure sensing and mechanotransduction., The work was supported by grants from the Spanish government (BFU2009-07835 and CONSOLIDER-INGENIO 2010 CSD2007-0023). A-L.C holds a JAE (Junta de Ampliación de Estudios) predoctoral fellowship from the Spanish Research Council, cofinanced by the European Social Fund.

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.

Insulin resistance and hyperinsulinemia are generally associated with obesity. Obese nondiabetic individuals develop a compensatory β-cell response to adjust insulin levels to the increased demand, maintaining euglycemia. Although several studies indicate that this compensation relies on structural changes, the existence of β-cell functional adaptations is incompletely understood. Here, we fed female mice with a high-fat diet (HFD) for 12 weeks. These animals became obese, hyperinsulinemic, insulin-resistant, and mildly glucose-intolerant while fed, and fasting glycemia was comparable in HFD and control mice. Islets from HFD animals exhibited increased β-cell mass and hypertrophy. Additionally, they had enhanced insulin gene expression and content and augmented glucose-induced insulin secretion. Electrophysiological examination of β-cells from both groups showed no differences in KATP channel open probability and conductance. However, action potentials elicited by glucose had larger amplitude in obese mice. Glucose-induced Ca2+ signals in intact islets, in isolated β-cells, and individual β-cells within islets were also increased in HFD mice. Additionally, a higher proportion of glucose-responsive cells was present in obese mice. In contrast, whole-cell Ca2+ current densities were similar in both groups. Capacitance measurements showed that depolarization-evoked exocytosis was enhanced in HFD β-cells compared with controls. Although this augment was not significant when capacitance increases of the whole β-cell population were normalized to cell size, the exocytotic output varied significantly when β-cells were distributed by size ranges. All these findings indicate that β-cell functional adaptations are present in the islet compensatory response to obesity. This work was supported by grants from the Ministerio de Ciencia e Innovación (BFU2010–21773; BFU2011–28358; SAF2010–19527), Generalitat Valenciana(PROMETEO/2011/080; ACOMP/2013/022), Generalitat de Catalunya (2009 SGR 1426) and European Foundation for the Study Diabetes (EFSD/BI Basic Programme). CIBERDEM is an initiative of the Instituto de Salud Carlos III.

The estrogen receptor β (ERβ) is emerging as an important player in the physiology of the endocrine pancreas. We evaluated the role and antidiabetic actions of the ERβ selective agonist WAY200070 as an insulinotropic molecule. We demonstrate that WAY200070 enhances glucose-stimulated insulin secretion both in mouse and human islets. In vivo experiments showed that a single administration of WAY200070 leads to an increase in plasma insulin levels with a concomitant improved response to a glucose load. Two-week treatment administration increased glucose-induced insulin release and pancreatic β-cell mass and improved glucose and insulin sensitivity. In addition, streptozotocin-nicotinamide–induced diabetic mice treated with WAY200070 exhibited a significant improvement in plasma insulin levels and glucose tolerance as well as a regeneration of pancreatic β-cell mass. Studies performed in db/db mice demonstrated that this compound restored first-phase insulin secretion and enhanced pancreatic β-cell mass. We conclude that ERβ agonists should be considered as new targets for the treatment of diabetes. This work was supported by Generalitat Valenciana grant PROMETEO/2011/080, Ministerio de Economia y Competitividad BFU2011-28358, and Ministerio de Economía y Competitividad BFU2010-21773 and BFU2008-1942; the Swedish Cancer Fund; the Emerging Technology Fund of Texas; and the Robert A. Welch Foundation (E-0004).

El receptor nicotínico de acetilcolina (nAChR) es el miembro mejor caracterizado de la superfamilia de canales iónicos activados por ligando (LGICs). Las disfunciones del nAChR se han relacionado con graves alteraciones fisiopatológicas del sistema nervioso, así como con alteraciones inflamatorias e inmunológicas y con el desarrollo de ciertos tipos de cáncer. Por ello, el conocimiento de las moléculas que modulan la función de este receptor, el estudio de sus mecanismos de acción y la dilucidación de sus lugares de unión son de gran interés científico, ya que son pasos fundamentales para avanzar en el desarrollo de nuevas moléculas de aplicación terapéutica, dirigidas al tratamiento de las patologías mencionadas. Esta revisión pretende describir los principales mecanismos de modulación del nAChR y exponer los efectos que diversas moléculas de interés clínico ejercen sobre el nAChR, algunas inhibiendo su actividad y otras potenciándola. Los autores están financiados por proyectos del MINECO CSD2008-00005 y BFU2012-31359.

En internet hay disponibles materiales educativos de calidad, pero encontrarlos es difícil por el enorme volumen de información y su heterogeneidad. Muchos de los contenidos son de dudosa fiabilidad, pues no siempre se conoce su origen. Por otro lado, el 83% de los usuarios de internet entre 18 y 29 años participa en algún tipo de red social, lo que genera un compromiso con la comunidad virtual. Con todos estos datos, parece interesante que los docentes utilicen las TICs para crear una red social de contenidos docentes seleccionados por el profesor; Esto requiere una inversión de tiempo y dinero muy inferior a los necesarios para el desarrollo de materiales específicos equivalentes. La elaboración de un tablero de selección de contenidos constituye una estrategia docente no presencial, que pone a disposición del alumno recursos docentes de excelencia que facilitan su aprendizaje. La herramienta SCLIPED permite formar redes sociales colaborativas de información seleccionada y establecer una comunicación dinámica multidireccional entre los usuarios. También fomenta la participación activa del alumnado en su aprendizaje, permitiéndoles una amplia autonomía temporal y espacial y les habitúa como futuros profesionales, a una gestión eficiente de la información disponible en internet, para transformarla en conocimiento.

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.

Bisphenol-A (BPA) is a widespread endocrine-disrupting chemical (EDC) used as the base compound in the manufacture of polycarbonate plastics. It alters pancreatic β-cell function and can be considered a risk factor for type 2 diabetes in rodents. Here we used ERβ−/− mice to study whether ERβ is involved in the rapid regulation of KATP channel activity, calcium signals and insulin release elicited by environmentally relevant doses of BPA (1 nM). We also investigated these effects of BPA in β-cells and whole islets of Langerhans from humans. 1 nM BPA rapidly decreased KATP channel activity, increased glucose-induced [Ca2+]i signals and insulin release in β-cells from WT mice but not in cells from ERβ−/− mice. The rapid reduction in the KATP channel activity and the insulinotropic effect was seen in human cells and islets. BPA actions were stronger in human islets compared to mouse islets when the same BPA concentration was used. Our findings suggest that BPA behaves as a strong estrogen via nuclear ERβ and indicate that results obtained with BPA in mouse β-cells may be extrapolated to humans. This supports that BPA should be considered as a risk factor for metabolic disorders in humans. This research was supported by grants from Ministerio de Ciencia e Innovación grants BFU2008-01492, BFU2011-28358 and BFU2010-21773, Generalitat Valenciana grants Prometeo/2011/080 and ACOMP/2010/113, the European Commission (Program PEOPLE). The “Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas” is an initiative of the Instituto de Salud Carlos III.