La comunicación celular es un proceso fundamental para la supervivencia de los organismos. Gracias a las distintas vías de comunicación, las células reciben e interpretan mensajes del exterior, los cuales inducen respuestas necesarias para el correcto funcionamiento de dichas células y del organismo que estas constituyen. Los canales iónicos activados por ligandos (LGIC) son proteínas integrales de membrana encargadas de transducir la señal proveniente del exterior hacia el interior celular. Sus vías de transducción son muy variadas, pero en general llevan a dos respuestas fundamentales según el receptor implicado, respuesta de excitación o respuesta de inhibición. Dentro del grupo LGIC existen tres familias de receptores, cada una conformada por varios miembros relacionados evolutivamente. Dichas familias se clasifican como: canales catiónicos activados por glutamato, canales activados por ATP y los receptores pertenecientes a la familia Cys-loop. En el presente trabajo de tesis doctoral estudiamos los mecanismos de activación de dos miembros de la familia de receptores Cys-loop, el receptor de acetilcolina de músculo adulto (AChR) y el receptor de serotonina homopentamérico tipo 3A (5-HT3AR). El AChR es considerado el modelo, tanto estructural como funcional, para todos los miembros de esta familia. Para un mejor entendimiento de los mecanismos que llevan al correcto funcionamiento de dichos receptores, es necesario: a) conocer su estructura molecular y los mecanismos que gobiernan su activación, y b) definir un modelo cinético que logre representar los estados en los cuales se encuentra el receptor y los pasos afectados por mutaciones o moduladores. En base a estudios de mutagénesis dirigida, determinamos el aporte de los residuos 15 de M1 de las subunidades ,  y  para el correcto funcionamiento del AChR. Además, definimos la relación entre el volumen del residuo en dicha posición y el efecto provocado sobre la eficiencia de gatillado del canal. Observamos que para la subunidad  el aumento del volumen del residuo en 15 lleva a una disminución en la constante de gatillado del canal. En cambio, para las otras subunidades, ocurre el efecto opuesto. Demostramos que los residuos 15 de M1 y 11 de M2 de la subunidad  interaccionan directamente. Dicha interacción explicaría la realción observada entre el volumen del residuo en 15 de M1 y la eficiencia del canal, donde la interacción 11-15 se vuelve más significativa al aumentar el volumen del residuo en 15, llevando a una reducción en la eficiencia del gatillado del canal. Debido a la baja conductancia del canal del 5-HT3AR, solo han sido propuestos hasta el momento modelos cinéticos basados en el análisis de corrientes macroscópicas. Utilizando el receptor de serotonina de alta conductancia (5-HT3AR-AC) obtuvimos corrientes macroscópicas y registros de canal único. En base a dichos registros, definimos un modelo cinético que describe con alto grado de exactitud los datos experimentales. Este es el primer modelo que, además de representar lo observado a nivel de corrientes macroscópicas, describe también la activación del receptor a nivel de canal único. Por otro lado, realizamos mutaciones sobre el 5-HT3AR-AC en los residuos 10 y 14 del segmento M4. Dichos residuos fueron demostrados como importantes en el gatillado del AChR y presentan un patrón de conservación particular entre las subunidades de estos dos receptores. Confirmamos que ambos residuos son importantes para el correcto funcionamiento del 5-HT3AR, donde las mutaciones en 10 afectaron la activación del receptor a nivel de canal único y las mutaciones en 14 solo mostraron efectos a nivel de las corrientes macroscópicas. Utilizando los datos obtenidos a partir del receptor mutado en 10 de M4, realizamos el análisis cinético en base al esquema propuesto. Determinamos que las velocidades afectadas fueron fundamentalmente de apertura y cierre del canal, similar a lo demostrado para el residuo equivalente del AChR. Nuestros resultados brindan importante información sobre la intervención de los segmentos transmembranales en el correcto funcionamiento de receptores de la familia Cys-loop. Asimismo, muestran cómo la función de determinados aminoácidos se ha conservado durante la evolución. Además, definimos el primer modelo cinético para el 5-HT3AR, el cual representa correctamente la activación de este receptor, tanto a nivel de corrientes macroscópicas como de canal único. La utilización de este modelo será de gran apoyo al entendimiento de los efectos generados por mutaciones o la acción de moduladores de la funcionalidad de dicho receptor. / Cellular communication is a fundamental process for survival of the organisms. Thanks to different signaling pathways, cells can receive messages from the environment which induce responses that allow appropriate functioning of these cells and the organism that they constitute. Ligand-gated ion channels (LGIC) are integral membrane proteins involve in transduction of signals from the external side of the cell. These signaling pathways are diverse, but in general, they can generate one of both responses: excitatory or inhibitory response. The LGIC group is composed by three different families of receptors: the glutamate-activated cationic channels, the ATP-gated channels, and the Cys-loop receptors. In the present thesis we studied the mechanism of activation of two members of the Cys-loop receptor family, the nicotinic acetylcholine receptor (AChR) and the homopentameric serotonin type 3A receptor (5-HT3AR). The AChR has been the structural and functional model of all members of this family. For a better understanding of the mechanisms that lead to the correct functioning of these receptors is necessary to: a) know its molecular structure and the mechanisms which govern its activation, and b) define a kinetic model that describes its activation and elucidate how mutations or modulators can affect the transitions between different conformational states. By combining site-directed mutagenesis with electrophysiological studies we determine the contribution of residues at position 15 of M1 in ,  and  subunits to the correct functioning of the AChR. We also define the relationship between the volume of the residue at this position and efficacy for channel gating. We show that the increase in the volume of residue at 15 of M1 of the  subunit impairs channel gating, whereas the opposite effect is observed for the same position in  and  subunits. Furthermore, we demonstrate that there is a direct interaction between residues at 15 of M1 and 11 of M2 of the  subunit. This explains the relationship between the volume of the residue at 15 of M1 and the efficiency of channel: the increase in the volume of the residue at 15 of M1 may restrict the movement of M2 through its interaction with the residue at 11 of M2, thus leading to a reduction in channel gating efficiency. Due to the low conductance of the 5-HT3AR, different kinetics models proposed until now have been based on macroscopic currents. Using the high conductance form of this receptor (5-HT3AR-HC) we recorded macroscopic currents and single-channel events. On the basis of these recordings we defined a kinetic model that closely describes the experimental data. In addition, we introduced mutations at positions 10 and 14 of the M4 transmembrane segment of the 5-HT3AR-HC. Residues at these positions have been shown to be important for the correct functioning of the AChR, and they show a particular conservation pattern among 5-HT3R and AChR subunits. We demonstrate that these residues are important for the appropriate functioning of the 5-HT3AR. Mutations at 10 of M4 affect the single-channel properties, and mutations at 14 of M4 affect the decay rate of macroscopic currents and the potency for activation. With the single-channel data obtained for 5-HT3AR-HC mutated at 10 of M4, we performed kinetic analysis on the basis of the scheme proposed in this thesis. The analysis reveals that mutations at 10 affect mainly opening and closing rates from the slowest open state. This result is similar to that previously reported for AChR, indicating that the function of this position is conserved among members of the same family. Our results provide important information about the involvement of transmembrane segments in the correct functioning of receptors from the Cys-loop superfamily. These results reveal how the function of some amino acids has been conserved along evolution. In conclusion, we defined the first kinetic model for the 5-HT3AR, which perfectly represents the activation of this receptor at both macroscopic and single-channel level. Moreover, our kinetic model provides a foundation for studying the contribution of residues to receptor function and for understanding molecular mechanisms of drug modulation.

Cys-loop receptors are ligand-gated ion channels that are involved in fast synaptic neurotransmission in the central and peripheral nervous system. The Cys-loop receptor RDL ('resistant to dieldrin') is a GABA-gated chloride channel from Drosophila melanogaster and is a major target site for insecticides. The aim of this dissertation was to characterise RDL receptors with particular focus on the agonist binding site. To assess the potency of a range of GABA analogues on RDL receptors, I expressed receptors in Xenopus oocytes and used voltage-clamp electrophysiology to detect receptor responses. I carried out computational modelling of these analogues to determine the dipole separation distances and atomic charges. Computational calculations and functional experiments revealed that agonists require a charged ammonium and an anionic centre, with the most potent agonists having a dipole separation distance of ~5 Å. I made a homology model of the extracellular domain of RDL and docked the active analogues into the putative binding site. I then conducted mutagenesis studies to test the accuracy of this model. Functional data from mutagenesis studies broadly support the location of GABA within this model. This model may be useful for further structure-activity studies and rational drug design. Natural compounds from the traditional Chinese medicine 'Ginkgo biloba' (ginkgolide A, ginkgolide B and bilobalide) have potent insecticidal properties and are similar in structure to picrotoxin. I tested the effect of these compounds on RDL receptor function using voltage-clamp electrophysiology. All compounds were found to inhibit RDL receptor function. I probed the binding site of these compounds using site-directed mutagenesis and electrophysiology. Mutations to the 2'A and 6'T channel-lining (M2) residues greatly reduced the potency of these compounds. I then made a homology model of the transmembrane domain of RDL and docked these compounds into the channel. Compounds docked into the channel pore close to the 2' and 6' channel-lining residues and H-bonding interactions were detected at these locations. Ginkgolides are therefore antagonists of RDL receptors, binding in the channel close to the 2' and 6' residues and this may be the mechanism underlying their potent insecticidal properties. The 5-HT3 receptor is a member of the Cys-loop receptor family and shows homology to RDL receptors. To explore different techniques for studying Cys-loop receptor function I assessed the functionality of two brain derived transcripts of the 5-HT3B subunit (Br1 and Br2) using single-channel electrophysiology and a fluorometric assay. Receptors containing Br1 were found to have a conductance identical to the 5-HT3B subunit whilst Br2 receptors were found not to be expressed. This finding has implications for 5-HT3 brain signalling, in which Br1 may play an important role. In conclusion, work here has described how agonists bind to and activate RDL GABA receptors and I have identified a candidate mechanism for the potent insecticidal properties of Ginkgo biloba extracts. I have also confirmed that 5-HT3 receptor brain transcript Br1 forms functional channels with similar properties to the 5-HT3B subunit.

Caenorhabditis elegans es un nematodo de vida libre utilizado como organismo modelo en diferentes disciplinas de la ciencia. Su tamaño reducido, plan corporal anatómicamente simple, ciclo de vida corto y amplio repertorio de comportamientos, lo han transformado en un organismo muy útil en investigación. Además, emerge como un modelo de interés en la industria farmacéutica para realizar ensayos in vivo rápidos y económicos, y para la detección de compuestos con actividad biológica. C. elegans comparte características fisiológicas y farmacológicas con nematodos parásitos y además es sensible a la mayoría de las drogas antiparasitarias que se utilizan en el hombre y en los animales. Dado que es difícil trabajar con nematodos parásitos en el laboratorio, C. elegans ha emergido como un excelente modelo de nematodo parásito y ha contribuido al conocimiento de los mecanismos de acción de diversos fármacos. C. elegans cuenta con la mayor familia de receptores Cys-loop. En sus músculos, posee tres receptores Cys-loop principales: dos receptores nicotínicos (nAChRs), el L-AChR y el N-AChR, y el receptor de GABA, UNC-49. Los nAChRs median la contracción de los músculos de la pared del cuerpo mientras que los receptores de GABA median la relajación muscular, permitiendo el movimiento sinusoidal típico del nematodo. Estos receptores son los blancos moleculares de drogas antihelmínticas. El levamisol, actuando como agonista del L-AChR, genera contracción sostenida de los músculos y finalmente la parálisis espástica del nematodo. La piperazina, actuando como agonista de los receptores de GABA, genera relajación muscular y parálisis flácida. Otros receptores Cys-loop presentes en el nematodo también son blancos de fármacos antihelmínticos. El receptor de glutamato permeable a cloruro (GluCl) presente en neuronas y células musculares es el blanco molecular de la ivermectina (IVM), uno de los antiparasitarios más utilizados a nivel mundial. En cuanto a los receptores Cys-loop, C. elegans no es más diferente a los nematodos parásitos de lo que cada especie individual de parásito lo es de otra. Esto se evidencia en la amplia diversidad de subunidades que generan receptores Cys-loop con diferente composición y propiedades farmacológicas en los nematodos y cuyas bases moleculares no se comprenden completamente. En esta Tesis se utilizó a C. elegans como modelo de nematodo parásito. Se estudiaron las propiedades antihelmínticas y los blancos de acción de diferentes compuestos químicos a través de ensayos de comportamiento. Para determinar sus mecanismos de acción se realizaron registros electrofisiológicos de corrientes unitarias y macroscópicas sobre receptores presentes en células musculares de C. elegans o expresados heterologamente en células de mamífero. En el Capítulo 1 se estudió el befenio, un antihelmíntico colinérgico cuyo modo de acción no se conocía completamente. Mediante ensayos de comportamiento se determinó que befenio genera parálisis espástica en nematodos salvajes adultos jóvenes. Utilizando cepas mutantes se determinó que el L-AChR es el blanco molecular involucrado en la actividad paralizante de befenio. Estos resultados sugieren que no existiría un receptor específico para befenio en los músculos de C. elegans. Cuando befenio fue combinado con levamisol el efecto paralizante fue aditivo. Esto es de importancia ya que la combinación de drogas es una buena estrategia para reducir la resistencia en nematodos parásitos. A nivel molecular, mediante registros de canal único, se determinó que befenio activa el L-AChR de C. elegans tanto en larvas L1 como L2, y a mayores concentraciones, actúa como un bloqueador de canal abierto de dicho receptor. Los estudios de docking molecular mostraron que befenio se une al sitio de unión ortostérico del agonista y forma las interacciones cation-π requeridas para la activación del receptor. Estos resultados podrían explicar la alta eficacia para activar el L-AChR. La selectividad de befenio por el nAChR muscular de mamífero fue estudiada mediante registros de canal único y de corrientes macroscópicas. Se determinó que befenio activa el nAChR pero actúa como un agonista muy débil y un bloqueador de canal potente. Según estudios de docking molecular, befenio generaría las interacciones necesarias para la activación solamente en uno de los dos sitios ortostéricos del receptor. Esto explicaría su baja eficacia en receptores de mamífero con respecto a los receptores de nematodos. Cepas mutantes de C. elegans que carecen de la subunidad LEV-8 podrían contener LAChRs formados por la subunidad ACR-8 en su reemplazo. Estos L-AChRs imitan un receptor de nematodo parásito, como el receptor de H. contortus, cuya subunidad ACR-8 podría mediar la actividad de befenio. Mediante ensayos de comportamiento con la cepa mutante se determinó que la subunidad ACR-8 no es requerida para el efecto paralizante de befenio en C. elegans. A nivel de canal único, los receptores que carecen de la subunidad LEV-8 también fueron activados por befenio y dicha droga, al igual que ACh, indujo una rápida desensibilización del receptor. En el Capítulo 2 se estudiaron tres terpenoides, carvacrol, timol y eugenol, presentes en plantas. Mediante ensayos de comportamiento utilizando nematodos salvajes, se determinó que los terpenoides paralizan rápidamente a C. elegans. El orden de potencia de parálisis fue: carvacrol>timol>eugenol. Las larvas fueron más sensibles que los nematodos adultos jóvenes. Además, los compuestos inhibieron irreversiblemente la eclosión de los huevos con el mismo orden de potencia. Estos hallazgos indican que los terpenoides producen efectos antihelmínticos a corto y largo plazo. Se evaluaron tres combinaciones de drogas: timol/levamisol, timol/piperazina y timol/ivermectina. El efecto paralizante de la combinación timol/levamisol fue sinérgico y dicha combinación también fue efectiva en la inhibición de la eclosión de huevos. Mediante ensayos de comportamiento con nematodos mutantes se determinó que los L-AChRs y los receptores de GABA son los blancos moleculares de los terpenoides. Los registros de corrientes macroscópicas revelaron que los compuestos no son capaces de activar los receptores, pero inhiben las corrientes evocadas por los agonistas. En registros de canal único, los terpenoides disminuyeron la actividad de L-AChRs generada por ACh y levamisol, redujeron la frecuencia de aperturas del L-AChR e indujeron un componente de estado cerrado más prolongado. Sin embargo, no afectaron las propiedades del canal como la conductancia y la duración de apertura. El análisis global indicó que los terpenoides ejercen su efecto antihelmíntico actuando como antagonistas no competitivos del L-AChR. En el Capítulo 3 se estudió la doxepinona, considerada una estructura química privilegiada. Mediante ensayos de comportamiento se demostró que la doxepinona ejerce su acción paralizante sobre nematodos salvajes adultos jóvenes actuando a través el GluCl, el blanco molecular de la IVM. Este compuesto sintético generó parálisis estacionaria en nematodos salvajes. La IVM actúa sobre GluCls presentes en la faringe del nematodo e inhibe el bombeo faríngeo. Doxepinona también redujo la velocidad de bombeo faríngeo en nematodos salvajes y el efecto fue mediado por los GluCls. Mediante registros de corrientes macroscópicas se caracterizaron las corrientes del receptor heteromérico GluCl α 1/GluClß de C. elegans evocadas por el agonista glutamato. Se determinó que la doxepinona no es un agonista de dicho receptor ya que no es capaz de activarlo. Mediante diferentes protocolos de aplicación de drogas, se determinó que la doxepinona actúa como un inhibidor alostérico de los GluCls. Se propuso a la inhibición del GluCl como un nuevo mecanismo antihelmíntico. En resumen, en esta Tesis Doctoral, utilizando a C. elegans como modelo de nematodo parásito, se identificaron los sitios y se descifraron los mecanismos de acción molecular de diferentes compuestos químicos, con actividad antihelmíntica. / Caenorhabditis elegans is a free-living nematode used as a model organism in different science disciplines. Its reduced size, anatomically simple body plan, short life cycle and broad repertoire of behaviours have turned it in a useful organism for research. It also emerges as an interesting model in the pharmaceutical industry for fast and cheap in vivo assays and for the detection of compounds with biological activity. C. elegans shares pharmacological and physiological characteristics with parasitic nematodes and is sensitive to most antiparasitic drugs used in humans and animals. Given that parasitic nematodes are difficult to work with in the laboratory, C. elegans has emerged as an excellent parasitic model and has contributed to the understanding of mechanisms of action of anthelmintic drugs. C. elegans has the largest Cys-loop receptor family. In its muscle, it has three main Cysloop receptors: two nicotinic receptors (nAChRs), L-AChR and N-AChR, and the UNC-49 GABA receptor. nAChRs mediate body wall muscle contraction while GABA receptors mediate muscle relaxation, thus allowing the typical sinusoidal movement of the nematode. These receptors are the molecular targets of anthelmintic drugs. Levamisole, acting as an L-AChR agonist, generates sustained muscle contraction which ends in spastic paralysis of the nematode. Piperazine, by acting as an agonist of GABA receptors, generates muscle relaxation and flaccid paralysis. Other Cys-loop receptors in the nematode are also targets of anthelmintic drugs. The glutamate-activated chloride channel (GluCl) present in neurons and muscle cells is the molecular target of ivermectin (IVM), which is one of the most used antiparasitic drug worldwide. Considering Cys-loop receptors, C. elegans is no more dissimilar to parasitic nematodes than each individual species of parasite is to another. This results from the wide subunit diversity that generates Cys-loop receptors with different compositions and pharmacological properties among nematodes; the molecular basis of this diversity remains not fully understood. In this Thesis, C. elegans was used as parasitic nematode model. The anthelmintic properties and molecular targets of different chemical compounds were studied through behavioural assays. To determine their mechanisms of action, electrophysiological recordings, single-channel and macroscopic current recordings, were carried out in C. elegans muscle cells or in mammalian cells heterologously expressing the receptor under study. In Chapter 1 bephenium was studied. It is a cholinergic anthelmintic drug whose mechanism of action was not fully understood. Through behavioural assays it was determined that bephenium generates spastic paralysis in young adult wild-type worms. By using different mutant strains, it was determined that L-AChR is the molecular target involved in the paralyzing activity of bephenium. The results suggested that there may not be a specific receptor for bephenium in C. elegans muscle. When bephenium was combined with levamisole, the paralyzing effects were additive; which is of significance since drug combination is a good strategy to reduce resistance in parasitic nematodes. At the molecular level, through single channel recordings, it was determined that bephenium activates L-AChR in larvae L1 and L2 C. elegans. At higher concentrations, it acted as an L-AChR open channel blocker. Molecular docking studies showed that bephenium binds to the orthostetic agonist binding site and forms the cation-π interactions required for receptor activation. This result may explain the high efficacy for L-AChR activation. Bephenium selectivity for the mammalian muscle nAChR was studied through singlechannel and macroscopic current recordings. Bephenium activated nAChRs, but it acted as a very weak agonist and a potent channel blocker. According to the molecular docking studies, bephenium would generate the necessary interactions for activation only in one of the two orthosteric sites of the receptor. This may explain the low efficacy in the mammalian receptor with respect to nematode receptors. C. elegans mutant strains that lack LEV-8 subunit may have L-AChRs containing the spare ACR-8 subunit in its replacement. These L-AChRs may mimic those in certain nematode parasites, like the H. contortus receptor, for which it was suggested that its ACR-8 subunit may mediate bephenium activity. Through behavioural assays in the mutant strain, it was determined that the ACR-8 subunit is not required for the paralyzing effects of bephenium on C. elegans. At the single channel level, the receptors that lack LEV-8 subunit were similarly activated by bephenium. Bephenium, like ACh, induced fast receptor desensitization. In the Chapter 2 terpenoids present in plants (carvacrol, thymol and eugenol) were studied. Through behavioural assays in wild-type nematodes, it was determined that terpenoids produced fast paralysis of the worms. The paralyzing potency order was: carvacrol > thymol > eugenol. The larvae were more sensitive than young adults. Also, the compounds irreversibly inhibited egg hatching with the same potency order. These findings indicate that terpenoids generate short- and long-term anthelmintic effects. Three drug combinations were evaluated: thymol/levamisole, thymol/piperazine and thymol/ivermectin. The paralyzing effect of thymol/levamisole combination was synergic, and this combination was effective in the inhibition of egg hatching too. Through behavioural assays in mutant nematodes, it was determined that L-AChRs and GABA receptors are the molecular targets of the terpenoids. The macroscopic current recordings revealed that the compounds could not activate the receptors but inhibited the currents evoked by the agonists. In single channel recordings, terpenoids reduced L-AChR activity generated by ACh and levamisole, reduced the frequency of L-AChR openings and induced a longer closed state component. However, terpenoids did not affect channel properties, such as conductance and open duration. The global analysis indicated that, terpenoids exert their anthelmintic effect, acting as L-AChR non-competitive antagonists. In the Chapter 3, doxepinone was studied. Doxepinone is considered a privileged chemical structure. Through behavioural assays, it was demonstrated that doxepinone exert the paralyzing action in wild-type young adult worms acting through GluCls, which are the molecular targets of IVM. The synthetic compound generated stationary paralysis on wild-type worms. IVM acts on nematode pharyngeal GluCls and inhibits pharyngeal pumping. Doxepinone also reduced the pharyngeal pumping rate in wild-type worms and the effect was mediated by GluCls. Through macroscopic current recordings, the responses of GluCl α1/GluClß receptors of C. elegans evoked by the agonist glutamate were characterized. It was determined that doxepinone is not a GluCl agonist because it is not capable of activating the receptor. Through different drug application protocols, it was determined that doxepinone acts as an allosteric inhibitor of GluCls. The inhibition of GluCls was proposed as a new anthelmintic mechanism. In summary, in this Doctoral Thesis, using C. elegans as a model of parasitic nematode, the target sites and mechanisms of action of different chemical compounds with anthelmintic activity were deciphered.

Los receptores cys-loop pertenecen a la familia de canales iónicos pentaméricos activados por ligandos (pLGICs). Se expresan ampliamente en el sistema nervioso, donde ejercen un rol vital en la comunicación neuronal. Están involucrados en los procesos de aprendizaje, memoria, movimiento, entre otros. Se han asociado alteraciones en la funcionalidad de estos receptores con una gran variedad de desórdenes neurológicos, tales como enfermedad de Alzheimer, enfermedad de Parkinson, epilepsia, síndromes miasténicos, esquizofrenia y depresión. Por ello, los receptores cys-loop son importantes blancos farmacológicos. En consecuencia, consideramos que el conocimiento de los mecanismos moleculares que conducen a su activación y disfunción es de suma relevancia. Los receptores Cys-loop están formados por un dominio extracelular, que contiene los sitios de unión de agonista, y un dominio transmembrana, que forma el poro iónico. La interfase entre ambos dominios, llamada región de acoplamiento, desempeña un rol clave en la propagación de los cambios conformacionales que se inician con la unión del agonista en la región extracelular y culminan con la apertura del poro iónico a nivel transmembranal. En este trabajo de Tesis Doctoral estudiamos dos regiones claves en el proceso de activación de los receptores Cys-loop: el sitio de unión de agonista, donde comienza la respuesta, y la interfase entre los dominios extracelular y transmembrana o región de acoplamiento. Utilizamos receptores homopentaméricos que por estar compuestos por cinco subunidades iguales, poseen cinco sitios de unión de agonista y cinco regiones de acoplamiento idénticas. Los receptores homoméricos surgieron más tempranamente en la escala evolutiva por lo que presentan características estructurales y funcionales comunes a todos los miembros Cys-loop, y son, por lo tanto, modelos útiles para el estudio de los receptores de esta familia. En el Capítulo I de esta Tesis determinamos el número de regiones de acoplamiento necesario para la activación de los receptores Cys-loop y su relación con los sitios de unión de agonista. Para ello, utilizamos como modelo de receptor homopentamérico al receptor quimérico a7-5HT3A, compuesto por secuencias del receptor a7 en su dominio extracelular y secuencias del receptor 5-HT3A en su dominio transmembrana, el que ha sido ampliamente utilizado como modelo de a7. Para conocer la contribución de cada una de las cinco regiones de acoplamiento a la estabilidad de canal abierto del receptor a7-5HT3A, empleamos nuestra estrategia experimental denominada electrical fingerprinting. Según esta estrategia, co-transfectamos células con una subunidad conteniendo la región de acoplamiento activa y otra subunidad conteniendo la región de acoplamiento inactiva, una de ellas conteniendo además mutaciones reporteras de conductancia. De esta forma, logramos expresar en membrana receptores con distinto número de regiones de acoplamiento funcionales que son identificados mediante registros de patch-clamp de canal único. Gracias a la presencia de las mutaciones reporteras de conductancia, la medición de la amplitud de cada apertura nos permitió conocer la estequiometria del receptor, es decir, el número de subunidades con región de acoplamiento funcional que tiene el receptor pentamérico que dio origen a esa apertura. Determinamos la duración de los eventos de apertura provenientes de receptores con distinto número de regiones de acoplamiento funcionales, que constituye una medida de la estabilidad de canal abierto. Encontramos que cada región de acoplamiento contribuye en forma independiente y simétrica a la estabilidad del canal abierto y que son necesarias las cinco regiones de acoplamiento funcionales para lograr la óptima activación del receptor. Demostramos además que la presencia de una sola región de acoplamiento funcional en el pentámero es suficiente para lograr la activación pero no permite mantener el canal abierto en su tiempo óptimo. Además generamos receptores a7-5HT3A mutantes, que contenían distinto número de sitios de unión de agonista y regiones de acoplamiento funcionales. Esta estrategia nos permitió establecer los requisitos estructurales mínimos que logran la activación del receptor, así como también los requerimientos estructurales que conducen a la máxima estabilidad del estado abierto. Encontramos que el receptor es capaz de responder al agonista mediante la ocupación de un único sitio si este se encuentra formado por dos subunidades con regiones de acoplamiento funcionales. Sin embargo, para lograr la óptima activación y duración máxima del canal abierto, el receptor modelo utilizado requiere de tres sitios de unión de agonista funcionales y sus cinco regiones de acoplamiento intactas. En el Capítulo II, estudiamos la activación del receptor neuronal a7 en condiciones de sub-ocupación de sus cinco sitios de unión de agonista. Este receptor se localiza principalmente en sitios distantes a los sitios de síntesis y liberación de acetilcolina (ACh), por lo que la ACh, o su producto colina, deben difundir y unirse a receptores a7 distantes. Este mecanismo colinérgico no sináptico predice que el grado de ocupación de los receptores a7 sería bajo en condiciones fisiológicas. Para estudiar la activación del receptor a7 en condiciones de sub-ocupación de sus sitios de agonista, realizamos ensayos electrofisiológicos y medimos la duración del canal abierto de receptores individuales que presentan un único sitio de unión de agonista funcional, y la comparamos con la de receptores que tienen sus cinco sitios funcionales. Para conocer el número de sitios de unión de agonista funcionales empleamos nuevamente la estrategia electrical fingerprinting. Esta estrategia requiere la medición exacta de la amplitud. Teniendo en cuenta que los receptores a7 presentan aperturas de duración breve que no permiten la resolución de su máxima amplitud, los estudios electrofisiológicos se realizaron sobre receptores a7 mutados o en presencia de potenciadores que aumentan la duración del canal abierto. En este trabajo, demostramos que la estabilidad del canal abierto de receptores a7 que presentan un único sitio de unión de agonista funcional es la misma que la de los receptores que presentan sus cinco sitios disponibles. Por otro lado, cuando reemplazamos el dominio transmembrana del receptor a7 por el del receptor 5-HT3A, encontramos que la duración del canal abierto se incrementa al aumentar el número de sitios ocupados por agonista. Este resultado demuestra por primera vez que el dominio extracelular no es el único determinante de la relación entre ocupación y estabilidad del canal abierto. Por lo tanto, en este trabajo demostramos la capacidad del receptor a7 de activarse y producir respuestas máximas con la ocupación de un solo sitio de unión de agonista, propiedad que es única y exclusiva de este receptor dentro de todos los miembros de la familia de receptores Cys-loop. Este resultado posee además relevancia fisiológica dado que esta propiedad le permitiría al receptor adaptarse al mecanismo de transmisión no sináptico. En su conjunto, los resultados que surgen de esta Tesis revelan una novedosa relación funcional entre dos dominios estructurales de estos receptores, el sitio de unión de agonista y la región de acoplamiento, y, además, contribuyen al conocimiento general del mecanismo de activación de los receptores de la familia Cys-loop. / Cys-loop receptors belong to the family of pentameric ligand-gated ion channels (pLGICs). They are widely expressed in the nervous system, where they exert a vital role in neuronal communication. They are involved in learning, memory, movement processes, among others. Functional disorders of these receptors have been associated with several neurological disorders, such as Alzheimer's disease, Parkinson's disease, epilepsy, myasthenic syndromes, schizophrenia and depression. Because Cys-loop receptors are important pharmacological targets for the development of therapies, the knowledge of the molecular mechanisms leading to activation and dysfunction of these receptors is of great importance. Cys-loop receptors contain an extracellular domain that carries the agonist binding sites and a transmembrane region that forms the ion pore. The interface between both domains, named as the coupling region, plays a key role in the propagation of the conformational changes from the binding site at the extracellular domain to the pore, located at the transmembrane region. In this Thesis, we studied two key regions that are essential for the activation process of Cys-loop receptors: the agonist binding site, where the response begins, and the interface between the extracellular and transmembrane domains or coupling region. We used homopentameric receptors that contain five identical subunits, and therefore five identical agonist binding sites and coupling regions. Because homomeric receptors appeared earlier on the evolutionary scale, they present structural and functional features that are common to all Cys-loop members, and are therefore useful models for the study of this receptor family. In Chapter I of this Thesis we studied the number of coupling regions necessary for Cys-loop receptor activation and evaluated the functional relationship of this domain with the agonist binding sites. To this end, we used a model of homopentameric receptor, the a7-5HT3A chimeric receptor, which contains a7 sequences in the extracellular domain and 5-HT3A sequences in the transmembrane domain. To determine the contribution of each of the five coupling regions to the stability of the open channel, we used our experimental strategy which is called electrical fingerprinting. For this strategy, cells were co-transfected with a subunit with an active coupling region and another subunit with an inactive coupling region, one of which carrying reporter conductance mutations, to generate receptors with different number of functional coupling regions. Next, we performed single-channel recordings to identify functional receptors using the patch-clamp technique. Due to the introduction of reporter conductance mutations, the measurement of the amplitude of each opening event allowed us to know receptor stoichiometry, i.e., the number of subunits with functional coupling region present in the pentameric receptor which originated the event. We measured open channel duration of receptors with different numbers of functional coupling regions, which indicates the open channel stability. We found that each coupling region contributes independently and symmetrically to open channel stability. We showed that five coupling regions are necessary to achieve optimal receptor activation and that the presence of only one functional coupling region is sufficient for receptor activation, but with reduced open channel duration. Furthermore, we constructed a7-5HT3A mutant receptors, containing different number of agonist binding sites and functional coupling regions. This strategy allowed us to establish the minimum structural requirements for receptor activation as well as the structural requirements for maximal open channel stability. We found that a7-5HT3A receptors are capable of responding to agonist by occupying a single agonist binding site, only if this site is formed by two subunits carrying functional coupling regions. However, to achieve optimal activation and maximal open channel duration, the model receptor requires three functional agonist binding sites and five functional coupling regions. In Chapter II, we studied a7 neuronal receptor activation under sub-occupancy conditions of its five agonist binding sites. In the brain, this receptor is mainly located at distant sites from the sites of synthesis and release of acetylcholine (ACh), so ACh, or its product choline, diffuse to bind distant a7 receptors. This non-synaptic cholinergic mechanism predicts that the degree of a7 receptor occupancy is low under physiological conditions. To study a7 activation under sub-occupancy conditions we performed single-channel recordings and measured open channel duration of receptors with only one functional agonist binding site, and compared it with that of receptors containing their five intact agonist binding sites. To know the number of agonist binding sites, we employed again the electrical fingerprinting strategy. This strategy requires accurate measurement of open channel amplitude. Because the brief duration of a7 opening events do not allow full amplitude resolution, single-channel recordings were performed in either a7 mutant receptors or in the presence of potentiators that increase open channel duration. In this work, we demonstrated that open channel stability of receptors with a single agonist binding site is the same as that of receptors containing five functional sites. Moreover, when we replaced the transmembrane domain of a7 receptors by that of 5-HT3A receptor, we found that open channel lifetime increases as the number of sites occupied by agonist increases. This result shows for the first time that the extracellular domain is not the only determinant of the relationship between occupancy and open channel stability. Therefore, in this work we demonstrated the ability of a7 receptor for activation and eliciting maximal responses with occupancy of only one agonist binding site, a property that is unique for a7 among all members of the Cys-loop family. This result has a physiological relevance since this property would allow a7 receptors to adapt to their non-synaptic mechanism. Taken together, the results that emerge from this Thesis reveal a novel functional relationship between two structural domains, the agonist binding site and the coupling region, and contribute to the general knowledge of the activation mechanism of Cys-loop receptors.

Cys-loop receptors are membrane proteins that are key players for the fast synaptic neurotransmission. Their ion transport initiates new nerve signals after activation by small agonist molecules, but this function is also highly sensitive to allosteric modulation by a number of compounds such as anesthetics, alcohol or anti-parasitic agents. For a long time, these modulators were believed to act primarily on the membrane, but the availability of high- resolution structures has made it possible to identify several binding sites in the transmembrane domains of the ion channels. It is known that ligand binding in the extracellular domain causes a conformational earthquake that interacts with the transmembrane domain, which leads to channel opening. The investigations carried out in this thesis aim at understanding the connection between ligand binding and channel opening. I present new models of the mammalian GABAA receptor based on the eukaryotic structure GluCl co-crystallized with an anti-parasitic agent, and show how these models can be used to study receptor-modulator interactions. I also show how removal of the bound modulator leads to gradual closing of the channel in molecular dynamics simulations. In contrast, simulations of the receptor with both the agonist and the modulator remain stable in an open-like conformation. This makes it possible to extract several key interactions, and I propose mechanisms for how the extracellular domain motion is initiated. The rapid increase in the number of cys-loop receptor structures the last few years has further made it possible to use principal component analysis (PCA) to create low-dimensional descriptions of the conformational landscape. By performing PCA on the crystal structure ensemble, I have been able to divide the structures into functional clusters and sample the transitions between them using various sampling methods. The studies presented in this thesis contribute to our understanding of the gating mechanism and the functional clustering of the cys-loop receptor structures, which both are important to design new allosteric modulator drugs that influence the channel function, in particular to treat neurological disorders. / <p>QC 20160518</p>

The glycine receptor (GlyR) is a ligand-gated ion channel member of the Cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Zinc is a potent allosteric modulator of GlyR function, enhancing GlyR activity at low nM to 10[mu]M concentrations while inhibiting GlyR activity at higher concentrations. We investigated sources of contaminating zinc, identifying low nM levels of zinc in ultrapure H₂O, powdered reagents used in the preparation of common electrophysiological buffers, and in polystyrene pipets. These low levels of zinc were capable of enhancing GlyR function. These findings suggest that without checking for this effect using a zinc-chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of zinc with drugs of abuse on taurine-activated GlyR to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by drugs of abuse, indicating no synergistic effects on efficacy when receptors are saturated with taurine as may be seen during synaptic events in vivo. Finally, we utilized phage display to identify novel peptide modulators of the GlyR. We tested 26 peptides against [alpha1beta] GlyRs, identifying peptides with various levels of activity on GlyR function. We demonstrated that these modulators were zinc-dependent, as their effects on GlyR activity were abolished in the presence of the zinc-chelating agent tricine. Together, these data indicate the importance of accounting for the effects of zinc when studying the function of the GlyR, as even low levels of zinc that can be found as contaminants in labware and buffers can affect GlyR function and responses to various allosteric modulators, including drugs of abuse. / text

El sistema nervioso consiste en una red muy compleja de billones de neuronas que se comunican entre sí. Las sinapsis químicas son primordiales para la comunicación neuronal rápida y eficiente, y están mediadas por la liberación de neurotransmisores a la hendidura sináptica para unirse a sus receptores específicos ubicados en otras células o neuronas postsinápticas. Entre los receptores de neurotransmisores se encuentran los receptores Cys-loop, que pertenecen a la superfamilia de canales pentaméricos activados por ligando (pLGIC). Son proteínas integrales de membrana que convierten la señal química en una respuesta eléctrica al permitir el paso de iones de un lado a otro de la membrana. Estos receptores se constituyen por 3 regiones fundamentales, el dominio extracelular, donde se encuentra el sitio de unión del neurotransmisor o sitio ortostérico; el dominio transmembrana, que conforma el poro del canal y contiene los sitios de unión para diferentes moduladores alostéricos; y el dominio intracelular que posee sitios de modulación y aminoácidos determinantes de la conductancia del canal. En vertebrados, esta familia de receptores está formada por los receptores nicotínicos de acetilcolina y el receptor de serotonina tipo 3 (5-HT3), los cuales son permeables a cationes, y por los receptores de ácido γ-aminobutírico tipo A (GABAA) y receptores de glicina, permeables a aniones. El repertorio de receptores Cys-loop en invertebrados es más variado y extenso, incluyendo entre ellos un canal aniónico activado por serotonina, denominado MOD-1. Los receptores Cys-loop son ampliamente estudiados por ser blancos moleculares terapéuticos en una extensa variedad de patologías. Es por ello por lo que ampliar el conocimiento de la función y modulación de estos receptores permitirá generar mejores estrategias farmacológicas. En esta Tesis Doctoral se realizaron estudios de la función y modulación de los receptores Cys-loop activados por serotonina humano, 5-HT3, y de Caenorhabditis elegans, MOD-1, para la búsqueda de nuevos fármacos y la generación de estrategias de reposicionamiento de medicamentos de uso clínico. En el Capítulo 1 se estudió la funcionalidad molecular del receptor 5-HT3A humano. Se logró descifrar las diferencias entre la activación ortostérica y la activación y modulación alostérica por terpenoides, utilizando la valiosa técnica de patch-clamp. Se definieron las bases mecanísticas de la activación de 5-HT3A por los agonistas ortostéricos serotonina y triptamina, y de la activación alostérica por los terpenoides timol y carvacrol. Se demostró que triptamina es un agonista de muy baja eficacia y potencia. Mediante registros de whole-cell, se demostró que los terpenoides potencian las corrientes evocadas por serotonina, pero también poseen capacidad para evocar alostéricamente respuestas macroscópicas, con mayor potencia y eficacia que triptamina, aunque de forma más lenta que serotonina. Mediante registros de canal único, se evidenció que dichos compuestos activan al receptor 5-HT3 como agonistas muy eficaces dado que generan episodios de activación más prolongados que serotonina. Nuestro estudio mostró la primera caracterización a nivel de canal único de la activación de 5-HT3A humano por ligandos alostéricos, de creciente interés como herramientas terapéuticas. En el Capítulo 2 se halló un nuevo blanco promisorio para la terapia antihelmíntica, estudiando ampliamente al receptor MOD-1 de C. elegans. Este nematodo es un buen modelo parasitario ya que comparte las características funcionales y farmacológicas con los nematodos parasitarios. Además C. elegans es sensible a la mayoría de los fármacos antiparasitarios que están dirigidos a los receptores Cys-loop de los parásitos. Dado que MOD-1 está ausente en vertebrados, se encuentra limitado al filo, y está presente en nematodos parasíticos, emerge como un blanco farmacológico promisorio. En este capítulo se revelaron las diferencias en la selectividad agonista y en los sitios ago-PAM entre MOD-1 y el receptor de serotonina humano 5-HT3. Se encontró que la triptamina, un agonista muy parcial de 5-HT3A, es un agonista eficaz de MOD-1, y se demostró además que posee una acción antihelmíntica. Por otro lado, se realizó un testeo de una variedad de compuestos en búsqueda de nuevos moduladores de MOD- 1. Se demostró, mediante estudios electrofisiológicos, que muscimol y piperazina (PZE) son antagonistas no competitivos de MOD-1. Mediante ensayos de comportamiento en gusanos se confirmó el efecto de PZE sobre MOD-1, hallando de esta manera un nuevo blanco molecular para este fármaco de uso antiparasitario. Estos estudios ampliaron el conocimiento de receptores Cys-loop de nematodos para el descubrimiento de nuevos compuestos con actividad nematicida. Por lo tanto, se concluye que, derivados de triptamina y PZE podrían ser explorados como nuevos compuestos con potencial actividad antiparasitaria. En el Capítulo 3, se comenzó a plantear estrategias para reposicionar medicamentos de uso clínico, para lograr superar las desventajas de generar un fármaco de novo, acortando los tiempos y recursos necesarios para realizar los estudios farmacológicos, famacocinéticos y de toxicidad requeridos para la producción de nuevos fármacos dirigidos a tratar patologías en las que los receptores estudiados en esta Tesis están involucrados. Con tal fin, se exploró el efecto de fármacos de uso comercial derivados de triptamina y PZE, debido a lo postulado en el capítulo 2, y se encontró que sumatriptan, un fármaco comercializado para tratar la migraña, sería un buen candidato para reposicionar para la terapia antiparasitaria. Además, PZE podría reutilizarse como modulador alostérico negativo de los receptores 5-HT3A humano abordando patologías en las que este receptor está involucrado, tales como náuseas y vómitos relacionados a la quimioterapia, radioterapia y anestesia, así como para mitigar los síntomas del síndrome de intestino irritable y para patologías del sistema nervioso como depresión, ansiedad y esquizofrenia, entre otras en las que este receptor está involucrado. En resumen, aprovechando la potencialidad que brindan los registros de canal único y de corrientes macroscópicas en el estudio de las bases moleculares del funcionamiento y modulación de estos receptores Cys-loop, se logró hallar un nuevo blanco antiparasitario, descifrar por primera vez las diferencias de activación ortostérica y alostérica del receptor 5-HT3A humano por timol y carvacrol, y generar estrategias de reposicionamiento de fármacos de uso clínico mediante la modulación alostérica de estos receptores tanto en C. elegans como en humano. Estos estudios amplían el conocimiento de la función y modulación de los receptores Cys-loop, generando una base para abordar patologías que involucran a estos receptores y para el desarrollo de nuevos y más selectivos fármacos. / The nervous system consists of a highly complex network of billions of neurons that communicate with each other. Chemical synapses are key to the rapid and efficient neuronal communication and are mediated by the release of neurotransmitters into the synaptic cleft to bind to their specific receptors located either on other cells or postsynaptic neurons. Among the neurotransmitter receptors are the Cys-loop receptors, which belong to the superfamily of ligand-gated pentameric channels (pLGIC). They are integral membrane proteins that convert the chemical signal into an electrical response by allowing ions to pass from one side of the membrane to the other. These receptors are made up of 3 fundamental regions: the extracellular domain, where the neurotransmitter binding site or orthosteric site is located; the transmembrane domain, which forms the channel pore and contains the binding sites for different allosteric modulators; and the intracellular domain which contains modulation sites and amino acids that determine channel conductance. In vertebrates, this family of receptors consists of the nicotinic acetylcholine receptors and the serotonin receptor type 3 (5- HT3), both of which are permeable to cations, and the receptors for γ-aminobutyric acid type A (GABAA) and glycine receptors, permeable to anions. The repertoire of Cys-loop receptors in invertebrates is more varied and extensive, including among them, a serotonin-activated anion channel called MOD-1. Cys-loop receptors have been extensively studied as they are therapeutic molecular targets in a wide variety of pathologies. Expanding our knowledge on the function and modulation of these receptors will therefore contribute to generating better pharmacological strategies for the treatment of these disorders. In this Ph. D. thesis studies were carried out on the function and modulation of the Cys-loop receptors activated by human serotonin, 5-HT3, and of Caenorhabditis elegans, MOD-1, for the search for new drugs and the generation of repositioning strategies for drugs for clinical use. In Chapter 1 the molecular functionality of the human 5-HT3A receptor was studied. It was possible to decipher the differences between orthosteric activation and allosteric activation and modulation by terpenoids, using the valuable patch-clamp technique. The mechanistic bases of 5-HT3A activation by the orthosteric agonists serotonin and tryptamine, and allosteric activation by the terpenoids thymol and carvacrol, were defined. It is revealed that tryptamine is an agonist with very low efficacy and potency. Using whole-cell recordings, they show that terpenoids not only potentiate the currents evoked by serotonin but also have the ability to allosterically evoke macroscopic responses, with greater power and efficiency than tryptamine, although more slowly than serotonin. Using single channel recordings, these compounds were shown to activate the 5-HT3A receptor as highly effective agonists, since they generate longer episodes of activation than serotonin. Our study showed the first characterization at the single channel level of the activation of human 5-HT3A by allosteric ligands, of increasing interest as therapeutic tools. In Chapter 2, a promising new target for anthelmintic therapy was found by extensively studying the MOD-1 receptor of C. elegans. This nematode is a good parasitic model since it shares functional and pharmacological characteristics with parasitic nematodes. In addition, C. elegans is sensitive to most antiparasitic drugs that are directed at the Cys-loop receptors of the parasites. Since MOD-1 is absent in vertebrates, limited to the phylum, and present in parasitic nematodes, it emerges as a promising drug target. In this chapter we revealed the differences in agonist selectivity and ago-PAM sites between MOD-1 and the human serotonin 5-HT3 receptor. Tryptamine, a very partial agonist of 5-HT3A, was found to be an effective agonist of MOD-1, and was further shown to have an anthelmintic action. On the other hand, we carried out a test of a variety of compounds in search of new modulators of MOD-1. We demonstrated by means of electrophysiological studies that muscimol and piperazine (PZE) are non- competitive antagonists of MOD-1. Also, through behavioral tests in worms, we confirmed the effect of PZE on MOD-1, thus finding a new molecular target for this drug for antiparasitic use. These studies have considerably expanded the knowledge of nematode Cys-loop receptors for the discovery of new compounds with nematicidal activity. We conclude that tryptamine and PZE derivatives could be explored as new compounds with potential antiparasitic activity. In Chapter 3, we began to propose strategies to reposition drugs for clinical use in order to overcome the disadvantages of generating a new drug, shortening the time and resources necessary to perform the pharmacological, pharmacokinetic, and toxicity studies required for the production of new drugs aimed at treating pathologies in which the receptors studied in this Ph. D. thesis are involved. To this end, the effect of drugs in commercial use derived from tryptamine and PZE was explored based on the results collected in Chapter 2. It was found that sumatriptan, a drug marketed to treat migraine, appears to be a good candidate to reposition for antiparasitic therapy. In addition, PZE could be reused as a negative allosteric modulator of human 5-HT3A receptors, addressing pathologies in which this receptor is involved, such as nausea and vomiting related to chemotherapy, radiotherapy, and anesthesia, as well as to alleviate the symptoms of irritable bowel syndrome and for pathologies of the nervous system, such as depression, anxiety and schizophrenia, among others in which this receptor is involved. Summing up, taking advantage of the potential offered by single channel recordings and macroscopic currents in the study of the molecular bases of the functioning and modulation of the Cys-loop receptors explored in this Ph. D. thesis, it was possible to: i) find a new antiparasitic target, ii) to decipher for the first time the differences of orthosteric and allosteric activation of the human 5-HT3A receptor by thymol and carvacrol, and iii) to generate drug repositioning strategies for clinical use through allosteric modulation of these receptors in both C. elegans and humans. These studies broaden the knowledge of the function and modulation of Cys- loop receptors, generating a basis for addressing pathologies that involve these receptors as well as for developing new and more selective drugs.

Le récepteur à la glycine est un récepteur neuronal qui appartient à la famille des canaux ligand-dépendants «cys-loop». Avec le récepteur ionotrope GABA ils fournissent la neurotransmission inhibitrice rapide dans le SNC des vertébrés grâce à leur perméabilité sélective au Cl-. Les récepteurs à la glycine participent à différents processus physiologiques comprenant le contrôle de l'activité motrice, la respiration, la sensation de douleur inflammatoire, la perception des stimuli visuels et auditifs. Le développement de modulateurs efficaces des récepteurs à la glycine permettra un contrôle précis de leur activité, ce qui est particulièrement important dans le cas des pathologies des récepteurs à la glycine, comme l'hyperekplexie. En utilisant l'analyse électrophysiologique, la mutagenèse dirigée et l'expression de protéines spécifiques dans un système hétérologue, nous avons identifié les acides ginkgoliques et niflumiques comme nouveaux modulateurs de récepteurs de la glycine, caractérisé leur action sur différentes sous-unités du récepteur et déterminé les sites importants pour la potentialisation ou l'inhibition des récepteurs à la glycine par ces composés. Cette approche est très prometteuse et ouvre de nouvelles voies vers des futures actions thérapeutiques. / Glycine receptor is a ligand-gated neuronal receptor that possesses an ion pore permeable for Cl- and represents an important component of inhibitory neurotransmission in CNS of vertebrates. Glycine receptors participate in the control of motor activity, respiration, inflammatory pain sensation, perception of visual and auditory stimuli. Development of efficient modulators of glycine receptors will allow a precise control of their activity, which is especially important in the case of glycine receptor pathologies, such as hyperekplexia. In the present work we have identified ginkgolic and niflumic acids as novel modulators of glycine receptors, characterized their action on different subunits of the receptor and determined the most probable sites of interaction of the compounds with glycine receptors.

Haemonchus contortus is a blood feeding parasitic nematode infecting ruminants causing anemia and poor health at great economic cost. The ability to pharmaceutically control infection has been challenged by the rapid development and spread of drug resistance. The discovery of new targets is therefore required for sustainable parasite control. UNC-49 is a nematode ligand-gated ion channel that plays an important role in muscle contraction required for normal locomotion. However, little is known regarding its sensitivity to different agonists and how they interact with the binding site. This thesis describes an investigation into the efficacy of a range of classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus oocytes. The results of our electrophysiological recordings indicate that there is a size requirement for full agonism of the Hco-UNC-49 binding site. Furthermore, a number of molecules that are known to act on vertebrate GABA receptors have no effect on Hco-UNC-49. This suggests that the binding site of nematode GABA receptors does exhibit some unique properties. These findings could possibly be exploited to develop new drugs that specifically target GABA receptors from parasitic nematodes. / UOIT

Glycine receptors (GlyRs) are ligand-gated ion channels (LGICs) that, along with other members of the cys-loop superfamily of receptors, mediate a considerable portion of fast neurotransmission in the central nervous system (CNS). GlyRs are pentameric channels, organized quasi-symmetrically around an ion-conducting pore. Opening of the integral ion pore depends on ligand binding and transduction of this binding signal to the channel gate. Research presented in this dissertation describes a number of critical electrostatic interactions that play a role in conserving the closed-state stability of the receptor in the absence of ligand, ensuring that receptor activation occurs only upon neurotransmitter binding. These amino acids, aspartic acid at position 97 (D97), lysine 116 (K116), arginine 119 (R119) and arginine R131 (R131) are charged residues that interact with one another through electrostatic attraction. When D97 is replaced with any other amino acid this destabilizes the closed state of the channel and causes spontaneous GlyR channel opening. I show that restoration of this electrostatic interaction in GlyR bearing double mutations in which the charges are swapped (D97R/R119E and D97R/R131D) markedly decreases this spontaneous current. In addition, I investigate how these residues that interact at the interfaces between receptor subunits affect the efficacies of GlyR partial agonists. My work shows that the partial agonist taurine is converted into a full agonist at both D97R and R131D receptors. Furthermore, I analyzed the structure of the more extracellular part of the transmembrane (TM) 2 segment that lines the ion channel pore, showing that it is unlikely that this fragment (stretching from T13’ to S18’) is constrained in a true alpha helical conformation. From this work, using disulfide trapping and whole cell electrophysiology, I conclude that a significant level of flexibility characterizes this part of the TM2 domain. This segment includes residue S267, previously shown to be significant for alcohol and anesthetic actions, as well as residue Q266 that, when mutated, produces a hyperekplexia-like phenotype. The range of movement of residues in this region may therefore play an important role not only in channel gating but also in how modulators of GlyR function exert their actions. / text