Generación de una línea celular estable en células HEK293, como modelo celular para el estudio de la actividad de canales BK.

Los canales de alta conductancia BK, son canales iónicos de potasio que pueden ser activados por voltaje y por calcio, lo cual hace que tengan un rol fisiológico importante en la repolarización de la membrana en respuesta a cambios en la concentración intracelular de calcio ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/0896-6273(95)90321-6","ISBN":"0896-6273 (Print)\\r0896-6273 (Linking)","ISSN":"08966273","PMID":"7695911","abstract":"Mammalian high conductance, calcium-activated potassium (maxi-K) channels are composed of two dissimilar subunits, α and β. We have examined the functional contribution of the β subunit to the properties of maxi-K channels expressed heterologously in Xenopus oocytes. Channels from oocytes injected with cRNAs encoding both a and β subunits were much more sensitive to activation by voltage and calcium than channels composed of the α subunit alone, while expression levels, single-channel conductance, and ionic selectivity appeared unaffected. Channels from oocytes expressing both subunits were sensitive to DHS-I, a potent agonist of native maxi-K channels, whereas channels composed of the α subunit alone were insensitive. Thus, α and β subunits together contribute to the functional properties of expressed maxi-K channels. Regulation of coassembly might contribute to the functional diversity noted among members of this family of potassium channels. © 1995.","author":[{"dropping-particle":"","family":"McManus","given":"Owen B.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Helms","given":"Lisa M H","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Pallanck","given":"Leo","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ganetzky","given":"Barry","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Swanson","given":"Richard","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Leonard","given":"Reid J.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Neuron","id":"ITEM-1","issue":"3","issued":{"date-parts":[["1995"]]},"page":"645-650","title":"Functional role of the β subunit of high conductance calcium-activated potassium channels","type":"article-journal","volume":"14"},"uris":["http://www.mendeley.com/documents/?uuid=b49e600e-dfa7-4d82-9af6-076ad0f24762"]}],"mendeley":{"formattedCitation":"(McManus et al., 1995)","plainTextFormattedCitation":"(McManus et al., 1995)","previouslyFormattedCitation":"(McManus et al., 1995)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(McManus et al., 1995; Orio et al., 2002). Estos canales están implicados en una gran diversidad de procesos que incluyen liberación de catecolaminas, regulación de la vasoconstricción, cáncer y dolor. Esto hace que sea importante la identificación de moléculas que puedan tener potencial terapéutico para patologías asociadas a la actividad de canales BK ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1073/pnas.2204620119","ISSN":"10916490","PMID":"35704760","abstract":"In neurosecretion, allosteric communication between voltage sensors and Ca2+ binding in BK channels is crucially involved in damping excitatory stimuli. Nevertheless, the voltage-sensing mechanism of BK channels is still under debate. Here, based on gating current measurements, we demonstrate that two arginines in the transmembrane segment S4 (R210 and R213) function as the BK gating charges. Significantly, the energy landscape of the gating particles is electrostatically tuned by a network of salt bridges contained in the voltage sensor domain (VSD). Molecular dynamics simulations and proton transport experiments in the hyperpolarization-activated R210H mutant suggest that the electric field drops off within a narrow septum whose boundaries are defined by the gating charges. Unlike Kv channels, the charge movement in BK appears to be limited to a small displacement of the guanidinium moieties of R210 and R213, without significant movement of the S4.","author":[{"dropping-particle":"","family":"Carrasquel-Ursulaez","given":"Willy","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Segura","given":"Ignacio","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Díaz-Franulic","given":"Ignacio","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Echeverría","given":"Felipe","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lorenzo-Ceballos","given":"Yenisleidy","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Espinoza","given":"Nicolás","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Rojas","given":"Maximiliano","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Garate","given":"Jose Antonio","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Perozo","given":"Eduardo","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Alvarez","given":"Osvaldo","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gonzalez-Nilo","given":"Fernando D.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Latorre","given":"Ramón","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Proceedings of the National Academy of Sciences of the United States of America","id":"ITEM-1","issue":"25","issued":{"date-parts":[["2022"]]},"page":"1-11","title":"Mechanism of voltage sensing in Ca2+- and voltage-activated K+ (BK) channels","type":"article-journal","volume":"119"},"uris":["http://www.mendeley.com/documents/?uuid=cbe09138-b836-42df-8ffa-bbea30670c9b"]}],"mendeley":{"formattedCitation":"(Carrasquel-Ursulaez et al., 2022)","plainTextFormattedCitation":"(Carrasquel-Ursulaez et al., 2022)","previouslyFormattedCitation":"(Carrasquel-Ursulaez et al., 2022)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Carrasquel-Ursulaez et al., 2022; Ancatén-González et al., 2023). Para el estudio de interacciones específicas entre estos canales y moléculas con potencial terapéutico es conveniente utilizar células que tengan una alta expresión de los canales de interés, lo cual se puede obtener utilizando sistemas de expresión heteróloga ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1152/physrev.00001.2016","ISSN":"15221210","abstract":"Large-conductance Ca2+- and voltage-activated K+ (BK) channels play many physiological roles ranging from the maintenance of smooth muscle tone to hearing and neurosecretion. BK channels are tetramers in which the pore-forming α subunit is coded by a single gene (Slowpoke, KCNMA1). In this review, we first highlight the physiological importance of this ubiquitous channel, emphasizing the role that BK channels play in different channelopathies. We next discuss the modular nature of BK channel-forming protein, in which the different modules (the voltage sensor and the Ca2+ binding sites) communicate with the pore gates allosterically. In this regard, we review in detail the allosteric models proposed to explain channel activation and how the models are related to channel structure. Considering their extremely large conductance and unique selectivity to K+, we also offer an account of how these two apparently paradoxical characteristics can be understood consistently in unison, and what we have learned about the conduction system and the activation gates using ions, blockers, and toxins. Attention is paid here to the molecular nature of the voltage sensor and the Ca2+ binding sites that are located in a gating ring of known crystal structure and constituted by four COOH termini. Despite the fact that BK channels are coded by a single gene, diversity is obtained by means of alternative splicing and modulatory β and γ subunits. We finish this review by describing how the association of the α subunit with β or with γ subunits can change the BK channel phenotype and pharmacology.","author":[{"dropping-particle":"","family":"Latorre","given":"Ramon","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Castillo","given":"Karen","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Carrasquel-Ursulaez","given":"Willy","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"V.","family":"Sepulveda","given":"Romina","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gonzalez-Nilo","given":"Fernando","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gonzalez","given":"Carlos","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Alvarez","given":"Osvaldo","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Physiological Reviews","id":"ITEM-1","issue":"1","issued":{"date-parts":[["2017"]]},"page":"39-87","title":"Molecular determinants of BK channel functional diversity and functioning","type":"article-journal","volume":"97"},"uris":["http://www.mendeley.com/documents/?uuid=d292d2d4-94d2-41f6-9a11-cdfbb2a8c4f8"]}],"mendeley":{"formattedCitation":"(Latorre et al., 2017)","plainTextFormattedCitation":"(Latorre et al., 2017)","previouslyFormattedCitation":"(Latorre et al., 2017)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Latorre et al., 2017; Pratt et al., 2017). Esto permite limitar la interferencia de otro tipo de canales y restringir los efectos observados a la actividad de los canales iónicos que se están estudiando. Adicionalmente, al tener una alta expresión de los canales de interés, es posible realizar ensayos en los cuales se requiere una alta concentración de esas moléculas. Para establecer el sistema de expresión heteróloga, se utilizan modelos celulares que tienen características ampliamente descritas y una alta capacidad de proliferación. El proceso implica realizar transfecciones utilizando agentes formados por lípidos catiónicos disponibles a nivel comercial, como la lipofectamina. La transfección puede ser estable o transitoria ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1371/journal.pone.0243645","ISBN":"1111111111","ISSN":"19326203","PMID":"33667217","abstract":"Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, CaV2.2 and NaV1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPRTetra and the automated patch clamp QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPRTetra assays. Lipofectamine 3000-mediated transfection produced the largest peak currents for automated patch clamp QPatch assays. However, the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch clamp configuration, medium to large peak currents, and higher rates of accomplished assays for both CaV2.2 and NaV1.7 channels. Our findings can facilitate the development of HT automated patch clamp assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharmacology of mutated channels.","author":[{"dropping-particle":"","family":"Hasan","given":"Md Mahadhi","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ragnarsson","given":"Lotten","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Cardoso","given":"Fernanda C.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lewis","given":"Richard J.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"PLoS ONE","id":"ITEM-1","issue":"3 March 2021","issued":{"date-parts":[["2021"]]},"page":"1-18","title":"Transfection methods for high-throughput cellular assays of voltage-gated calcium and sodium channels involved in pain","type":"article-journal","volume":"16"},"uris":["http://www.mendeley.com/documents/?uuid=540b7183-7b92-46f6-b88d-02086ca1cb05"]}],"mendeley":{"formattedCitation":"(Hasan et al., 2021)","plainTextFormattedCitation":"(Hasan et al., 2021)","previouslyFormattedCitation":"(Hasan et al., 2021)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(Hasan et al., 2021). En este último caso, es necesario realizar el proceso cada vez que se requiera el uso de las células, lo que incrementa significativamente tanto el costo de los ensayos como el tiempo dedicado a los mismos. Por tal motivo, es conveniente trabajar con una línea celular que exprese de forma estable los canales de interés. De acuerdo con lo anterior, en este trabajo se propone obtener células HEK293 que expresen de forma estable los canales BK. Esto permitirá tener un sistema de expresión estable que será utilizado para realizar ensayos de electrofisiología, en los cuales es posible evaluar el efecto de diferentes moléculas sobre la actividad de canales BK. Aunque este proceso no es novedoso, pues se ha realizado para el estudio de diversos tipos de canales, sí brinda una ventaja para los ensayos asociados a los proyectos que llevamos a cabo en el grupo de investigación, a los cuales se espera vincular a corto plazo a los miembros del semillero de investigación.