Este trabajo fue financiado por los Institutos Nacionales de Salud subvenciones R01-R01-HL70393 y NS060706 a JCJC es un profesor de ingeniería biomédica de la Fundación Spencer T. Olin.

1. La inyección de ARNm en los oocitos <ol><li> Inyectar 0,05 a 50 ng de ARN mensajero que se transcribe in vitro en ovocitos de Xenopus laevis (fase V-VI) con Nanoject II Auto-Inyector nanolitros (Drummond Scientific Company, modelo 3-000-204). Repetir la inyección en una docena de ovocitos de cada ARNm. </li><li> Enjuague los ovocitos inyectados dos veces con solución de ND-96 (96 mM de cloruro de sodio (NaCl, el Sr. 58,44 g / mol), 2 mM de cloruro de potasio (KCl, el Sr. 74,56 g / mol), 1,8 mM de cloruro de calcio (CaCl 2 • 2H 2 O , el Sr. 147.02), 1 mM de cloruro de magnesio (MgCl 2 • 6H 2 O, el Sr. 203,31 g / mol), 5 mm 4 - (2-hidroxietil)-1-piperazineethanesulfonic ácido (HEPES, el Sr. 238,31 g / mol), 2,5 mM piruvato de sodio (Sr. 110,04 g / mol), y 1 de penicilina-estreptomicina. pH 7,6), y luego colocarlos en una placa de cultivo y se incuba a 18 ° C durante 2 días +. </li></ol> 2. Preparación del sistema de perfusión <img src="/files/ftp_upload/2269/2269fig1.jpg" alt="Figura 1" /> Ilustración del sistema de Automatización de ValveLink perfusión 16. El sistema de perfusión utiliza nitrógeno a presión para empujar las soluciones de perfusión de los embalses de solución, a través de los tubos de perfusión, y el lápiz de perfusión y la propina. El flujo de cada flujo de solución de perfusión es controlada por la válvula de un depósito y una válvula electrónica. En este protocolo, uno de los reservorios no está presurizado, y funciona como un recolector de residuos, así como un mecanismo de liberación de presión. (Imagen adaptada de http://www.autom8.com sitio web del proveedor) <ol><li> Conectar los tubos para el lápiz de perfusión. Encienda el controlador de la válvula electrónica y abrir las válvulas electrónicas. </li><li> Para asegurar que los tubos y el lápiz de la perfusión es libre de burbujas y tapar el aire, empuje desionizada (DI) a través de cada tubo con una jeringa llena de agua desionizada. </li><li> Conectar los tubos a los depósitos de solución y abrir la válvula del cilindro de gas a aplicar nitrógeno a presión. </li><li> Abra la válvula del depósito para llenar la tubería con una solución de reserva. Flick de la válvula de reservorio para eliminar las burbujas en la solución si es necesario. Cierre la válvula después de que el tubo se llena con la solución. </li><li> Llenar la punta de perfusión con agua y apriete los tornillos en el lápiz de la perfusión. Tenga cuidado de no atrapar las burbujas cuando se conecta la punta. </li><li> Fijar el lápiz de perfusión a la etapa de baño usando arcilla. Asegúrese de que la punta de la perfusión en el espacio de baño. El sistema de perfusión ya está configurado. </li><li> Añadir agua desionizada en el baño. Asegúrese de que la punta de la perfusión se sumerge en el agua. </li><li> Prueba de que cada solución de perfusión que sale de la punta de la perfusión correctamente. Cierre la válvula electrónica conectada a la tubería del colector de residuos y abrir la válvula para la solución de perfusión de uno en uno (140 mM de hidróxido de potasio (KOH, M r 56,1 g / mol), 20 mM de 4 - (2-hidroxietil) -1 - piperazineethanesulfonic ácido (HEPES, M r 238,31 g / mol), 2 mM de cloruro de potasio (KCl, M r 74,56 g / mol), 1 mM de etilenglicol-bis (2-aminoethylether)-N, N, N &#39;, N&#39;- tetraacético (EGTA, M r 380,35 g / mol), se ajusta el pH a 7,1-7,2 por ácido metanosulfónico (MESO 3, M r 96,1 g / mol), Ca 2 + o Mg2 + se suma a la concentración deseada cuando sea necesario). Bajo el microscopio, observar el chorro de líquido de perfusión que sale de la punta de la perfusión. Cierre la válvula de solución de perfusión y abrir la válvula para el recolector. El chorro de la perfusión debe casi desaparecer. Repita la misma prueba para todas las soluciones para infusión. </li><li> Cuando haya verificado que todas las soluciones de flujo de perfusión correctamente, reemplace el agua DI en el baño con una solución de baño (140 mM de hidróxido de potasio (KOH, M r 56,1 g / mol), 20 mM de 4 - (2-hidroxietil)-1-piperazineethanesulfonic ácido (HEPES, M r 238,31 g / mol), 2 mM de cloruro de potasio (KCl, M r 74,56 g / mol), se ajusta el pH a 7,1-7,2 por ácido metanosulfónico (MESO 3, M r 96,1 g / mol)). </li></ol> 3. Patch clamp <ol><li> Recubrir el electrodo de registro Ag con AgCl antes de cada sesión de patch clamp. En primer lugar, quitar el cable del electrodo del titular de la pipeta y sumergir la media punta de la aguja electrodo en un frasco que contiene lejía fresca durante al menos 15 minutos. Esto deposita una capa de AgCl en el cable. </li><li> Enjuague el alambre del electrodo con agua destilada y secar. Luego de instalar el electrodo de Ag / AgCl de nuevo en el soporte de la pipeta. </li><li> Conecte el baño (de referencia) del electrodo a la headstage y colocarlo en el baño. Esto prepara a los electrodos para la grabación. </li><li> Ahora a prepararse para la adquisición de datos. Encienda el ordenador y conectar la llave de hardware en el puerto de impresora de su ordenador. La llave de hardware debe estar conectado para poder utilizar el software de adquisición de datos, que es HEKA pulso. </li><li> Encienda el amplificador (Axon Instruments, artefactos explosivos abandonadosPARCHE 200B) y empezar a HEKA pulso. Cargar el archivo de protocolo y los ajustes de configuración, como la Escala de estímulo. El objetivo para el ajuste de los parámetros de configuración es asegurar que el potencial de la prueba es exactamente igual a la capacidad de mando. Esto prepara el equipo de adquisición de datos. </li><li> Prepare los ovocitos. Sumerja el ovocito en la solución de extracción (200 mM de N-metil-D-glucamina (NMG, M r 195,22 g / mol), 200 mM aspartato (no K +) (M r 133,1 g / mol), 2 mM de cloruro de potasio (KCl , M r 74,56 g / mol), 10 mM de etilenglicol-bis (2-aminoethylether)-N, N, N &#39;, N&#39;-tetraacético (EGTA, M r 380,35 g / mol), 1 mM de cloruro de magnesio (MgCl 2 • 6H 2 O, M r 203,31 g / mol), 10 mM de 4 - (2-hidroxietil)-1-piperazineethanesulfonic ácido (HEPES, M r 238,31 g / mol), se ajusta el pH a 7,4 con hidróxido de sodio (NaOH, M r 40,0 g / mol) de 5 - 10 min. La solución de extracción se separa la membrana vitelina de la membrana plasmática que permite despojar a la membrana vitelina. </li><li> Suavemente tira de la membrana vitelina del ovocito utilizando dos pares de pinzas. Un ovocito devitellinized es extremadamente frágil y, por tanto, moverse tan poco como sea necesario. </li><li> Teniendo cuidado de evitar la exposición de los ovocitos devitellinized de aire o burbujas, utilizar una pipeta de vidrio llena con una solución suficiente para transferir el óvulo de la bañera. Esto prepara al ovocito para la grabación. </li><li> Prepare pipetas parche. Tire de la pipetas de vidrio después de la inserción de un tubo capilar de vidrio (VWR International, Cat # 53432 a 921) en un Sutter P-97 Flaming / Brown Micropipeta extractor. Observe las pipetas bajo el microscopio para determinar la forma de la punta y el diámetro de apertura. El diámetro de la abertura debe ser 4.2 micrómetros. </li><li> Para reducir la corriente capacitiva durante la grabación y para asegurar una punta suave, cubra la punta con cera y luego el fuego de uñas. </li><li> Llenar la pipeta mediante la colocación de la punta de la pipeta dentro de la solución de la pipeta (140 mM de hidróxido de potasio (KOH, M r 56,1 g / mol), 20 mM de 4 - (2-hidroxietil)-1-piperazineethanesulfonic ácido (HEPES, M r 238,31 g / mol), 2 mM de cloruro de potasio (KCl, M r 74,56 g / mol), 2 mM de cloruro de magnesio (MgCl 2 • 6H 2 O, M r 203,31 g / mol), se ajusta el pH a 7,1-7,2 por metano ácido (MESO 3, M r 96,1 g / mol)) y el dibujo del émbolo. Este moja la punta. </li><li> Llenar la pipeta 1 / 3 del total con una solución de pipeta con una jeringa y coloque la pipeta en el soporte de la pipeta con el interior del electrodo Ag / AgCl y el contacto con la solución de la pipeta. Esto prepara el parche pipeta. </li><li> Para extirpar un parche de dentro a fuera de los ovocitos preparados, encuentre la clara ventaja de los ovocitos bajo el microscopio. Mueva el parche pipeta cerca del ovocito utilizando manipulador. Registrar la resistencia de serie de la pipeta. La resistencia en serie ideal de la pipeta parche está entre 1-1.5 mW cuando se llena con solución de la pipeta y se sumergen en una solución de baño. </li><li> Vuelva a introducir con la pipeta contra el ovocito hasta que la resistencia se duplica. </li><li> Aplicar succión suave a la membrana por vía oral a través de un tubo de aspiración que está conectado al titular de la pipeta hasta que un sello de giga-ohm se obtiene. Estabilizar el parche mediante la celebración de la tensión en mV -30 para un par de minutos. </li><li> Para obtener un parche de adentro hacia fuera, los impuestos especiales por el parche rápidamente empujando aún más la pipeta en el ovocito y luego tirando de él hacia fuera. </li><li> El parche de adentro hacia afuera ya está listo para la sujeción. Mueva la pipeta de sujeción del parche de adentro hacia fuera a cerca de 100 micras de distancia de la abertura de la punta de la perfusión. </li><li> Cierre la válvula electrónica de recogida de residuos y abra la válvula de reservorio para la solución de perfusión deseado </li><li> Comenzar a grabar las corrientes a través del parche. Cambiar las soluciones de tensión y de la perfusión como se desee. </li><li> Cuando haya terminado de grabar, limpie la perfusión de tubos, lápiz y punta empujando a través de agua destilada para evitar obstrucciones. </li></ol> 4. Resultados representante Durante la preparación de los oocitos de patch clamp, el tratamiento de 5-10 minutos de la solución de extracción se separan de la membrana vitelina de la membrana plasmática, lo que hace posible extracción de la membrana vitelina. La resistencia en serie ideal de la pipeta parche está entre 1-1.5 mW cuando se llena con solución de la pipeta y se sumergen en una solución de baño. A continuación se presenta un registro representativo de tipo salvaje canales BK en un parche de membrana del ovocito. En la parte superior izquierda, las ondas cuadradas indican el voltaje aplicado a la mancha - el segundo paso de los aumentos de voltaje de 50 mV a 200 mV con un incremento de 50 mV. A continuación se muestra las huellas correspondientes actual cuando la solución de perfusión ha nominal 0 Ca 2 + (libre [Ca 2 +] es de aproximadamente 0,5 nM). Enaumento de la amplitud de la corriente indica que la probabilidad de abrir los canales BK de los incrementos de voltaje. En el lado derecho, el mismo parche es perfundidos con 1,0 M de Ca 2 + cuando se aplica con el protocolo mismo voltaje. La amplitud de la corriente se incrementa más en el mismo voltaje, lo que indica que la probabilidad aumenta con la abierta [Ca 2 +]. <img src="/files/ftp_upload/2269/2269fig2.jpg" alt="Figura 2" />

<ol>
	<li>Methfessel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+measurements+on+Xenopus+laevis+oocytes:+currents+through+endogenous+channels+and+implanted+acetylcholine+receptor+and+sodium+channels.">Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels.</a> Pfl&#252;gers Arch. 407, 577-588 (1986).</li><li>Toro, L., Wallner, M., Meera, P., Tanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maxi-K+(Ca),+Unique+Member+of+the+Voltage-Gated+K+Channel+Superfamily.">Maxi-K (Ca), Unique Member of the Voltage-Gated K Channel Superfamily.</a> News Physiol. Sci. 13, 112-117 (1998).</li><li>Fettiplace, R., Fuchs, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+hair+cell+tuning.">Mechanisms of hair cell tuning.</a> Annu. Rev. Physiol. 61, 809-834 (1999).</li><li>Du, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-sensitive+potassium+channelopathy+in+human+epilepsy+and+paroxysmal+movement+disorder.">Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder.</a> Nat. Genet. 37, 733-738 (2005).</li><li>Ledoux, J., Werner, M. E., Brayden, J. E., Nelson, M. T., T, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-activated+potassium+channels+and+the+regulation+of+vascular+tone.">Calcium-activated potassium channels and the regulation of vascular tone.</a> Physiology (Bethesda). 21, 69-78 (2006).</li><li>Salkoff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-conductance+potassium+channels+of+the+SLO+family.">High-conductance potassium channels of the SLO family.</a> Nat. Rev. Neurosci. 7, 921-931 (2006).</li><li>Shi, J., Cui, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Mg2++enhances+the+function+of+BK-type+Ca2+-activated+K++channels.">Intracellular Mg2+ enhances the function of BK-type Ca2+-activated K+ channels.</a> J. Gen. Physiol. 118, 589-606 (2001).</li><li>Magleby, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gating+mechanism+of+BK+(Slo1)+channels:+so+near,+yet+so+far.">Gating mechanism of BK (Slo1) channels: so near, yet so far.</a> J. Gen. Physiol. 121, 81-96 (2003).</li><li>Latorre, R., Brauchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+conductance+Ca2+-activated+K++(BK)+channel:+activation+by+Ca2++and+voltage.">Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage.</a> Biol. Res. 39, 385-401 (2006).</li><li>Cox, D. H., Cui, J., Aldrich, R. W., W, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+gating+of+a+large+conductance+Ca-activated+K++channel.">Allosteric gating of a large conductance Ca-activated K+ channel.</a> J. Gen. Physiol. 110, 257-281 (1997).</li><li>Cui, J., Aldrich, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+linkage+between+voltage+and+Ca2+-dependent+activation+of+BK-type+mslo1.">Allosteric linkage between voltage and Ca2+-dependent activation of BK-type mslo1.</a> K+ channels. Biochemistry. 39, 15612-15619 (2000).</li><li>Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Slo1+BK+channels+by+Mg2++coordinated+between+the+voltage+sensor+and+RCK1.">Activation of Slo1 BK channels by Mg2+ coordinated between the voltage sensor and RCK1.</a> 15, 1152-1159 (2008).</li><li>Lee, U. S., Cui, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term={beta}+subunit-specific+modulations+of+BK+channel+function+by+a+mutation+associated+with+epilepsy+and+dyskinesia.">{beta} subunit-specific modulations of BK channel function by a mutation associated with epilepsy and dyskinesia.</a> J. Physiol. , 587-1481 (2009).</li></ol>

El sistema de expresión de ovocitos es ideal para la caracterización electrofisiológica de los canales iónicos dependientes de voltaje debido a la relativamente baja de fondo de los canales endógenos. Además, dado que se trata de un sistema de expresión transitoria, que proporciona un método eficiente de llevar a cabo el estudio de mutagénesis de estos canales. Sin embargo, cabe señalar que el sistema de expresión de los ovocitos es diferente de la de células de mamíferos, por lo tanto puede haber diferenci...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Nanoject II Auto-Nanoliter Injector</td>
<td>&nbsp;</td>
<td>Drummond Scientific Company</td>
<td>3-000-204</td>
<td>&nbsp;</td>
<tr>
<td>P-97 Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Perfusion System and Electronic Controller</td>
<td>&nbsp;</td>
<td>AutoMate Scientific, Inc.</td>
<td>ValveLink 16</td>
<td>Inner diameter of perfusion tip: 100 microns</td>
</tr>
<tr>
<td>Inverted Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>CKX31</td>
<td>&nbsp;</td>
<tr>
<td>Glass Pipettes</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>53432-921</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument Co.</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Amplifier</td>
<td>&nbsp;</td>
<td>Axon Instruments</td>
<td>AXOPATCH 200B</td>
<td>&nbsp;</td>
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<td>Computer Interface</td>
<td>&nbsp;</td>
<td>INSTRUTECH Corporation</td>
<td>ITC-18</td>
<td>&nbsp;</td>
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<td>Headstage</td>
<td>&nbsp;</td>
<td>Axon Instruments</td>
<td>CV 203BU</td>
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patch clamp and perfusion techniques for studying ion channels expressed in xenopus oocytes

El protocolo que aquí se presenta está diseñado para estudiar la activación de la gran conductancia, el voltaje y Ca 2 +-K + activado (BK) canales. El protocolo también puede ser usado para estudiar la relación estructura-función de otros canales iónicos y receptores de los neurotransmisores 1. Canales BK se expresan ampliamente en diferentes tejidos y se han implicado en muchas funciones fisiológicas, incluyendo la regulación de la contracción del músculo liso, Ajuste de la frecuencia de las células ciliadas internas y la regulación de la liberación de neurotransmisores 2-6. Canales BK son activados por despolarización de la membrana y por intracelular de Ca 2 + y Mg 2 + 9.6. Por lo tanto, el protocolo está diseñado para controlar tanto el voltaje de la membrana y la solución intracelular. En este protocolo, el ARN mensajero de los canales BK se inyecta en ovocitos de Xenopus laevis (fase V-VI), seguido de 2-5 días de incubación a 18 ° C 10-13. Parches de membrana que contienen canales BK uno o varios son extirpados con la configuración de adentro hacia afuera utilizando técnicas de patch clamp 10-13. La parte intracelular del parche es perfundidos con soluciones deseadas durante la grabación para que la activación del canal en condiciones diferentes pueden ser examinados. En resumen, el ARNm de los canales BK se inyecta en ovocitos de Xenopus laevis que expresan proteínas de canales en la membrana del ovocito, las técnicas de patch clamp se utilizan para registrar las corrientes que fluyen a través de los canales de bajo voltaje controlado y soluciones intracelulares.

Watch this Scientific Journal Video about Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes at JoVE.com

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Corriente iónica de los canales BK se registraron con la técnica de patch clamp. Canales BK se expresan en<em&gt; Oocitos de Xenopus</em&gt; Mediante la inyección de ARN mensajero. La solución intracelular durante las grabaciones de patch clamp es controlado por un sistema de perfusión.

Patch clamp y técnicas de perfusión para el estudio de los canales iónicos expresados ​​en Xenopus Ovocitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

patch-clamp-perfusion-techniques-for-studying-ion-channels-expressed

Research

JoVE Journal

Biology

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

20.8K vistas.  Washington University in St. Louis. Corriente iónica de los canales BK se registraron con la técnica de patch clamp. Canales BK se expresan en Oocitos de Xenopus Mediante la inyección de ARN mensajero. La solución intracelular durante las grabaciones de patch clamp es controlado por un sistema de perfusión.

Video: Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

Métodos para grabaciones de parche Capacidad de la pinza del cáliz

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Investigación

Biología

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Parche de grabación Clamp de canales iónicos expresados ​​en ovocitos de Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

Pulido a presión para la Mejora de pipetas de patch-clamp grabación

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinyección de oocitos de Xenopus laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualizar la localización del ARN en Xenopus Los ovocitos

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagénesis y análisis funcional de los canales iónicos heteróloga se expresa en células de mamífero

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

Preparación de liposomas gigantes de la Imagen y Patch-Clamp Electrofisiología

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Un canal celular conectado a Patch-clamp grabación

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Reconstitución de una proteína transmembrana, el canal iónico dependiente de voltaje, KvAP, en Gigante vesículas unilamelares para Microscopía y Patch Clamp Estudios

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

Técnicas de Imagen y Prometafase metafase de la meiosis I en fijo<em&gt; Drosophila</emLos ovocitos&gt;

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

Patch clamp y técnicas de perfusión para el estudio de los canales iónicos expresados ​​en Xenopus Ovocitos

Patch clamp y técnicas de perfusión para el estudio de los canales iónicos expresados en Xenopus Ovocitos