Este trabalho foi financiado pelo National Institutes of Health bolsas R01-R01-HL70393 e NS060706 para JCJC é um professor de engenharia biomédica na T. Spencer Olin Endowment.

1. Injeção de mRNA em oócitos <ol><li> Injetar 0,05-50 ng de RNA mensageiro que foi transcrita in vitro em oócitos Xenopus laevis (estágio V-VI), utilizando Nanoject II Auto-nanolitros Injector (Drummond empresa Scientific, modelo 3-000-204). Repetir a injecção em uma dúzia de ovócitos para cada mRNA. </li><li> Enxágüe os oócitos injetados duas vezes com solução ND-96 (96 mM de cloreto de sódio (NaCl, deputado 58,44 g / mol), 2 mM de cloreto de potássio (KCl, deputado 74,56 g / mol), 1,8 mM de cloreto de cálcio (CaCl 2 • 2H 2 O , o Sr. 147,02), 1 mM de cloreto de magnésio (MgCl 2 • 6H 2 O, o Sr. 203,31 g / mol), 5 mM 4 - (2-hidroxietil)-1-piperazineethanesulfonic ácido (HEPES, deputado 238,31 g / mol), 2,5 mM piruvato de sódio (Mr 110,04 g / mol), pH e 1x penicilina-estreptomicina. 7.6), em seguida, colocá-los em uma placa de cultura e incubar a 18 ° C por 2 dias +. </li></ol> 2. Preparando o sistema de perfusão <img src="/files/ftp_upload/2269/2269fig1.jpg" alt="Figura 1" /> Ilustração do sistema de perfusão Automatize ValveLink 16. O sistema de perfusão usa nitrogênio pressurizado para empurrar soluções para perfusão fora dos reservatórios de solução, através da perfusão tubulações, e para o lápis de perfusão e gorjeta. O fluxo de cada fluxo de solução de perfusão é controlada por uma válvula do reservatório e uma válvula eletrônica. Neste protocolo, um dos reservatórios não é pressurizado e funciona como um coletor de resíduos, bem como um mecanismo de liberação de pressão. (Imagem adaptada de http://www.autom8.com fornecedor website) <ol><li> Conectar as tubulações para o lápis de perfusão. Ligue o controlador de válvula eletrônica e abrir as válvulas eletrônicas. </li><li> Para assegurar que as tubulações e os lápis de perfusão são livres de bolhas de entupir e ar, empurrar a água (DI) deionizada através de cada tubo usando uma seringa com água deionizada. </li><li> Conecte o tubulações aos reservatórios solução e abrir a válvula do cilindro de gás nitrogênio pressurizado para aplicar. </li><li> Abrir a válvula do reservatório para encher o tubo com a solução do reservatório. Flick da válvula do reservatório para remover as bolhas na solução, se necessário. Feche a válvula após o tubo é preenchido com a solução. </li><li> Preencha a ponta de perfusão com água e aperte-o para o lápis de perfusão. Tenha cuidado para não prender as bolhas ao se conectar a ponta. </li><li> Fixar o lápis de perfusão para o palco banho usando massa de modelar. Certifique-se que a ponta de perfusão é no espaço do banho. O sistema de perfusão está agora configurado. </li><li> Adicione a água DI no banho. Certifique-se que a ponta de perfusão está submerso na água. </li><li> Teste que cada solução de perfusão sai da ponta de perfusão corretamente. Fechar a válvula eletrônico conectado à tubulação do coletor de resíduos e abrir a válvula para uma solução de perfusão de cada vez (140 mM de hidróxido de potássio (KOH, M r 56,1 g / mol), 20 mM 4 - (2-hidroxietil) -1 - piperazineethanesulfonic ácido (HEPES, M r 238,31 g / mol), 2 mM de cloreto de potássio (KCl, M r 74,56 g / mol), 1 mM de etileno glicol bis (2-aminoethylether)-N, N, N &#39;, N&#39;- tetraacetic ácido (EGTA, M r 380,35 g / mol), pH é ajustado para 7,1-7,2 por metanosulfonico ácido (meso 3, M r 96,1 g / mol), Ca 2 + ou Mg2 + é adicionado à concentração desejada quando necessário). Sob o microscópio, observe o jato de líquido de perfusão que sai da ponta de perfusão. Fechar a válvula para solução de perfusão e abrir a válvula para o coletor de resíduos. O jato de perfusão deve quase desaparecer. Repita o mesmo teste para todas as soluções para perfusão. </li><li> Quando você tiver verificado que todas as soluções de fluxo de perfusão corretamente, substituir a água DI no banho com uma solução de banho (140 mM de hidróxido de potássio (KOH, M r 56,1 g / mol), 20 mM 4 - (2-hidroxietil)-1-piperazineethanesulfonic ácido (HEPES, M r 238,31 g / mol), 2 mM de cloreto de potássio (KCl, M r 74,56 g / mol), pH é ajustado para 7,1-7,2 por metanosulfonico ácido (meso 3, M r 96,1 g / mol)). </li></ol> 3. Patch clamping <ol><li> Recobrimento do eletrodo de registro Ag com AgCl antes de cada patch clamping sessão. Primeiro, remova o fio de eletrodo do porta-pipeta e submergir o meia ponta do arame-eletrodo em um frasco contendo lixívia fresca pelo menos durante 15 minutos. Este depósitos uma camada de AgCl sobre o fio. </li><li> Lavar o arame eletrodo com água deionizada e secar. Em seguida, instalar o eletrodo Ag / AgCl de volta no suporte da pipeta. </li><li> Conecte o banho de eletrodo (referência) para o headstage e colocá-lo no banho. Isso prepara os eletrodos para gravação. </li><li> Agora preparar para aquisição de dados. Ligue o computador e ligue a chave de hardware na porta da impressora de seu computador. A chave de hardware deve ser ligada para que você use o software de aquisição de dados, que é Heka pulso. </li><li> Ligue o amplificador (Axon Instruments, AXOPATCH 200B) e começar a Heka Pulse. Carregar o arquivo de protocolo e ajustar as definições de configuração, tais como a Escala de estímulo. A meta para ajustar as definições de configuração é garantir que o potencial de teste é exatamente igual ao potencial de comando. Isso prepara o equipamento de aquisição de dados. </li><li> Prepare oócitos. Oócito submergir o stripping em solução (200 mM N-Metil-D-glucamina (NMG, M r 195,22 g / mol), 200 mM Aspartato (sem K +) (M r 133,1 g / mol), 2 mM de cloreto de potássio (KCl , M r 74,56 g / mol), 10 mM de etileno glicol bis (2-aminoethylether)-N, N, N &#39;, N&#39;-tetraacetic ácido (EGTA, M r 380,35 g / mol), 1 mM de cloreto de magnésio (MgCl 2 • 6H 2 O, M r 203,31 g / mol), 10 mM 4 - (2-hidroxietil)-1-piperazineethanesulfonic ácido (HEPES, M r 238,31 g / mol), pH é ajustado para 7,4 por hidróxido de sódio (NaOH, M r 40,0 g / mol) por 5 -. 10 min A solução stripping destaca a membrana vitelina da membrana plasmática que torna possível para tirar a membrana vitelina. </li><li> Gentilmente tira a membrana vitelina do oócito usando dois pares de fórceps. Um ovócito devitellinized é extremamente frágil e, portanto, deve ser movido o mínimo necessário. </li><li> Tendo o cuidado de evitar expor o ovócito devitellinized ao ar ou bolhas, use uma pipeta de vidro preenchidos com solução suficiente para transferir o ovócito ao banho. Isso prepara o óvulo para a gravação. </li><li> Prepare pipetas patch. Puxe a pipetas de vidro após a inserção de um tubo de vidro capilar (VWR International, Cat # 53432-921) em um Sutter P-97 Flaming / Brown Micropipeta Puller. Observe as pipetas ao microscópio para determinar a forma da ponta e diâmetro de abertura. O diâmetro de abertura deve ser 2-4 micrômetros. </li><li> Para reduzir a corrente capacitiva durante a gravação e para garantir uma ponta lisa, o revestimento da ponta com cera e depois do fogo-polonês-lo. </li><li> Encha a ponta da pipeta, colocando a ponta da pipeta dentro da solução de pipeta (140 mM de hidróxido de potássio (KOH, M r 56,1 g / mol), 20 mM 4 - (2-hidroxietil)-1-piperazineethanesulfonic ácido (HEPES, M r 238,31 g / mol), 2 mM de cloreto de potássio (KCl, M r 74,56 g / mol), 2 mM de cloreto de magnésio (MgCl 2 • 6H 2 O, M r 203,31 g / mol), pH é ajustado para 7,1-7,2 por metanosulfonico ácido (meso 3, M r 96,1 g / mol)) e desenho o êmbolo. Este molha a ponta. </li><li> Preencha pipeta 03/01 completa com pipeta solução utilizando uma seringa e coloque a pipeta no suporte da pipeta com a parte interna do eletrodo Ag / AgCl e entrar em contato com a solução da pipeta. Isto prepara a pipeta patch. </li><li> Para extirpar um patch de dentro para fora do oócito preparado, encontrar a clara vantagem do ovócito sob o microscópio. Mover a pipeta de patch perto do oócito usando manipulador. Anote a resistência série do pipeta. A resistência série ideal da pipeta patch é entre 1-1,5 mohms quando preenchido com solução de pipeta e submerso em solução de banho. </li><li> Empurre lentamente a pipeta contra o ovócito até a resistência aproximadamente dobra. </li><li> Aplicar sucção suave para a membrana por via oral, através de um tubo de sucção que é conectado ao titular da pipeta até um selo giga-ohm é obtido. Estabilizar o patch, segurando-o em tensão -30 mV para um par de minutos. </li><li> Para obter uma mancha de dentro para fora sobre o consumo, o patch rapidamente por empurrar a pipeta mais para o ovócito e, em seguida, puxando-o para fora. </li><li> O patch de dentro para fora agora está pronto para fixação. Mover a pipeta holding o patch de dentro para fora para cerca de 100 mícrons de distância da abertura da ponta de perfusão. </li><li> Fechar a válvula eletrônica para a recolha de resíduos e abrir a válvula do reservatório para a solução de perfusão desejado </li><li> Começar a gravar correntes em todo o patch. Mudar a tensão e soluções de perfusão, como desejado. </li><li> Quando você terminar a gravação, limpa a perfusão tubulações lápis, e empurrando a ponta através da água deionizada para evitar obstruções. </li></ol> 4. Resultados representante Durante a preparação dos ovócitos para patch clamp, o tratamento 10/05 min de solução stripping seria separar a membrana vitelina da membrana plasmática, o que torna possível stripping a membrana vitelina. A resistência série ideal da pipeta patch é entre 1-1,5 mohms quando preenchido com solução de pipeta e submerso em solução de banho. Segue-se uma gravação representante de canais do tipo selvagem BK em um remendo de membrana de ovócitos. No canto superior esquerdo, as ondas quadradas indicam a tensão aplicada ao patch - a segunda etapa do aumentos de tensão de 50 mV a 200 mV com incremento de 50 mV. Abaixo está os traços correspondentes atuais, quando a solução de perfusão foi nominal 0 Ca 2 + (livre [Ca 2 +] é cerca de 0,5 nM). O emaumento da amplitude da corrente indica que a probabilidade de abrir canais BK aumenta com a tensão. No lado direito, o patch mesmo é perfundido com 1,0 mM Ca 2 +, quando aplicado com o protocolo mesma tensão. A amplitude da corrente aumenta mais sob a mesma tensão, indicando que a probabilidade aumenta com open [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>

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O sistema de expressão de ovócitos é ideal para a caracterização eletrofisiológica de canais voltagem-dependentes de íons devido ao fundo relativamente baixo de canais endógena. Além disso, uma vez que é um sistema de expressão transitória, ela fornece um método eficiente de realizar estudo de mutagênese destes canais. No entanto, deve-se notar que o sistema de expressão de ovócitos é diferente do que em células de mamíferos, assim, pode haver diferenças na modificação pós-traducional e associaç?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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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>
<tr>
<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>
<tr>
<td>Amplifier</td>
<td>&nbsp;</td>
<td>Axon Instruments</td>
<td>AXOPATCH 200B</td>
<td>&nbsp;</td>
<tr>
<td>Computer Interface</td>
<td>&nbsp;</td>
<td>INSTRUTECH Corporation</td>
<td>ITC-18</td>
<td>&nbsp;</td>
<tr>
<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

O protocolo aqui apresentado foi concebido para estudar a ativação da condutância grande tensão e Ca 2 +-K + ativados (BK) canais. O protocolo também pode ser usado para estudar a relação estrutura-função de canais iônicos e outros receptores de neurotransmissores 1. Canais BK são amplamente expressos em diferentes tecidos e têm sido implicados em muitas funções fisiológicas, incluindo a regulação da contração do músculo liso, Afinação da frequência de células ciliadas internas e regulação da liberação de neurotransmissores 2-6. BK canais são ativados por despolarização da membrana e por Ca 2 + intracelular e Mg 2 + 6-9. Portanto, o protocolo é projetado para controlar a tensão de membrana ea solução intracelular. Neste protocolo, o RNA mensageiro dos canais BK é injetado dentro de oócitos Xenopus laevis (estágio V-VI), seguido de 2-5 dias de incubação a 18 ° C 10-13. Remendos da membrana que contêm um ou vários canais BK são excisadas com a configuração de dentro para fora usando técnicas de patch-clamp 10-13. O lado intracelular do patch é perfundido com soluções desejadas durante a gravação de modo que a ativação de canais em diferentes condições podem ser examinados. Para resumir, o mRNA de canais BK é injetado dentro de oócitos Xenopus laevis para expressar proteínas canal na membrana do oócito; técnicas de patch clamp são usados ​​para gravar correntes que fluem através dos canais sob tensão controlada e soluções intracelular.

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

Corrente iônica de canais BK é gravado usando técnicas de patch clamp. Canais BK são expressos em<em&gt; Oócitos Xenopus</em&gt; Através da injeção de RNA mensageiro. A solução intracelular durante gravações de patch clamp é controlado por um sistema de perfusão.

Grampo patch e Técnicas de Perfusão para estudar canais iônicos Expresso em Xenopus Oócitos

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

20.9K visualizações.  Washington University in St. Louis. Corrente iônica de canais BK é gravado usando técnicas de patch clamp. Canais BK são expressos em Oócitos Xenopus Através da injeção de RNA mensageiro. A solução intracelular durante gravações de patch clamp é controlado por um sistema de perfusão.

Vídeo: 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 Recordings patch-clamp Capacitância da Calyx

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

Biologia

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.

Gravação de patch-clamp de canais iônicos Expresso em Oócitos 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.

Pressão polimento Pipetas para Gravação de patch-clamp melhorada

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.

Microinjeção de Oócitos Xenopus laevis

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

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Técnicas de microinjeção para estudar na Mitose Drosophila melanogaster Embrião Sincicial

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

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.

Visualizando Localização RNA em Xenopus Oócitos

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.

Mutagênese e Análise Funcional de canais iônicos Heterologously Expresso em células de mamíferos

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.

Preparação de lipossomas gigantes por Imagem e patch-clamp Eletrofisiologia

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

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

O<em&gt; Xenopus</em&gt; Oocyte Cut-aberto vaselina Gap Técnica Voltage-clamp Com Fluorometria

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

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.

Um canal de gravação de patch-clamp anexado-Cell

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