Ce travail a été soutenu par les instituts nationaux de subventions de la santé et la R01-R01-HL70393 NS060706 d&#39;JCJC est un professeur de génie biomédical de la dotation Spencer T. Olin.

1. L&#39;injection d&#39;ARNm dans des ovocytes <ol><li> Injecter 0,05 à 50 ng d&#39;ARN messager qui a été transcrit in vitro dans des ovocytes de Xenopus laevis (stade V-VI) en utilisant Nanoject II Auto-injecteur nanolitres (Drummond Scientific Company, modèle 3-000-204). Répéter l&#39;injection sur une douzaine d&#39;ovocytes pour chaque ARNm. </li><li> Rincez les ovocytes injectés à deux reprises avec ND-96 solution (96 mM de chlorure de sodium (NaCl, M. 58,44 g / mol), 2 mM de chlorure de potassium (KCl, M. 74,56 g / mol), 1,8 mM de chlorure de calcium (CaCl 2 • 2H 2 O , M. 147.02), 1 mM de chlorure de magnésium (MgCl 2 • 6H 2 O, M. 203,31 g / mol), 5 mM de 4 - (2-hydroxyéthyl)-1-pipérazineéthanesulfonique (HEPES, M. 238,31 g / mol), 2,5 mM pyruvate de sodium (M. 110,04 g / mol), et 1x pénicilline-streptomycine. pH 7,6), puis les placer dans une plaque de culture et incuber à 18 ° C pendant 2 jours +. </li></ol> 2. Préparer le système de perfusion <img src="/files/ftp_upload/2269/2269fig1.jpg" alt="Figure 1" /> Illustration du système Automatiser ValveLink perfusion 16. Le système de perfusion utilise de l&#39;azote sous pression pour pousser les solutions de perfusion de la solution de réservoirs, à travers les tubulures de perfusion, et le crayon de perfusion et de pointe. Le débit de chaque flux de solution de perfusion est contrôlé par une valve du réservoir et une valve électronique. Dans ce protocole, l&#39;un des réservoirs n&#39;est pas sous pression et fonctionne comme un collecteur de déchets ainsi que d&#39;un mécanisme de pression à la libération. (Image adapté de http://www.autom8.com site du vendeur) <ol><li> Connectez les tuyaux pour le crayon de perfusion. Mettez le contrôleur de valve électronique et ouvrir les vannes électroniques. </li><li> Afin d&#39;assurer que les tuyaux et le crayon de perfusion sont libres de bulles d&#39;air et d&#39;obstruer, poussez déminéralisée (DI) de l&#39;eau à travers chaque tube à l&#39;aide d&#39;une seringue remplie d&#39;eau déminéralisée. </li><li> Connectez les tuyaux aux réservoirs de solution et ouvrir la vanne de gaz en bouteilles pour appliquer l&#39;azote sous pression. </li><li> Ouvrez le robinet de réservoir pour remplir le tube avec une solution réservoir. Flick la valve du réservoir pour éliminer les bulles dans la solution si nécessaire. Fermer le robinet après que le tuyau est rempli de solution. </li><li> Remplissez la pointe de perfusion avec de l&#39;eau et de le visser sur le crayon de perfusion. Soyez prudent de ne pas emprisonner les bulles lors de la connexion de la pointe. </li><li> Fixer le crayon de perfusion à l&#39;étape de bain à l&#39;aide de pâte à modeler. Assurez-vous que la pointe de perfusion est dans l&#39;espace bain. Le système de perfusion est maintenant mis en place. </li><li> Ajouter de l&#39;eau DI dans le bain. Assurez-vous que la pointe de perfusion est immergé dans l&#39;eau. </li><li> Testez que chaque solution de perfusion sort de la pointe de perfusion correctement. Fermer le robinet électronique relié à la tubulure du collecteur de déchets et ouvrir la vanne pour une solution de perfusion à un moment (140 mM d&#39;hydroxyde de potassium (KOH, M r 56,1 g / mol), 20 mm 4 - (2-hydroxyéthyl) -1 - pipérazineéthanesulfonique (HEPES, M r 238,31 g / mol), 2 mM de chlorure de potassium (KCl, M r 74,56 g / mol), 1 mM éthylène glycol-bis (2-aminoethylether)-N, N, N &#39;, N&#39;- tétraacétique (EGTA, M r 380,35 g / mol), le pH est ajusté à 7.1 à 7.2 par l&#39;acide méthane sulfonique (MESO 3, M r 96,1 g / mol), Ca 2 + ou Mg 2 + est ajouté à la concentration voulue en cas de besoin). Sous le microscope, observer le jet de liquide de perfusion sortant de la pointe de perfusion. Fermer le robinet pour solution pour perfusion et ouvrir la vanne du collecteur de déchets. Le jet de perfusion devrait presque disparaître. Répétez le même test pour toutes les solutions de perfusion. </li><li> Lorsque vous avez vérifié que toutes les solutions de flux de perfusion correctement, remplacer l&#39;eau DI dans le bain avec une solution de bain (140 mM d&#39;hydroxyde de potassium (KOH, M r 56,1 g / mol), 20 mm 4 - (2-hydroxyéthyl)-1-pipérazineéthanesulfonique acide (HEPES, M r 238,31 g / mol), 2 mM de chlorure de potassium (KCl, M r 74,56 g / mol), le pH est ajusté à 7.1 à 7.2 par l&#39;acide méthane sulfonique (MESO 3, M r 96,1 g / mol)). </li></ol> 3. Patch clamp <ol><li> Recouvrir l&#39;électrode d&#39;enregistrement Ag avec AgCl avant chaque session de patch clamp. D&#39;abord, enlever le fil électrode du porte-pipette et submerger la moitié extrémité du fil électrode dans un flacon contenant l&#39;eau de Javel fraîche pendant au moins 15 minutes. Cette dépose une couche de AgCl sur le fil. </li><li> Rincez le fil électrode avec de l&#39;eau déminéralisée et sécher. Ensuite, installez l&#39;électrode Ag / AgCl retour dans le porte-pipette. </li><li> Connectez le bain (de référence) électrode à l&#39;headstage et le placer dans le bain. Cela prépare les électrodes pour l&#39;enregistrement. </li><li> Maintenant préparer à l&#39;acquisition de données. Allumez votre ordinateur et branchez la clé matérielle sur le port imprimante de votre ordinateur. La clé matérielle doit être branché pour vous d&#39;utiliser le logiciel d&#39;acquisition de données, ce qui est HEKA pouls. </li><li> Allumez l&#39;amplificateur (Axon Instruments, AXOPATCH 200B) et commencer HEKA Pulse. Chargez le fichier de protocole et d&#39;ajuster les paramètres de configuration, tels que l&#39;échelle de stimulation. L&#39;objectif pour ajuster les paramètres de configuration est d&#39;assurer que le potentiel de test est exactement égal au potentiel de commande. Cela prépare le matériel d&#39;acquisition de données. </li><li> Préparer ovocytes. Immerger l&#39;ovocyte dans la solution de décapage (200 mM de N-méthyl-D-glucamine (NMG, M r 195,22 g / mol), 200 mM d&#39;aspartate (pas de K +) (133,1 M r g / mol), 2 mM de chlorure de potassium (KCl , M r 74,56 g / mol), 10 mM d&#39;éthylène glycol-bis (2-aminoethylether)-N, N, N &#39;, N&#39;-tétraacétique (EGTA, M r 380,35 g / mol), 1 mM de chlorure de magnésium (MgCl 2 • 6H 2 O, M r 203,31 g / mol), 10 mM de 4 - (2-hydroxyéthyl)-1-pipérazineéthanesulfonique (HEPES, M r 238,31 g / mol), le pH est ajusté à 7,4 par l&#39;hydroxyde de sodium (NaOH, M r 40,0 g / mol) pour 5 - 10 min. La solution de décapage se détache de la membrane vitelline de la membrane plasmique qui permet de dépouiller la membrane vitelline. </li><li> Doucement la bande de la membrane vitelline de l&#39;ovocyte à l&#39;aide de deux paires de pinces. Un ovocyte devitellinized est extrêmement fragile et doit donc être déplacé aussi peu que nécessaire. </li><li> Prendre soin d&#39;éviter d&#39;exposer l&#39;ovocyte devitellinized à l&#39;air ou de bulles, utiliser une pipette en verre remplie d&#39;assez de solution pour transférer l&#39;ovocyte dans le bain. Cela prépare l&#39;ovocyte pour l&#39;enregistrement. </li><li> Préparer les pipettes de patch. Tirez les pipettes en verre après l&#39;insertion d&#39;un tube capillaire en verre (VWR International, Cat # 53432-921) dans un Sutter P-97 Flaming / Brown Micropipette Puller. Respecter les pipettes sous microscope pour déterminer la forme de la pointe et le diamètre d&#39;ouverture. Le diamètre d&#39;ouverture devrait être 2-4 micromètres. </li><li> Afin de réduire le courant capacitif pendant l&#39;enregistrement et à assurer une astuce lisse, enduire la pointe avec de la cire, puis le feu-polonaise. </li><li> Remplissez la pipette en plaçant la pointe de la pipette à l&#39;intérieur de la solution de pipette (140 mM d&#39;hydroxyde de potassium (KOH, M r 56,1 g / mol), 20 mm 4 - (2-hydroxyéthyl)-1-pipérazineéthanesulfonique (HEPES, M r 238,31 g / mol), 2 mM de chlorure de potassium (KCl, M r 74,56 g / mol), 2 mM de chlorure de magnésium (MgCl 2 • 6H 2 O, M r 203,31 g / mol), le pH est ajusté à 7.1 à 7.2 par méthanesulfonique l&#39;acide (MESO 3, M r 96,1 g / mol)) et le dessin du piston. Cette mouille le bout. </li><li> Remplissez la pipette 1 / 3 plein avec une solution de pipette à l&#39;aide d&#39;une seringue et le lieu de la pipette dans le porte-pipette avec l&#39;intérieur électrode Ag / AgCl et contact avec la solution pipette. Cela prépare la pipette de patch. </li><li> Pour d&#39;accise d&#39;un patch à l&#39;envers de l&#39;ovocyte prêt, trouver le bord claire de l&#39;ovocyte sous microscope. Déplacer la pipette de patch proche de l&#39;ovocyte à l&#39;aide manipulateur. Enregistrement de la résistance série de la pipette. La résistance série idéale de la pipette de patch est entre 1-1,5 MQ quand il est rempli avec une solution de pipette et submergés dans une solution de bain. </li><li> Poussez lentement la pipette contre l&#39;ovocyte jusqu&#39;à la résistance à double approximativement. </li><li> Appliquer une aspiration douce à la membrane par la bouche à travers un tube d&#39;aspiration qui est relié au porte-pipettes jusqu&#39;à un giga-ohms joint est obtenu. Stabiliser le patch en le tenant à la tension -30 mV pour un couple de minutes. </li><li> Pour obtenir un dedans-dehors de patch, le patch en accise rapidement pousser plus loin la pipette dans l&#39;ovocyte et puis doucement le tirant. </li><li> Le patch inside-out est maintenant prêt pour le serrage. Déplacer la pipette tenant le patch à l&#39;envers à environ 100 microns loin de l&#39;ouverture de la pointe de perfusion. </li><li> Fermer le robinet électronique pour la collecte des déchets et ouvrez le robinet de réservoir pour la solution de perfusion désirée </li><li> Commencez l&#39;enregistrement des courants à travers le patch. Changer les solutions de tension et de la perfusion comme désiré. </li><li> Lorsque vous avez terminé l&#39;enregistrement, nettoyer les tubulures de perfusion, le crayon et la pointe en poussant dans l&#39;eau déminéralisée pour éviter sabots. </li></ol> 4. Les résultats représentatifs Pendant la préparation d&#39;ovocytes pour le patch-clamp, le traitement 5-10 min d&#39;une solution de décapage serait détachent de la membrane vitelline de la membrane plasmique, ce qui rend possible de décapage de la membrane vitelline. La résistance série idéale de la pipette de patch est entre 1-1,5 MQ quand il est rempli avec une solution de pipette et submergés dans une solution de bain. Voici un enregistrement représentant de canaux BK de type sauvage sur un patch de membrane ovocytaire. En haut à gauche, les ondes carrées indiquent la tension appliquée au patch - la deuxième étape de la tension augmente de 50 mV à 200 mV avec incrément de 50 mV. Ci-dessous, les traces correspondant courant lorsque la solution de perfusion a nominale 0 Ca 2 + (libre [Ca 2 +] est d&#39;environ 0,5 nM). L&#39;aupli de l&#39;amplitude du courant indique que la probabilité d&#39;ouverture des canaux BK augmente avec la tension. Sur le côté droit, le même patch est perfusé avec 1,0 uM de Ca 2 + lorsqu&#39;elle est appliquée avec le protocole même tension. L&#39;amplitude du courant augmente plus sous la même tension, ce qui indique que la probabilité augmente avec l&#39;ouverture [Ca 2 +]. <img src="/files/ftp_upload/2269/2269fig2.jpg" alt="Figure 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>

Le système d&#39;expression ovocyte est idéal pour la caractérisation électrophysiologique des canaux ioniques voltage-dépendante due à fond relativement faible des canaux endogènes. Par ailleurs, puisque c&#39;est un système d&#39;expression transitoire, il fournit une méthode efficace de réaliser l&#39;étude de mutagenèse de ces canaux. Cependant, il convient de noter que le système d&#39;expression d&#39;ovocytes est différente de celle des cellules de mammifères, donc il peut y avoir des différences ...

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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>
		</thead>
		<tbody>
		
<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

Le protocole présenté ici est conçu pour étudier l&#39;activation de la conductance grande tension et de Ca 2 +-K + activé (BK) canaux. Le protocole peut également être utilisé pour étudier la relation structure-fonction pour d&#39;autres canaux ioniques et les récepteurs des neurotransmetteurs 1. Canaux BK sont largement exprimés dans différents tissus et ont été impliqués dans de nombreuses fonctions physiologiques, y compris la réglementation de la contraction des muscles lisses, la fréquence d&#39;accord de cellules ciliées internes et la réglementation de libération des neurotransmetteurs 2-6. Canaux BK sont activés par dépolarisation de la membrane et par intracellulaire de Ca 2 + et Mg 2 + 6-9. Par conséquent, le protocole est conçu pour contrôler à la fois la tension de membrane et la solution intracellulaire. Dans ce protocole, l&#39;ARN messager de canaux BK est injecté dans des ovocytes de Xenopus laevis (stade V-VI), suivi de 2-5 jours d&#39;incubation à 18 ° C 10-13. Patches membranaires qui contiennent un ou plusieurs canaux BK sont excisées avec la configuration inside-out en utilisant des techniques de patch-clamp 10-13. Le côté intracellulaire du patch est perfusé avec des solutions souhaitées lors de l&#39;enregistrement de telle sorte que l&#39;activation du canal dans des conditions différentes peuvent être examinés. Pour résumer, l&#39;ARNm de canaux BK est injecté dans des ovocytes de Xenopus laevis pour exprimer des protéines de canal sur la membrane ovocytaire, les techniques de patch-clamp sont utilisés pour enregistrer les courants circulant dans les canaux sous tension contrôlée et des solutions intracellulaires.

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

Courant ionique des canaux BK est enregistrée en utilisant des techniques de patch-clamp. Canaux BK sont exprimés en<em&gt; Ovocytes de Xenopus</em&gt; En injectant de l&#39;ARN messager. La solution intracellulaire pendant les enregistrements de patch-clamp est contrôlé par un système de perfusion.

Patch Clamp et des techniques de perfusion pour étudier les canaux ioniques Exprimé en Xenopus Ovocytes

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

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Biology

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

20.8K vues.  Washington University in St. Louis. Courant ionique des canaux BK est enregistrée en utilisant des techniques de patch-clamp. Canaux BK sont exprimés en Ovocytes de Xenopus En injectant de l'ARN messager. La solution intracellulaire pendant les enregistrements de patch-clamp est contrôlé par un système de perfusion.

Vidéo: 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.

Méthodes pour les enregistrements patch clamp Capacité du calice

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

Recherche

Biologie

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.

Enregistrement Patch Clamp des canaux ioniques exprimés dans les oocytes 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.

Pression de polissage Pipettes Amélioration patch-clamp

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.

Microinjection d&#39;ovocytes 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.

Visualisation de localisation de l&#39;ARN Xenopus Les ovocytes

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.

Mutagenèse et analyse fonctionnelle des canaux ioniques hétérologue exprimée dans les cellules de mammifères

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.

Préparation des liposomes de géant pour l&#39;imagerie et de patch-clamp électrophysiologie

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 cellulaire attaché patch-clamp enregistrement

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.

Reconstitution d&#39;une protéine transmembranaire, le Ion canal de voltage-dépendants, KvAP, en géant vésicules unilamellaires pour la microscopie et Patch études Clamp

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.

Techniques for Imaging Prométaphase et Metaphase de méiose I fixe<em&gt; Drosophila</em&gt; ovocytes

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