Tutti i membri della Washington University di St. Louis Cardiac molecolare Engineering Lab. Un Burroughs Welcome Fondo Premio alla Carriera al Interface scientifico - 1010299 (JS).

<ol>
	<li>Kalstrup, T., Blunck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+internal+pore+opening+in+KV+channels+probed+by+a+fluorescent+unnatural+amino+acid.">Dynamics of internal pore opening in KV channels probed by a fluorescent unnatural amino acid.</a> Proc. Natl. Acad. Sci. U.S.A. 110, 8272-8277 (2013).</li><li>Stefani, E., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cut-open+oocyte+voltage-clamp+technique.">Cut-open oocyte voltage-clamp technique.</a> Methods Enzymol. 293, 300-318 (1998).</li><li>Muroi, Y., Chanda, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+anesthetics+disrupt+energetic+coupling+between+the+voltage-sensing+segments+of+a+sodium+channel.">Local anesthetics disrupt energetic coupling between the voltage-sensing segments of a sodium channel.</a> J. Gen. Physiol. 133, 1-15 (2009).</li><li>Stefani, E., Toro, L., Perozo, E., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gating+of+Shaker+K++channels:+I.+Ionic+and+gating+currents.">Gating of Shaker K+ channels: I. Ionic and gating currents.</a> Biophys. J. 66, 996-1010 (1994).</li><li>Wang, S., Liu, S., Morales, M. J., Strauss, H. C., Rasmusson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+analysis+of+the+activation+and+inactivation+kinetics+of+HERG+expressed+in+Xenopus+oocytes.">A quantitative analysis of the activation and inactivation kinetics of HERG expressed in Xenopus oocytes.</a> J. Physiolt. 502 (Pt 1), 45-60 (1997).</li><li>Neely, A., Garcia-Olivares, J., Voswinkel, S., Horstkott, H., Hidalgo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+active+calcium+channel+beta(1b)+-subunit+by+size-exclusion+chromatography+and+its+role+on+channel+function.">Folding of active calcium channel beta(1b) -subunit by size-exclusion chromatography and its role on channel function.</a> J. Biol. Chem. 279, 21689-21694 (2004).</li><li>Silva, J. R., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensor+movements+describe+slow+inactivation+of+voltage-gated+sodium+channels+I:+wild-type+skeletal+muscle.">Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels I: wild-type skeletal muscle.</a> Na(V)1.4. J. Gen. Physiol. 141, 309-321 (2013).</li><li>Silva, J. R., Goldstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-sensor+movements+describe+slow+inactivation+of+voltage-gated+sodium+channels+II:+a+periodic+paralysis+mutation+in+Na(V)1.4+(L689I).">Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: a periodic paralysis mutation in Na(V)1.4 (L689I).</a> J. Gen. Physiol. 141, 323-334 (2013).</li><li>Taglialatela, M., Toro, L., Stefani, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+voltage+clamp+to+record+small,+fast+currents+from+ion+channels+expressed+in+Xenopus+oocytes.">Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes.</a> Biophys. J. 61, 78-82 (1992).</li><li>Clare, J. J., Trezise, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Expression and analysis of recombinant ion channels : from structural studies to pharmacological screening. , (2006).</li><li>Cha, A., Zerangue, N., Kavanaugh, M., Bezanilla, F., Susan, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in enzymology. 296, 566-578 (1998).</li><li>Lakowicz, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of fluorescence spectroscopy. 3rd edn. , (2006).</li><li>Cha, A., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+voltage-dependent+conformational+changes+in+the+Shaker+K++channel+with+fluorescence.">Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence.</a> Neuron. 19, 1127-1140 (1997).</li><li>Richards, R., Dempski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+the+conformational+dynamics+of+membrane+proteins+in+situ+with+site-directed+fluorescence+labeling.">Examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling.</a> J. Vis. Exp. , (2011).</li><li>Cohen, S., Au, S., Pante, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> J. Vis. Exp. , (2009).</li><li>Raynauld, J. P., Laviolette, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+silver-silver+chloride+electrode:+a+possible+generator+of+offset+voltages+and+currents.">The silver-silver chloride electrode: a possible generator of offset voltages and currents.</a> J. Neurosci. Methods. 19, 249-255 (1987).</li><li>Gagnon, D. G., Bissonnette, P., Lapointe, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+disulfide+bridge+linking+the+fourth+and+the+seventh+extracellular+loops+of+the+Na+/glucose+cotransporter.">Identification of a disulfide bridge linking the fourth and the seventh extracellular loops of the Na+/glucose cotransporter.</a> J. Gen. Physiol. 127, 145-158 (2006).</li><li>Pantazis, A., Olcese, R., Roberts, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cut-Open Oocyte Voltage-Clamp Technique. In: Roberts G. (Ed.) Encyclopedia of Biophysics: SpringerReference. , (2013).</li></ol>

Gli autori non hanno nulla da rivelare.

L&#39;ovocita vaselina tensione gap clamp cut-open consente una rapida risoluzione dei dati, a basso rumore, aumentato il controllo sulla soluzione interna e la composizione soluzione esterna, e registrazioni stabili nel relativamente lunghi protocolli 19. Questi vantaggi impostate questa tecnica a parte il voltage clamp a due elettrodi standard e le tecniche di patch clamp. Sebbene attrezzature specializzate è necessaria e il protocollo è relativamente difficile, molto pochi problemi si verificano quando i...

Tecniche tensione di serraggio specializzati permettono la registrazione di correnti ioniche a potenziali di membrana controllate. Ampiamente usato voltage clamp a due elettrodi (TEVC) e tecniche di patch clamp forniscono informazioni elettrofisiologiche attendibili sulle proprietà di molti canali ionici. Tuttavia, entrambi questi metodi hanno inconvenienti che impediscono l&#39;acquisizione di dati affidabili per i canali del sodio voltaggio-dipendenti veloci e altri canali attivanti veloce nelle membrane come quelle di ovociti di Xenopus. I laboratori Bezanilla e Stefani quindi sviluppato la tecnica del clamp di tensione gap vaselina cut-open (COVG) per ovociti 2. La tecnica è stata ampiamente applicata per registrare, Na +, K + e Ca 2 + canali 3-8. Durante la registrazione COVG, una membrana dell&#39;oocita-proteina eterologa esprimere è diviso in tre regioni. I dati corrente ionica viene registrata dalla regione superiore del ovocita comebagno circonda la regione superiore è fissata ad un potenziale di comando, che può essere cambiata facilmente e rapidamente. Le guardie regione del Medio contro le correnti di dispersione di essere bloccato allo stesso potenziale come la regione top 9. La regione inferiore è dove apertura ovocita (cut-open) avviene mediante l&#39;utilizzo di una soluzione di saponina o una cannula. Chimica o apertura manuale della membrana nella regione inferiore permette il controllo del potenziale interno, che viene fissato al terreno, e rende l&#39;interno della cellula contigua con la soluzione camera inferiore. Perfusione di soluzioni nella camera inferiore può regolare le proprietà dell&#39;ambiente interno, mentre lo scambio di soluzione nella camera superiore altera l&#39;ambiente esterno. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51040/51040fig1highres.jpg" src="/files/ftp_upload/51040/51040fig1.jpg" width="500px" /> Figura 1. Ovociti Cut-Open Voltage-Clamp Bath Diagramma di installazione. (A) Topdown vista di tre bagni separati l&#39;uno dall&#39;altro. Le dimensioni delle camere per COVG vengono visualizzati sulla figura. (B) Vista laterale della configurazione bagni in posizione di prova. <a href="http://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51040/51040fig1highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> . I vantaggi della tecnica COVG includono basso rumore corrente (1 nA a 3 kHz), il controllo della composizione ionica del supporto esterno, la capacità di modulare il supporto interno, risoluzione temporale veloce (20-100 costante msec tempo di decadimento della capacità transitorio), e le registrazioni stabili per diverse ore 9. Gli svantaggi sono che richiede attrezzature specializzate ed è più difficile da eseguire rispetto a due serraggio tensione elettrodo (TEVC) 10. Mentre l&#39;approccio COVG richiede attrezzature altamente specializzate e elementi procedurali complessi, può consentire l&#39;acquisizione di Valudati elettrofisiologici grado. Questi dati, come gating correnti con cinetica veloce e correnti di coda 4, può essere registrata senza alcuni dei problemi associati con altri protocolli tensione di chiusura comprese canale run-down. Piccole modifiche alla configurazione COVG possono consentire l&#39;uso di regolatori di temperatura e morsetto di tensione fluorometria (VCF). L&#39;inclusione di tensione morsetto elementi fluorometria all&#39;interno dell&#39;assembly COVG può aumentare output di dati conferendo la possibilità di monitorare cambiamenti conformazionali proteiche e contemporaneamente la registrazione corrente 11-13.

<table><tbody><tr><td>External Solution</td><td>Brand</td><td>Catalog Number</td><td>[Final], weight, or volume</td></tr><tr><td>N-methyl-D-glucamine (NMDG)</td><td>Sigma-Aldrich</td><td>M2004</td><td>25mM</td></tr><tr><td>MES Sodium Salt</td><td>Sigma-Aldrich</td><td>M5057</td><td>90mM</td></tr><tr><td>HEPES</td><td>Research Products International</td><td>H75030</td><td>20mM</td></tr><tr><td>Calcium hydroxide</td><td>Sigma-Aldrich</td><td>239232</td><td>2mM</td></tr><tr><td>MES Hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td>variable (pH to 7.4)</td></tr><tr><td>Internal Solution</td><td></td><td></td><td></td></tr><tr><td>N-methyl-D-glucamine (NMDG)</td><td>Sigma-Aldrich</td><td>M2004</td><td>105mM</td></tr><tr><td>MES Sodium Salt</td><td>Sigma-Aldrich</td><td>M5057</td><td>10mM</td></tr><tr><td>HEPES</td><td>Research Products International</td><td>H75030</td><td>20mM</td></tr><tr><td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#039;,N&#039;-tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>E4378</td><td>2mM</td></tr><tr><td>MES Hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td>variable (pH to 7.4)</td></tr><tr><td>Depolarizing Solution</td><td></td><td></td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>221473</td><td>110mM</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M8266</td><td>1.5mM</td></tr><tr><td>Calcium Chloride</td><td>Caisson</td><td>C021</td><td>0.8mM</td></tr><tr><td>HEPES</td><td>Research Products International</td><td>H75030</td><td>10mM</td></tr><tr><td>Pipet Solution</td><td></td><td></td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>221473</td><td>3M</td></tr><tr><td>Saponin Solution</td><td></td><td></td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td>0.125g</td></tr><tr><td>Internal Solution</td><td>See above</td><td></td><td>50mL</td></tr><tr><td>Agar Bridge Solution</td><td></td><td></td><td></td></tr><tr><td>N-methyl-D-glucamine (NMDG)</td><td>Sigma-Aldrich</td><td>M2004</td><td>100ml of 1M</td></tr><tr><td>HEPES</td><td>Research Products International</td><td>H75030</td><td>1.2g</td></tr><tr><td>MES Hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td>variable (pH to 7.4)</td></tr><tr><td>Granulated Agar</td><td>Research Products International</td><td>A20250</td><td>3%</td></tr><tr><td>NMDG Storage Solution</td><td></td><td></td><td></td></tr><tr><td>NMDG, HEPES, MES Hydrate solution</td><td>see above</td><td></td><td>40ml</td></tr><tr><td>Water</td><td></td><td></td><td>60ml</td></tr><tr><td>Name of Material/ Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>High Performance Oocyte Clamp</td><td>Dagan</td><td>CA-1B</td><td></td></tr><tr><td>Data Acquisition System</td><td>Axon CNS</td><td>&amp;nbsp;Digidata 1440A</td><td></td></tr><tr><td>Oscilloscope</td><td>Tektronix&amp;nbsp;</td><td>TDS 210</td><td></td></tr><tr><td>Rack Power Filter</td><td>APC&amp;nbsp;</td><td>G5</td><td></td></tr><tr><td>Heating/Cooling Bath Temperature Controller</td><td>Dagan</td><td>HCC-100A</td><td></td></tr><tr><td>PC</td><td>Dell</td><td>Optiplex 990</td><td></td></tr><tr><td>pCLAMP 10.3 Voltage Clamp Software</td><td>Molecular Devices, LLC</td><td>pCLAMP10.3</td><td></td></tr><tr><td>TMC Vibration Control TableTop Platform</td><td>TMC</td><td>64 SERIES</td><td></td></tr><tr><td>TMC Vibration Control Air Table</td><td>TMC</td><td>&lt;a name=&quot;RANGE!C10&quot;&gt;20 Series&amp;nbsp;&lt;/a&gt;</td><td></td></tr><tr><td>V1/I Electrode Data Collector</td><td>Dagan</td><td>part of CA-1B</td><td></td></tr><tr><td>MX10L Micromanipulator</td><td>Siskiyou</td><td>MX10L</td><td></td></tr><tr><td>Bath/Guard (I/V) Headstage (with appropriate connectors)</td><td>Dagan</td><td>part of CA-1B</td><td></td></tr><tr><td>Microscope</td><td>Omano</td><td>OM2300S-JW11</td><td></td></tr><tr><td>Temperature Control Bath</td><td>Custom or Dagan</td><td>Custom or HE-204C</td><td>Custom chamber made from materials from Cool Polymers (D-series). Dagan also provides a prefeabricated stage (HE-204C).</td></tr><tr><td>Custom AgCl Pellet Container</td><td>Custom</td><td>Custom</td><td>Custom machined</td></tr><tr><td>Ag/AgCl electrode, pellet, 2.0 mm</td><td>Warner</td><td>E-206</td><td></td></tr><tr><td>External Oocyte Bath</td><td>Custom or Dagan</td><td>Custom or CC-1-T-LB</td><td>Custom machined or purchased from Dagan</td></tr><tr><td>Internal Oocyte Bath</td><td>Custom or Dagan</td><td>Custom or CC-TG-ND</td><td>Custom machined or purchased from Dagan</td></tr><tr><td>Capillaries for Agar Bridges and Pulled Electrodes</td><td>Warner</td><td>G150T-4</td><td></td></tr><tr><td>Rotatable Mounts for the Microscope, Micromanipulator, and Bath</td><td>Siskiyou</td><td>SD-1280P</td><td></td></tr><tr><td>Fiber-Lite</td><td>Dolan-Jenner</td><td>LMI-600</td><td></td></tr><tr><td>Regular Bleach</td><td>Clorox</td><td>470174-764</td><td></td></tr><tr><td>Xenopus laevis Oocytes</td><td>Nasco</td><td>LM535M (sexually mature females)</td><td></td></tr><tr><td>90 Na&lt;sup&gt;+&lt;/sup&gt; External Solution</td><td>See Solutions sheet</td><td></td><td></td></tr><tr><td>10 Na&lt;sup&gt;+&lt;/sup&gt; Internal Solution</td><td>See Solutions sheet</td><td></td><td></td></tr><tr><td>3 M KCL</td><td>See Solutions sheet</td><td></td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>NMDG Storage Solution</td><td>See Solutions sheet</td><td></td><td></td></tr><tr><td>5mL transfer pipets</td><td>SciMart</td><td>GS-52</td><td></td></tr><tr><td>Modified KCl electrode injector</td><td>BD</td><td>309659</td><td>Plastic syringe tip melted to allow for injection of solution into electrodes. Alternatively, a Microfil by WPI can be purchased.</td></tr><tr><td>Microvaccum</td><td>Custom</td><td>Custom</td><td></td></tr><tr><td>Forceps</td><td>VWR</td><td>63040-458</td><td></td></tr><tr><td>Oocyte Handling Tools (Pipette Pump)</td><td>VWR</td><td>53502-222</td><td></td></tr><tr><td>Deionized Water Squirt Bottle</td><td>VWR</td><td>16649-911</td><td></td></tr><tr><td>Vaseline Petroleum Jelly</td><td>Fisher Scientific</td><td>19-086-291&amp;nbsp;</td><td></td></tr><tr><td>Additional Materials Required for VCF Recordings:</td><td></td><td></td><td></td></tr><tr><td>VCF Microscope</td><td>Nikon</td><td>Eclipse FN1</td><td></td></tr><tr><td>Nikon CFI APO 40XW NIR Objective</td><td>Nikon</td><td>N40X-NIR</td><td></td></tr><tr><td>X-Y Translator System for Fixed-Stage Upright Microscopes</td><td>Sutter Instruments</td><td>MT500-586</td><td></td></tr><tr><td>External VCF Oocyte Bath</td><td>Custom</td><td></td><td>Custom machined. The chamber dimensions are 2.7 x 1.9 x 0.4 cm.</td></tr><tr><td>Internal VCF Oocyte Bath</td><td>Custom</td><td></td><td>Custom machined. The chamber dimensions are 1.6 x 1.6 x 0.4 cm.</td></tr><tr><td>Modified Temperature Control Bath</td><td>Custom</td><td></td><td>Custom chamber made from materials from Cool Polymers (D-series). The chamber dimensions of the modified temperature controller bath are 2.7 x 1.9 x 0.3 cm for the horizontal chamber, and 1 x 2.5 x 0.5 cm for the vertical chamber.</td></tr></tbody></table>

the xenopus oocyte cut-open vaseline gap voltage-clamp technique with fluorometry

L&#39;ovocita vaselina gap cut-open (COVG) clamp di tensione permette l&#39;analisi delle proprietà elettrofisiologiche e cinetiche dei canali ionici eterologa in oociti. Registrazioni dalla configurazione cut-open sono particolarmente utili per risolvere basse correnti di magnitudo gating, rapida attivazione corrente ionica e disattivazione. I principali vantaggi rispetto al morsetto di tensione a due elettrodi (TEVC) tecnica includono maggiore velocità morsetto, miglior rapporto segnale-rumore, e la capacità di modulare la intracellulare ed extracellulare. Qui, impieghiamo canale del sodio cardiaco umano (HNA 1,5 V), espresso in oociti di Xenopus, per dimostrare la configurazione e protocollo cut-open nonché modifiche necessarie per aggiungere capacità fluorometria morsetto di tensione. Le proprietà dei canali ionici veloci attivanti, come HNA 1,5 V, non possono essere completamente risolti a temperatura ambiente usando TEVC, in which la totalità della membrana dell&#39;oocita è serrato, rendendo difficile il controllo della tensione. Tuttavia, nella tecnica cut-aperto, separazione di solo una piccola porzione della membrana cellulare consente il bloccaggio rapido necessaria per registrare accuratamente cinetica veloce evitando canale run-down associato a tecniche di patch clamp. In concomitanza con la tecnica COVG, cinetica del canale ionico e proprietà elettrofisiologiche possono essere ulteriormente analizzati mediante morsetto fluorometria tensione, in cui il movimento proteina viene monitorato tramite cisteina coniugazione di fluorofori applicate extracellulare, inserimento di proteine ​​fluorescenti geneticamente codificati, o l&#39;incorporazione di amminoacidi non naturali nella regione di interesse 1. Questi dati aggiuntivi produce informazioni cinetiche su voltaggio-dipendenti riarrangiamenti conformazionali della proteina attraverso cambiamenti nel microambiente circostante la molecola fluorescente.

Figura 4 mostra la variazione di permeabilità del ovocita come soluzione saponina viene applicata alla parte inferiore della ovocita. Figura 5 mostra il tasso di conversione soluzione intracellulare per diffusione seguente permeabilizzazione saponina. 20-40 min sono tenuti a venire a uno stato stazionario 2,18. Figura 6A mostra tracce generate dal protocollo di registrazione. La figura mostra correnti ioniche (dopo P/-8 perdita s...

Watch this Scientific Journal Video about The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry at JoVE.com

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

L&#39;approccio gap vaselina cut-aperto viene utilizzato per ottenere bassi registrazioni sonore di correnti ioniche e gating di canali ionici voltaggio-dipendenti espressi in oociti di Xenopus con alta risoluzione di canale cinetiche veloci. Con piccola modifica, morsetto di tensione fluorometria può essere accoppiato al protocollo ovocita cut-open.

Il<em&gt; Xenopus</em&gt; Ovociti Cut-open vaselina Gap Tecnica Voltage-clamp Con fluorometria

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

the-xenopus-oocyte-cut-open-vaseline-gap-voltage-clamp-technique-with

Universidad de Cartagena UDEC

Research

Education

JoVE Core

JoVE Journal

Electrical Engineering

Biology

Unit 1

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

Root-Locus Method

SCIENTISTS IN ACTION

18.3K Views.  Washington University in St. Louis.  L'approccio gap vaselina cut-aperto viene utilizzato per ottenere bassi registrazioni sonore di correnti ioniche e gating di canali ionici voltaggio-dipendenti espressi in oociti di Xenopus con alta risoluzione di canale cinetiche veloci. Con piccola modifica, morsetto di tensione fluorometria può essere accoppiato al protocollo ovocita cut-open. 

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

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Fluorescenza a tensione di serraggio in<em&gt; Xenopus</em&gt; Oociti che utilizzano aminoacidi innaturali fluorescenti

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Ricerca

Biochimica

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.

Patch di registrazione pinza dei canali ionici Espressa in ovociti di Xenopus

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

Biologia

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

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

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

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

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

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Esaminando la dinamica conformazionale delle proteine ​​di membrana In situ Con il sito-diretta Etichettatura fluorescenza

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Metodo elettrofisiologico per le registrazioni a morsetto di tensione a cellule intere da<em&gt; Drosophila</em&gt; Fotorecettori

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Neuroscienze

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Misurazione del potenziale d'azione ottico a singola cellula in cardiomiociti derivati da cellule staminali pluripotenti indotte umane

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Bioingegneria

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Misurazione di trasporto cationico da Na, K e H, K-ATPasi<em&gt; Xenopus</em&gt; Gli oociti per spettrofotometria di assorbimento atomico: una alternativa a saggi radioisotopi

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

Lievito luminometrici e<em&gt; Xenopus</em&gt; ovocita elettrofisiologiche Esami del mechanosensitivity molecolare del TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

Xenopus ovociti: Metodi ottimizzate per microiniezione, la rimozione di strati di cellule follicolari e cambiamenti soluzione veloce in esperimenti di elettrofisiologia

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

Recording Gap Junction Current from Xenopus Oocytes

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions (GJs). However, this approach is technically challenging because it requires a differential voltage clamp of two opposed oocytes sharing a common ground. Although a small number of labs have succeeded in performing this technique, essentially all of them have used either homemade amplifiers or commercial amplifiers that were designed for single-oocyte recordings. It is often challenging for other labs to implement this technique. Although a high side current measuring mode has been incorporated into a commercial amplifier for dual oocyte voltage-clamp recordings, there had been no report for its application until our recent study. We have made the high side current measuring approach more practical and convenient by introducing several technical modifications, including the construction of a magnetically based recording platform that allows precise placement of oocytes and various electrodes, use of the bath solution as a conductor in voltage differential electrodes, adoption of a commercial low-leakage KCl electrode as the reference electrode, fabrication of current and voltage electrodes from thin-wall glass capillaries, and positioning of all the electrodes using magnetically based devices. The method described here allows convenient and robust recordings of junctional current (Ij) between two opposed Xenopus oocytes.

Recording Gap Junction Current from Xenopus Oocytes

Here we present a protocol to express gap junction proteins in Xenopus oocytes and record junctional current between two apposed oocytes using a commercial amplifier designed for dual oocyte voltage-clamp recordings in a high side current measuring mode.

Registrazione della corrente di giunzione gap dagli ovociti xenopus

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions ...

Il

Riepilogo

Esplora Altri Video

Capitoli in questo video

Fluorescenza a tensione di serraggio in

Patch di registrazione pinza dei canali ionici Espressa in ovociti di Xenopus

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

Esaminando la dinamica conformazionale delle proteine di membrana In situ Con il sito-diretta Etichettatura fluorescenza

Metodo elettrofisiologico per le registrazioni a morsetto di tensione a cellule intere da

Misurazione del potenziale d'azione ottico a singola cellula in cardiomiociti derivati da cellule staminali pluripotenti indotte umane

Misurazione di trasporto cationico da Na, K e H, K-ATPasi

Lievito luminometrici e

Xenopus ovociti: Metodi ottimizzate per microiniezione, la rimozione di strati di cellule follicolari e cambiamenti soluzione veloce in esperimenti di elettrofisiologia

Registrazione della corrente di giunzione gap dagli ovociti xenopus

Il

Riepilogo

Esplora Altri Video

Capitoli in questo video

Fluorescenza a tensione di serraggio in

Patch di registrazione pinza dei canali ionici Espressa in ovociti di Xenopus

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

Esaminando la dinamica conformazionale delle proteine ​​di membrana In situ Con il sito-diretta Etichettatura fluorescenza

Metodo elettrofisiologico per le registrazioni a morsetto di tensione a cellule intere da

Misurazione del potenziale d'azione ottico a singola cellula in cardiomiociti derivati da cellule staminali pluripotenti indotte umane

Misurazione di trasporto cationico da Na, K e H, K-ATPasi

Lievito luminometrici e

Xenopus ovociti: Metodi ottimizzate per microiniezione, la rimozione di strati di cellule follicolari e cambiamenti soluzione veloce in esperimenti di elettrofisiologia

Registrazione della corrente di giunzione gap dagli ovociti xenopus

Esaminando la dinamica conformazionale delle proteine di membrana In situ Con il sito-diretta Etichettatura fluorescenza