Name: whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins
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Description: Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins at JoVE.com

Wir danken Maila Reh, Tharsana Tharmalingam und vor allem Altina Klein für hervorragende technische Unterstützung. Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft (DFG) (SFB1078 B2, FOR1279 SPP1665 bis PH) und dem Exzellenzcluster Vereinigungskonzepte in der Katalyse, UniCat, BIG-NSE (JV) und E4 (PH) unterstützt.

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The authors have nothing to disclose.

Die Bestimmung von Umkehrpotentialen bei definierten ionischen und elektrischen Bedingungen liefert Informationen über die nach der Lichtaktivierung von ChRs transportierten Ionenspezies. Wenn nur eine Ionenspezies in einem komplexen physiologischen Medium variiert wird und die erhaltenen Umkehrpotentialverschiebungen nach dem theoretischen Nernstpotential sind, ist diese Ionensorte die einzige transportierte. Für ChRs sind jedoch Umkehrpotentialverschiebungen in der Regel weniger ausgeprägt als erwartet aus der Nernst-Gleichung aufgrund der Permeabilität gegenüber verschiedenen ionischen Spezies sowie der Konkurrenz unter den geführten Ionen. In diesem Fall wird die Situation komplexer und alle potentiell geführten Ionen müssen separat ausgetauscht werden, um eine Vielzahl von Messlösungen anzufordern. Darüber hinaus sind die meisten CCRs und die ersten künstlichen ACRs 9 , 10 , 21 für Protonen leitend, aber variierende ProtonenkonzentrationErfordert eine besonders gründliche Analyse, da die Protonenkonzentration nicht nur Umkehrpotentiale verschieben kann, sondern auch die Ionenleitfähigkeit 9 , die Photoströme Kinetik 21 , 36 und die spektrale Lichtempfindlichkeit 11 , 37 aufgrund einer Veränderung der Wasserstoffbindungsnetzwerke, des Protonierungszustandes von Individuelle Rückstände und sekundäre Strukturänderungen. Die Protonenleitfähigkeit kann durch Verringerung der Konzentrationen aller anderen potentiell durchgeführten Ionen wie Na &amp; spplus ; , Ca &amp; sub2; &amp; spplus ; oder Cl &amp; rgr; adressiert werden, während der pH-Wert konstant gehalten wird, um die oben erwähnten Proteinveränderungen zu vermeiden. Nicht durchgeführte Substitute sind meist sperrige, geladene Moleküle wie NMG + für Kationen 12 und Gluconat, 2- ( N- Morpholino) ethansulfonat (MES-) oder Aspartat für Anionen. Vergleich von Umkehrpotentialen in verschiedenen iOnic-Lösungen erlaubt dann die Berechnung der relativen Ionenpermeabilitäten bei Gleichgewichtsbedingungen durch die Goldman-Hodgkin-Katz-Spannungsgleichung. Die Quantifizierung des exakten Beitrags verschiedener Ionen zu den Netto-Photostraten unter Berücksichtigung des Ionenkonzers über Gleichgewichtsbedingungen erfordert jedoch komplexe mathematische Modelle 12 , 38 . Die Kenntnis der Zusammensetzung von Ionen, die von ChRs transportiert werden, hilft, mögliche Nebenwirkungen, die sich aus der ChR-Anwendung ergeben, besonders in einem neurophysiologischen Kontext zu erhellen. Zum Beispiel kann eine verlängerte ChR-Aktivierung eine intrazelluläre Säuerung verursachen 39 oder durchgeführt-Ca 2+ kann die synaptische Freisetzung und die zweiten Messengerwege auslösen 40 . Da die Zellen in der Regel fest in Gewebe gepackt sind, kann die Aktivierung von mikrobiellen Rhodopsinen nicht nur die intrazelluläre Ionenzusammensetzung beeinflussen, sondern auch das extrazelluläre Lumen, wodurch nBenachbarten Zellen 41 . Trotz Ionenselektivität sind andere biophysikalische Merkmale von ChRs wie Photostromamplituden, Inaktivierung, Spektral- oder Lichtempfindlichkeit und kinetische Eigenschaften oft von Interesse, um Experimente mit optogenetischen Techniken durchzuführen, zu entwerfen und zu interpretieren. Einige dieser Eigenschaften können auch aus dem oben beschriebenen Protokoll , wie an anderer Stelle 18 , 37 , 42 beschrieben , bestimmt werden . Um die Lichtempfindlichkeit der ChR-exprimierenden Zelle zu untersuchen, kann die Lichtintensität durch Einstellen der Leistung der Lichtquelle oder durch Dämpfen des Aktivierungslichts mit Neutral-Dichte-Filtern variiert werden. Um die spektrale Empfindlichkeit zu erhalten, wird eine polychromatische Lichtquelle benötigt und die Intensitäten müssen auf den gleichen Photonenfluss und die Bandbreite für alle Wellenlängen eingestellt werden. Hochintensitätsspektren ähneln nur Absorptionsspektren des Photorezeptors, wenn kurze Lichtimpulse oderLaser-Blitze werden verwendet, ansonsten können Anpassungsphänomene und photochemische Rückreaktionen auftreten. Die Erholung von teilweise inaktiviertem bis voll aktivem ChR (Gesamt-Photocycle-Umschlagszeit) kann durch Anlegen eines Doppelpuls-Protokolls mit zunehmenden Dunkelintervallen zwischen den beiden Lichtpulsen untersucht werden. Die oben diskutierten biophysikalischen Eigenschaften von ChRs sind wichtig für die Auswahl eines geeigneten ChR, der den experimentellen Bedürfnissen entspricht 40 . Die Ableitung dieser biophysikalischen Eigenschaften durch elektrophysiologische Techniken ähnelt direkt der Proteinfunktion, die kaum durch spektroskopische Methoden abgedeckt ist. Nach erfolgreicher Etablierung von Spannungsklemmmessungen kann die Technik mit Protein-Engineering-Ansätzen wie ortsgerichtete Mutagenese 9 , 11 , 43 , 44 , Helix-Shuffling 37 , <supClass = &quot;xref&quot;&gt; 45, Einführung von Targeting Sequenzen 46 oder Fusion mit anderen Proteinen 41 , 47 . Dies hat das molekulare Wissen über ChRs und ihre Funktionsweise erhöht und eine hohe Diversität von ChR-Varianten mit veränderter Kinetik, Leitfähigkeit, spektraler Empfindlichkeit und Ionenselektivität 40 erzeugt .

Channelrhodopsine (ChR) sind lichtgesteuerte Ionenkanäle, die im Augenfleck von beweglichen Grünalgen vorkommen und als primäre Photosensoren für Phototaxis und phobische Reaktionen dienen 1 . Seit ihrer ersten Beschreibung im Jahr 2002 haben die ChRs den Weg für das entstehende Feld der Optogenetik geebnet und können in einer Vielzahl von erregbaren Zellen angewendet werden, z. B. in Skelettmuskeln, im Herzen oder im Gehirn 3 , 4 , 5 . Die Expression von ChRs in Zielzellen führt zu einer lichtsteuerbaren Ionenpermeabilität der jeweiligen Zelle. In einem neuronalen Kontext ermöglicht dies die Aktivierung 6 , 7 , 8 oder die Hemmung 9 , 10 des Aktionspotentials (AP), die - abhängig vom geführten Ion - mit dem räumlichen und zeitlichen abfeuertPräzision des Lichts, die hervorhebt, wie die Ionenselektivität einer ChR-Variante ihre optogenetische Anwendung bestimmt. Die ersten entdeckten ChRs von Chlamydomonas reinhardtii und Volvox carteri sind für Protonen durchlässig, aber auch für monovalente Kationen wie Natrium, Kalium und in geringerem Maße zu zweiwertigen Kationen wie Calcium und Magnesium 11 , 12 , 13 . Heute sind mehr als 70 natürliche kationenleitende Kanalrhodopsine (CCRs) 14 , 15 , 16 , 17 und mehrere konstruierte Varianten 18 , 19 , 20 mit unterschiedlichen Eigenschaften wie Photostromgröße, spektrale Empfindlichkeit, Kinetik und Kationenselektivität stehen zur Verfügung. Während in der Neurowissenschaften, CCRs aRe verwendet, um Zellen zu aktivieren und APs auszulösen, waren lichtgetriebene mikrobielle Pumpen die einzigen verfügbaren Antagonisten für die Stummschaltung von Neuronen seit Jahren. Im Jahr 2014 zeigten zwei Gruppen gleichzeitig, dass CCRs durch Veränderung der Polarität entlang der putativen Ionen leitenden Pore über molekulare Technik 9 , 21 in anion-leitende Kanalrhodopsine (ACRs) umgewandelt werden können . Anschließend wurden natürliche ACRs in mehreren Kryptophytenalgen 22 , 23 , 24 identifiziert. Am wichtigsten ist, dass die Lichtaktivierung von ACRs Chloridströme in erwachsenen Neuronen vermittelt, was eine Hemmung der neuronalen Aktivität bei viel geringeren Lichtintensitäten ermöglicht als mikrobielle Pumpen, die nur einzelne Ladungen pro absorbiertem Photon transportieren. Die ChR-Aktivität kann direkt durch elektrophysiologische Patch-Clamp-Aufnahmen von lichtinduzierten Strömen in HEK293-Zellen adressiert werden. Die Patch-ClampTechnik wurde ursprünglich in den späten 1970er Jahren entwickelt und weiter verbessert von Hamill et al. , Wodurch die Aufzeichnung der Entität von Strömen aus einer kleinen Zelle (Ganzzellenmodus) mit hoher Stromauflösung und direkter Steuerung der Membranspannung 26 ermöglicht wird. In der Zellkultur angewendet, bietet diese Technik eine genaue Kontrolle sowohl der ionischen als auch der elektrischen Aufzeichnungsbedingungen und ermöglicht die Untersuchung der Ionenselektivität zusammen mit dem relativen Beitrag der Ionen zum Gesamtstrom. Hier veranschaulichen wir die Untersuchung der Ionenselektivität für das anion-leitende Kanalrhodopsin von Proteomonas sulcata ( Ps ACR1) 22 , 23 über die Aufzeichnung von Strom-Spannungs-Beziehungen unter verschiedenen extrazellulären Chlorid-Konzentrationen, um eine hohe Chlorid-Leitfähigkeit zu beweisen.

<table><tbody><tr><td>HEK293 cells</td><td>Sigma Aldrich</td><td>85120602</td><td>Human embryonic kidney cells</td></tr><tr><td>Retinal</td><td>Sigma Aldrich</td><td>R2500</td><td>all-trans retinal</td></tr><tr><td>FuGENE HD</td><td>Promega</td><td>E2312</td><td>Transfection reagent</td></tr><tr><td>DMEM</td><td>Biochrome</td><td>FG 0445</td><td>Dulbecco&#039;s Modified Eagle Medium</td></tr><tr><td>Agarose</td><td>Roth</td><td>3810</td><td>Agar bridges</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>5239</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>CsCl</td><td>Biomol</td><td>2452</td><td></td></tr><tr><td>EGTA</td><td>Roth</td><td>3054</td><td></td></tr><tr><td>FBS</td><td>Biochrome</td><td>S0615</td><td>Cell culture</td></tr><tr><td>Glucose</td><td>Roth</td><td>HN06</td><td>D(+)-Glucose</td></tr><tr><td>KCl</td><td>Roth</td><td>6781</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td>2189</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; 6H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>NaCl</td><td>Roth</td><td>3957</td><td></td></tr><tr><td>NMG</td><td>Sigma Aldrich</td><td>M2004</td><td>N-Methyl-D-glucamine</td></tr><tr><td>Na-Aspartate</td><td>Sigma Aldrich</td><td>A6683</td><td>L-Aspartic acid sodium salt monohydrate</td></tr><tr><td>Citric acid</td><td>Roth</td><td>6490</td><td></td></tr><tr><td>AgeI</td><td>ThermoFischerScientific</td><td>ER1462</td><td>Restriction enzyme</td></tr><tr><td>XhoI</td><td>ThermoFischerScientific</td><td>ER0695</td><td>Restriction enzyme</td></tr><tr><td>NheI</td><td>ThermoFischerScientific</td><td>ER0975</td><td>Restriction enzyme</td></tr><tr><td>XL1Blue &lt;em&gt;E. coli&lt;/em&gt;</td><td>Agilent Technologies</td><td>200249</td><td>Chemocompetent &lt;em&gt;E. coli&lt;/em&gt;</td></tr><tr><td>Kanamycin</td><td>Roth</td><td>T832</td><td></td></tr><tr><td>Lysogeny broth medium</td><td>Roth</td><td>X964</td><td></td></tr><tr><td>Agar-Agar</td><td>Roth</td><td>6494</td><td>Agar plates</td></tr><tr><td>Plasmid purification kit</td><td>Marchery-Nagel</td><td>740727.25</td><td></td></tr><tr><td>Penicilin/Streptomycin</td><td>Biochrome</td><td>A 2213</td><td>Cell culture</td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma Aldrich</td><td>P6407-5MG</td><td>Cover slip coating</td></tr><tr><td>Microforge</td><td>Custom made</td><td></td><td>Fire polishing</td></tr><tr><td>Serological pipettes</td><td>TPP</td><td></td><td>Different sizes</td></tr><tr><td>Clean bench</td><td>Kojair</td><td>Biowizard SL130</td><td></td></tr><tr><td>Stirrer</td><td>IKA</td><td>RCT classic</td><td></td></tr><tr><td>Silver wire</td><td>Science Products</td><td>AG-T25; AG-T10</td><td>Electrodes, 0.64 mm (bath); 0.25 mm (electrode)</td></tr><tr><td>pH-meter</td><td>Knick</td><td>765 Calimetric</td><td></td></tr><tr><td>Osmometer</td><td>Vogel</td><td>OM 815</td><td></td></tr><tr><td>Microscope</td><td>Carl Zeiss</td><td>ID03</td><td>Fire polishing</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Binder</td><td>CB150</td><td></td></tr><tr><td>Cell culture dishes</td><td>TPP</td><td>93040</td><td>34 mm internal diameter</td></tr><tr><td>Cover slips</td><td>Roth</td><td>P232</td><td>15 mm diameter</td></tr><tr><td>Thermometer</td><td>R&amp;ouml;ssel Messtechnik</td><td>MTM12</td><td></td></tr><tr><td>Beamsplitter</td><td>Chroma</td><td>21011</td><td>90/10 transmission</td></tr><tr><td>Pipette holder</td><td>ALA Scientific Instruments</td><td>PPH-1P-AXU-0-1.5</td><td></td></tr><tr><td>Headstage</td><td>Molecular Devices</td><td>CV203BU</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>AxoPatch200B</td><td></td></tr><tr><td>Digitizer</td><td>Molecular Devices</td><td>DigiData1400</td><td>Digital analog converter</td></tr><tr><td>Lightsource</td><td>TILL Photonics</td><td>Polychrome V</td><td>Set to 540 nm full intensity</td></tr><tr><td>Microscope</td><td>Carl Zeiss</td><td>Axiovert 100</td><td></td></tr><tr><td>Shutter</td><td>Vincent Associates</td><td>VS25</td><td></td></tr><tr><td>Shutter driver</td><td>Vincent Associates</td><td>VCM-D1</td><td></td></tr><tr><td>Glass capilarries</td><td>Warner Instruments</td><td>G150F-3</td><td>Boresilicate capillaries with fire polished ends OD 1.5 mm ID&amp;nbsp; 0.86 mm</td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>P1000</td><td></td></tr><tr><td>Bath handler</td><td>Lorenz Messger&amp;auml;tebau</td><td>MPCU</td><td></td></tr><tr><td>Tripleband filterset</td><td>Chroma</td><td>69008</td><td>Fluorescence filter&amp;nbsp; ECFP/EYFP/mCherry&amp;nbsp;</td></tr><tr><td>CCD camera</td><td>Watec</td><td>Wat-221SCCD</td><td></td></tr><tr><td>Optometer</td><td>Gigahertz Optik</td><td>P9710</td><td>Measure light intensities</td></tr><tr><td>Objective</td><td>Carl Zeiss</td><td>421462-9900-000</td><td>W Plan-Apochromat 40X/1.0 DIC</td></tr><tr><td>Micromanipulator</td><td>Scientifica</td><td>PatchStar</td><td></td></tr><tr><td>Recording chamber</td><td>Custom made</td><td></td><td></td></tr><tr><td>Power supply</td><td>Manson</td><td>HCS-3202</td><td>Avoids electrical noise from microscope built-in power supply</td></tr><tr><td>Vibration isolated table</td><td>Newport</td><td>M-VW-3636-OPT-01</td><td></td></tr><tr><td>Faraday cage</td><td>Custom made or any commercial matching table</td><td></td><td></td></tr><tr><td>Hoses</td><td>Any comercial; e.g. Roth</td><td></td><td>Different sizes and materials for bath handling and application of pipette pressure; agar bridges</td></tr><tr><td>Linear shaker</td><td>Sunlab Instruments</td><td>SU 1000</td><td></td></tr><tr><td>Liquid junction potential calculator</td><td>Molecular Devices or directly from Peter H. Barry</td><td></td><td>Program is included in the Clampex aquisition software or can be obtained from&amp;nbsp; p.barry@unsw.edu.au</td></tr><tr><td>Data acquisition software</td><td>Molecular Devices</td><td>Clampex 10.X</td><td></td></tr><tr><td>Data evaluation software</td><td>Molecular Devices</td><td>Clampfit 10.X</td><td></td></tr><tr><td>&lt;em&gt;Ps&lt;/em&gt;ACR1</td><td>GenBank or Addgene</td><td>KF992074.1 or Addgene plasmid #85465</td><td>Gene encoding for &lt;em&gt;Ps&lt;/em&gt;ACR1</td></tr><tr><td>Amplifier guide</td><td>Molecular Devices</td><td>The Axon Guide</td><td></td></tr></tbody></table>

whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins

In den letzten zehn Jahren wurden Channelrhodopsine in der neurowissenschaftlichen Forschung unentbehrlich, wo sie als Werkzeuge zur nicht-invasiven Manipulation elektrischer Prozesse in Zielzellen verwendet werden. In diesem Zusammenhang ist die Ionenselektivität eines Kanalrhodopsins von besonderer Bedeutung. Dieser Artikel beschreibt die Untersuchung der Chloridselektivität für ein kürzlich identifiziertes anion-leitendes Kanalrhodopsin von Proteomonas sulcata über elektrophysiologische Patch-Clamp-Aufnahmen auf HEK293-Zellen. Das experimentelle Verfahren zur Messung von lichtgesteuerten Photoströmen erfordert eine schnell umschaltbare - idealerweise monochromatische - Lichtquelle, die in das Mikroskop eines ansonsten konventionellen Patch-Clamp-Setups eingekoppelt ist. Vorbereitende Verfahren vor dem Experiment werden unter Berücksichtigung von gepufferten Lösungen, Überlegungen über Flüssigkeitsübergangspotentiale, Seeding und Transfektion von Zellen und Ziehen von Patchpipetten skizziert. Die tatsächliche Aufzeichnung der Strom-Spannungs-BeziehungS zur Bestimmung der Umkehrpotentiale für verschiedene Chloridkonzentrationen findet nach der Transfektion 24 h bis 48 h statt. Schließlich werden elektrophysiologische Daten in Bezug auf theoretische Überlegungen zur Chloridleitung analysiert.

<html> Fig. 2 zeigt repräsentative Ergebnisse, die aus Messungen nach dem beschriebenen Protokoll erhalten wurden. Bei der Beleuchtung mit grünem Licht weist der Ps ACR1 einen schnellen Übergangsstrom auf, der schnell auf einen stationären Strompegel abfällt. Nach dem Ausschalten des Lichtes zerfallen die Photoströme innerhalb von Millisekunden auf Null (Abbildung 2A ). Der Austausch der extrazellulären Chloridkonzentration bewirkt eine Verschiebung des Umkehrpotentials, das direkt in den erworbenen Photostromspuren beobachtet werden kann. Die Auswertung von Umkehrpotentialen aus mehreren Messungen quantifiziert die dramatische Umkehrpotentialverschiebung, die durch die Variation der äußeren Chloridkonzentration verursacht wird (Abbildung 2B ). Auffallend entsprechen die gemessenen Umkehrpotentiale direkt den berechneten Nernstpotentialen für Chlorid (Abbildung 2C ), die die hohe Chloridselektivität von Ps ACR1 bestätigen. T &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="Figur 2" src="/files/ftp_upload/55497/55497fig2.jpg" /> Abbildung 2: Chloridselektivität von Ps ACR1 . ( A ) Repräsentative Stromspuren von Ps ACR1 in HEK293-Zellen. Die intrazelluläre Chloridkonzentration wurde bei 120 mM gehalten, während die extrazelluläre Konzentration von 150 mM (grün) bis 10 mM (lila) Chlorid durch teilweises Ersetzen von NaCl mit Na-Aspartat variierte. Das Haltungspotential wurde in 20 mV Schritten von -80 mV auf +40 mV (LJP korrigiert) erhöht. ( B ) Durchschnittliche Strom-Spannungs-Relationen (n = 6 entsprechend den Bedingungen in A). ( C ) Umkehrpotentiale (Mittellinien zeigen Medianen, Feldgrenzen zeigen die 25. und 75. Perzentile an, wobei die Whisker das 2-fache der Standardabweichung und die Kreise auf einzelne Datenpunkte hindeuten, die aus den Strom-Spannungs-Relationen extrahiert werden. Für die hohe Chlorid-Kon-(Grün) wurde der Wert zwischen -20 mV und 0 mV Haltepotential linear interpoliert, während der Wert linear über +40 mV (lila, gestrichelte Linie, Tafel B) extrapoliert wurde. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55497/55497fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a>

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins at JoVE.com

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Ganzzellige Patch-Clamp-Aufnahmen zur elektrophysiologischen Bestimmung der Ionenselektivität in Channelrhodopsinen

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

whole-cell-patch-clamp-recordings-for-electrophysiological

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

17.0K Aufrufe.  Humboldt-Universität zu Berlin. This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Serie: Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

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 Clamp Recording von Ionenkanälen in Xenopus Oozyten exprimiert

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

Forschung

Biologie

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

Gleichzeitige Ganzzellableitungen von Photorezeptoren und Neuronen zweiter Ordnung in eine Amphibie Retinal Scheibe Vorbereitung

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Neurowissenschaften

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.

Afferent sensory neurons signal sensory information from the periphery to the central nervous system. Identifying specific afferent neurons will help in understanding their physiology. We describe a method of retrograde labeling to identify afferent neurons, and study the voltage-gated ion channels in these neurons using patch clamp electrophysiology and immunocytochemistry.

Identifikation von spezifischen sensorischen Neuronenpopulationen für das Studium der Ausgedrückt Ionenkanäle

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ...

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-und Perfusion Techniken zur Untersuchung von Ionenkanälen ausgedrückt in Xenopus Oozyten

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

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

Optimierte Transfektion Strategie für Expression und elektrophysiologische Ableitung von rekombinantem Voltage-Gated Ion Channels in HEK-293T-Zellen

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

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.

Elektrophysiologische Methode für Ganzzell-Spannungsklemmen Aufnahmen von<em&gt; Drosophila</em&gt; Fotorezeptoren

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

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.

Ein-Kanal-Cell-Attached-Patch-Clamp-Aufnahme

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

Optogenetic Electrophysiology Recording of Cells with Light-Dependent Ion Channels

Source: Grimm, C., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55497">Whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins.</a> J. Vis. Exp. (2017)
In this video, embryonic kidney cell cultures expressing light-dependent ion channel proteins fused with a fluorescent reporter demonstrate the effect of light on ion movement. Whole-cell patch clamp is used to monitor the movement of ions.

Optogenetische Elektrophysiologie Erfassung von Zellen mit lichtabhängigen Ionenkanälen

1. Setup Prior to Recordings

	Prepare the recording solutions with ion concentrations as indicated in Table 1.&#8203;
	
		Prepare 15 mL of 2 M stock ...

JoVE Encyclopedia of Experiments

Neurophysiology

Imaging and Optical Techniques

Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptors

Source:&#160; Katz, B., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55627">Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors.</a> J. Vis. Exp. (2017)
The video demonstrates recording electrical activity in the photoreceptor neurons of Drosophila&#39;s ommatidium by establishing a whole-cell configuration. It highlights the activation of transient receptor potential channels by light pulses, resulting in measurable quantum bumps.

Ganzzell-Spannungsklemmen-Aufzeichnungen von Drosophila-Photorezeptoren

NOTE: During all of the following steps, use only dim red light illumination and ensure that the ommatidia&#39;s exposure to light is minimal (i.e., work ...

Electrophysiology Techniques

Patch Clamp

Many fundamental cell functions such as muscle contraction and nerve transmission rely on the electrical signals produced by the movement of positively and negatively charged ions across the cell membrane. One competent method to record current flowing across the whole cell or single ion channel is the patch-clamp technique.
In this method, a glass micropipette containing electrolyte solution is tightly sealed against a small portion of the cell membrane. As a result, a patch of the cell membrane is electrically isolated, ensuring that the ions moving across the channels flow only into the micropipette. Further, this tight sealing of the cell membrane prevents ions from escaping into the bath solution in which the cell is suspended.
Different Patch-clamp Methods:
Depending upon the research interest, several variations of the patch-clamp method can be used for measuring the cell&#39;s biophysical properties. For example, researchers can clamp or control the membrane voltage and measure the current passing through it. Alternatively, they can clamp the current and measure any variation in the membrane voltage.
In cell-attached mode, the membrane patch containing a single or a few ion channels remains intact. As a result, the current flowing through the membrane patch alone can be measured. In contrast, the whole-cell method involves disruption of the membrane patch by briefly applying strong suction. Consequently, the interior of the pipette becomes continuous with the cytoplasm. This mode enables the measurement of electrical current and voltage from the entire cell.
Another patch-clamp method requires gentle retraction of the attached pipette. As a result, the membrane patch is excised without affecting the tight seal. In this inside-out configuration, the intracellular portion of the membrane is exposed to the bath solution, allowing the study of intracellular factors affecting the channel functions.

Many fundamental cell functions such as muscle contraction and nerve transmission rely on the electrical signals produced by the movement of positively ...