We thank Maila Reh, Tharsana Tharmalingam and especially Altina Klein for excellent technical assistance. This work was supported by the German Research Foundation (DFG) (SFB1078 B2, FOR1279 SPP1665 to P. H.) and the Cluster of Excellence Unifying Concepts in Catalysis, UniCat, BIG-NSE (J.V.) and E4 (P.H.).

<img alt="Figure 1" src="/files/ftp_upload/55497/55497fig1.jpg" /> 
Figure 1: Patch-clamp Setup. (1) Light source, (2) optic fiber, (3) programmable shutter, (4) digitizer, (5) shutter driver, (6) amplifier, (7) perfusion system, (8) personal computer, (9) monitor, (10) faraday cage, (11) microscope stage, (12) perfusion inlet, (13) perfusion outlet, (14) recording chamber, (15) fluid level sensor, (16) bath electrode with agar bridge, (17) pipette holder, (18) heads...

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The authors have nothing to disclose.

Determination of reversal potentials at defined ionic and electrical conditions provides information about the ion species transported after light activation of ChRs. If exclusively one ion species is varied in a complex physiological medium and the obtained reversal potential shifts according to the theoretical Nernst potential, this ion species is the only transported one.
However, for ChRs, reversal potential shifts are usually less pronounced than expected from the Nernst equation due to p...

Channelrhodopsins (ChR) are light-gated ion channels that occur in the eye spot of motile green algae, and serve as primary photosensors for phototaxis and phobic responses1. Since their first description in 20022, ChRs have paved the way for the emerging field of optogenetics and can be applied in a variety of excitable cells e.g. within skeletal muscles, the heart, or the brain3,4,5. Expression of ChRs in target cells results in light-controllable ion permeability of the respective cell. In a neuronal context, this allows activation6,7,8 or inhibition9,10 of action potential (AP) firing &#8211; depending on the conducted ion &#8211; with the spatial and temporal precision of light emphasizing how the ion selectivity of a ChR variant determines its optogenetic application.
The first discovered ChRs from Chlamydomonas reinhardtii and Volvox carteri are permeable to protons, but also to monovalent cations like sodium, potassium, and to a lesser extent to divalent cations such as calcium and magnesium11,12,13. Today, more than 70 natural cation-conducting channelrhodopsins (CCRs)14,15,16,17 and several engineered variants18,19,20 with different properties such as photocurrent size, spectral sensitivity, kinetics, and cation selectivity are available. Whereas in neuroscience, CCRs are used to activate cells and trigger APs, light-driven microbial pumps were the only available antagonists for silencing neurons for years. In 2014, two groups simultaneously showed that CCRs can be converted into anion-conducting channelrhodopsins (ACRs) by alteration of the polarity along the putative ion conducting pore via molecular engineering9,21. Subsequently, natural ACRs were identified in several cryptophyte alga22,23,24. Most importantly, light activation of ACRs mediates chloride currents in adult neurons allowing inhibition of neuronal activity at much lower light intensities than microbial pumps that only transport single charges per absorbed photon.
ChR activity can be directly addressed by electrophysiological patch-clamp recordings of light-induced currents in HEK293 cells. The patch-clamp technique was originally developed in the late 1970s25 and further improved by Hamill et al., allowing the recording of the entity of currents from a small cell (whole cell mode) with high current resolution and direct control of the membrane voltage26. Applied in cell culture, this technique provides accurate control of the ionic as well as electrical recording conditions, and enables studying ion selectivity along with the relative contribution of the ions to the total current. Here we exemplify the examination of ion selectivity for the anion-conducting channelrhodopsin of Proteomonas sulcata (PsACR1)22,23 via the recording of current-voltage relations under various extracellular chloride concentrations to prove high chloride conductance.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;s Modified Eagle Medium</td>
		</tr>
		<tr >
			<td >Agarose</td>
			<td >Roth</td>
			<td>3810</td>
			<td>Agar bridges</td>
		</tr>
		<tr >
			<td >CaCl2</td>
			<td >Roth</td>
			<td>5239</td>
			<td>CaCl2 2H2O</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 >MgCl2</td>
			<td >Roth</td>
			<td>2189</td>
			<td>MgCl2 6H2O</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 E.coli/</td>
			<td >Agilent Technologies</td>
			<td>200249</td>
			<td>Chemocompetent E.coli</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 >CO2 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&#246;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&#160; 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&#228;tebau</td>
			<td >MPCU</td>
			<td></td>
		</tr>
		<tr >
			<td >Tripleband filterset</td>
			<td >Chroma</td>
			<td>69008</td>
			<td>Fluorescence filter&#160; ECFP/EYFP/mCherry&#160;</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&#160; 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 >PsACR1</td>
			<td>GenBank or Addgene</td>
			<td >KF992074.1 or Addgene plasmid #85465</td>
			<td>Gene encoding for PsACR1</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

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

Figure 2 shows representative results obtained from measurements following the described protocol. During illumination with green light, PsACR1 features a fast transient current which rapidly decays to a stationary current level. After light is switched off, photocurrents decay to zero within milliseconds (Figure 2A). Exchanging the extracellular chloride concentration causes a shift of the reversal potential that can be directly seen in the acqu...

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.

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

The overall goal of this experiment is to determine chloride selectivity of new light-gated ion channels. This method can answer key questions in the biophysics of channelrhodopsins such as photocurrent amplitudes, kinetics and ion selectivity. The main advantage of this technique is the direct readout of biophysical channel activity that is fast and easy to replicate.Generally individuals new to this method will struggle because each step of the experimental procedure from the preparation of HEK cells to the recording of photocurrents has to be optimized. Seeding and transection of the HEK cells will be demonstrated by Altina Klein. To begin this procedure, weigh the solid components for 100 milliliters of the intracellular and 500 milliliters of the extracellular buffered solutions in separate beakers.And then add the respective amount of the prepared stock solutions. Next add ultrapure water and carefully adjust the pH using acids and bases. Then adjust the osmolarity with glucose to 290 milliosmoles and 320 milliosmoles for the intracellular and extracellular solutions, respectively.In this procedure place up to three lysine-coated glass cover slips side-by-side in a 35-millimeter Petri dish. Then seed 1.5 times 10 to the fifth HEK cells in two milliliters of standard DMEM supplemented with 10%FBS and one micromolar retinol in each dish. To transfect the cells for each dish, prepare a solution of two micrograms of plasmid DNA in 250 microliters of DMEM without FBS and six microliters of transfection reagent.After 15 minutes of incubation, gently add the solution to the cells. Before measuring, pull some low resistance patch pipettes using a micropipette puller and fire polish them. Store the pipettes in a dust-free container for the day of measurement.30 minutes prior to recordings, turn on all the setup components to heat up to the working temperature. Ensure that the buffers are at room temperature. To begin, seal the measuring chamber with silicon to prevent leakage of the external buffer.Then place one coverslip into the chamber and close it. Fill the chamber carefully with extracellular buffer to prevent the cells from detaching. Store the Petri dish with other cover slips in an incubator for the next set of experiments.Then place the measuring chamber under the microscope using the 40X objective for cell visualization. Next put an Auger bridge over the bath electrode and place it into the chamber together with the fluid level sensor and the perfusion outlet of the bath handler. Exchange the extracellular solution twice with one milliliter of fresh external solution to remove the residual culture medium and the detached cells.Bring the cells into focus and search for a transfected cell which needs to be isolated from other cells. Then use a triple band filter set and orange light to excite and visualize mCherry. Next, fill one of the pulled pipettes with the intracellular solution.And remove the air bubbles by flipping the pipette several times. Afterward mount the pipette on the pipette holder and apply some positive pressure to prevent tip clogging. Locate the tip of the patch pipette under the microscope and navigate it close to the cell using the micromanipulator.Within the data acquisition software start the membrane test in Bath mode and apply a voltage step. Check if the pipette resistance is in the desired range of 1.3 to 3.0 megaohms. Then zero the offset currents and adjust the pipette potential by turning the pipette offset knob on the amplifier.To establish a patch in the whole-cell configuration, slowly approach the cell with the patch pipette from the top and release the positive pressure right before touching the cell. Sometimes the cell-attached configuration forms itself after that. After approaching the cell, change the membrane test from Bath mode to Patch mode.Compensate the pipette capacitance by turning the pipette capacitance compensation knob to get a flat response of the test pulse. Switch the membrane test to Cell mode. Then rupture the patch without destroying the seal by applying short pulses of negative pressure or negative pressure with increasing strength to obtain whole-cell confirmation.Start series resistance compensation by setting the two whole-cell parameters, cell capacitance and series resistance. In the series resistance compensation panel turn on prediction and correction up to 90%Then turn the whole-cell compensation switch on and adjust the whole-cell parameters in an iterative way to obtain an ideally flat test seal response. After establishing whole-cell configuration, wait at least two minutes before recording to ensure sufficient replacement of intracellular solution through the patch pipette.Record light-induced currents at different holding potentials. Next exchange the extracellular buffer to low chloride in the chamber at least five times without destroying the patch and record another current voltage relation at the new chloride concentration. This figure shows that during the illumination with green light, PsACR1 features a fast transient current which rapidly decays to a stationary current level.After the light is switched off, photocurrents decay to zero within milliseconds. Reducing the extracellular chloride concentration completely abolishes outward directed photo currents. Exchanging the extracellular chloride concentration causes a shift of the reversal potential that can be deduced from a current voltage plot.The evaluation of reversal potentials from multiple measurements quantifies the dramatic reversal potential shift caused by the variation of external chloride concentration. Strikingly, the measured reversal potentials directly correspond to the calculated Nernst potentials for chloride which confirms the high chloride selectivity of PsACR1. After watching this video, you should have a good understanding of how to perform electrophysiological recordings of light-induced photocurrents on HEK cells expressing channelrhodopsin.Once mastered, the whole procedure can be done in four days while the recording of photocurrents take several hours. Complementing this procedure, site-directed mutagenesis and spectroscopic methods can be performed to further elucidate structural features and cellular mechanisms of channelrhodopsins. The characterization and molecular engineering of channelrhodopsin paved the way for researchers in neuroscience to manipulate the excitability of selected neurons by light.

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

Research

JoVE Journal

Neuroscience

16.8K Görüntüleme.  Humboldt-Universität zu Berlin. The overall goal of this experiment is to determine chloride selectivity of new light-gated ion channels. This method can answer key questions in the biophysics of channelrhodopsins such as photocurrent amplitudes, kinetics and ion selectivity. The main advantage of this technique is the direct readout of biophysical channel activity that is fast and easy to replicate.Generally individuals new to this method will struggle because each step of the experimental procedure from the prepara...