Name: Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells (Video) | JoVE Journal
Uploaded: 2010-10-01T17:00:00.000+00:00
Duration: 15 min 28 s
Description: Learn site-directed mutagenesis and patch clamp analysis to study GRK ion channel function in mammalian cells. This method reveals how point mutations affect alcohol and G-protein activation.

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5 minutes

Our work was made possible by financial support from McKnight Endowment Fund for Neuroscience (PAS), the National Institute on Drug Abuse (R01 DA019022; PAS) and a pre-doctoral NIH NRSA fellowship to P.A. (F31AA017042) from the National Institute on Alcohol Abuse and Alcoholism. The GIRK2- L257W mutant was provided by Dr. Prafulla Aryal. Fluorescence microscope and sponsorship kindly provided by Leica Instruments.

1. Site Directed Mutagenesis
We use mammalian expression plasmid pcDNA3.1 encoding a cDNA for GIRK2 and introduce a point mutation using Stratagene (Agilent Technologies) Quikchange XL II site-directed mutagenesis kit, according to the manufacturer's instructions.
Primers sequence can be easily generated using online primer design program available at: <a href="https://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Tool&amp;SubPageType=ToolQCPD&amp;PageID=15">https://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Tool&amp;SubPageType=ToolQCPD&amp;PageID=15</a> Note: We use standard Eppendorf 1.5 mL tubes in place of BD 14 mL tubes described in the protocol. However, the use of XL10-Gold ultracompetent E. coli pretreated with β-mercaptoethanol, 30s heat shock time and NZY+ (or NZYM+) media are critical for the successful mutagenesis. If possible, engineering an additional silent mutation that creates a new restriction site can be convenient for screening mutant colonies below.
2. Miniprep, DNA Sequencing and Maxiprep
2.1 Qiagen miniprep and sequencing.
Individual bacterial colonies are picked and grown in LB broth with ampicillin (AMP). We follow the manufacturer's instructions for purifying the cDNA. A simple restriction digest can be used confirm the successful introduction of the mutation, if a restriction site is incorporated in Step 1 above. Automated DNA sequencing is done off-site, and is essential to confirm the presence of the mutation in the cDNA, as well as assess whether there are any unwanted mutations. This step is very important. We sequence the entire GIRK coding sequence using forward T7 promoter and reverse BGH sequencing primers that flank multiple cloning site of the pcDNA3.1 plasmid. Note: It is important to carefully inspect the sequencing electropherogram to assure sufficient quality of the sequencing data. High quality electropherogram has sharp, non-overlapping peaks with little background noise.
2.2 Qiagen maxiprep.
We follow the manufacturer's instructions. Note: This step is included in our experiments to improve the purity of the cDNA, which we find is important for efficient transfection.
3. Culture and Transfection of HEK293T Cells
All of these steps should be done in a sterile tissue culture hood.
3.1 Cell culture
HEK293T cells are convenient to use because they are easy to culture (37°C., 5%CO2 humidified incubator), have fast replication time, high transfection efficiency and are amenable to whole-cell patch-clamp electrophysiology. HEK293T cells express SV40 large T antigen that ensures episomal replication of pcDNA3.1 plasmid.
HEK293T cells are cultured in low-glucose DMEM media supplemented with L-glutamine and 10 % Fetal Bovine Serum (FBS). For passaging cells, add 0.25% trypsin/EDTA to confluent (90-95%) HEK293T cells, wait until cells detach from the tissue dish, then stop the enzyme activity by addition of fresh media containing FBS/. Plate ~1 X 105 cells/well into a 12 well cell culture dish together with 1 mL of antibiotic-free culture media per well. (1 X 105 cells is ~ 350 μL of cell suspension obtained from confluent T25 flask resuspended with 10 mL media).
3.2 Transfection
After 8-24 hrs, HEK cells will adhere to the plastic and are ready for transfection. Transfect cells with 0.2 μg channel cDNA, 0.4 μg M2R receptor cDNA and 0.04 μg YFP cDNA using Invitrogen Lipofectamine 2000. Note: we use a small amount of YFP cDNA to determine which cells are successfully transfected (green cells will likely contain both GIRK channels and M2 receptors). The concentration of cDNA to use will need to be adjusted for each clone.
3.3 Fluorescence visualization of transfected cells.
Place 12-well plate on stage of an inverted fluorescence microscope, and examine cells for YFP expression. Compare to DIC image and make note of approximate number of green cells. This provides an estimate of the transfection efficiency.
3.4 Preparation of 24-well dishes and Poly-D-lysine coated cover slips
Clean 12 mm glass cover slips (Warner, CS-12R) by shaking them overnight with Radiacwash, followed by washing with deionized water and overnight shaking in 95% ethanol. Store the clean cover slips in 70% ethanol until use.
Place cover slips in a Petri dish with ethanol, flame off ethanol using forceps and burner, and place in one well of a sterile 24 well plate.
Cover each well with 250 μL of poly-D-lysine (PDL) solution [0.2 mg/mL] and let the cover slips sit overnight immersed in PDL solution under sterile hood. Wrap the 24 well plate in Parafilm to avoid evaporation.
Next morning, aspirate PDL and wash 2x with sterile water. Leave plates open to dry under the hood, then cover. The 24 well dishes with coated cover slips can be wrapped in with sterile aluminum foil and stored at room temperature for weeks.
3.5 Plating transfected HEK293T cells on the cover slips
24h after transfection, split cells into 24-well dish (~4 X 103 cells/well) containing glass cover slips coated with poly-D-lysine. Note: transfected cells can be used up to 24-72 hrs post transfection. The cells are split to provide individual cover slips for patch clamp electrophysiology experiment.	
4. Electrophysiology
4.1 Preparing Solutions
Extracellular (bath) solution: 20 mM KCl, 140 mM NaCl, 0.5 mM CaCl2, 2 mM MgCl2 and 10 mM HEPES (pH 7.4 set with NaOH).
Aliquot 20K bath solution and add appropriate modulators
<ul>
	<li>20K + Ba2+: solution containing 1 mM BaCl2: a specific inhibitor of inward rectifying currents</li>
	<li>20K + Carbachol: solution with 5 μM Carbachol: a specific M2R agonist; M2R couples to G-proteins that open GIRK channels</li>
	<li>20K + Ethanol: bath solution containing 100 mM EtOH to activate GIRK channels directly</li>
</ul>
Place bath solutions into syringes (solution containers) of the rapid exchange perfusion system. Note: We use a Warner pinch valve system to control the bath solutions. PE tubing is recommended.
Internal/electrode solution: 140 mM KCl, 20 mM NaCl, 5 mM EGTA, 5.4 mM MgCl2, 10 mM HEPES (pH 7.4) with 2.5 mM K2ATP and 0.3 μM Li2GTP.
We make the electrode solution without ATP and GTP. We separately prepare aliquots of ATP and GTP, which are stored at -80°C, and add to the electrode solution on the day of the experiment. We keep syringe with final electrode solution on ice to preserve the ATP/GTP. We sterile filter (0.2 μm) the internal/external solutions to inhibit microbial growth and reduce possibility of clogging the tubes and the electrode.
4.2 Pulling electrodes
We use Narishige two-step vertical puller and borosilicate glass electrode from Warner Instrument (1.5 mm outer diameter, 0.86 mm inner diameter and 7.5 cm length) to obtain electrodes with 3-7 MΩ of resistance when filled with intracellular solution.
Note: It is necessary to experimentally determine parameters for the electrode puller and type of electrode glass in your lab. For our vertical puller, we use a heat setting of 60.2 for the first pull and ~43 for the second pull.
4.3 Whole-cell patch clamping
<ol>
	<li>Mount cover slip in a 35 mm dish using small amount of vaseline, cover with external solution and place dish on stage of an inverted fluorescence microscope. Use DIC to focus and find cells, switch to YFP fluorescence to locate YFP positive cells.</li>
	<li>Position tip of perfusion manifold near a green cell (20-30 μm from the cell of interest=2-3 cell length apart).</li>
	<li>Fill electrodes with solution, attach it to the headstage of Axopatch 200B amplifier (Axon Instruments) with the following initial settings: Gain 1, Configuration: whole-cell β=1, mode V-clamp.</li>
	<li>Apply positive pressure on electrode to prevent clogging of the pipette, and use the manipulator to place electrode tip just above the cell.</li>
	<li>To Zero the pipette potential, select the Seal Test button to deliver a test voltage step. The 5 mV rectangular voltage step will appear in the Seal Test window of Clampex. Adjust the Pipette Offset potentiometer on the amplifier to bring the base-line current to 0 nA. Alternatively, zero the pipette potential by switching the amplifier mode selector to V-track mode and the meter knob to Vtrack and use the Pipette Offset potentiometer on the amplifier to bring the voltage on the amplifier meter display to "0.00".</li>
	<li>Check resistance of electrode using seal test command of the Clampex 8.2 software (should be 3-7 MΩ).</li>
	<li>Open a saved protocol file or create a new protocol. To open protocol file select from the main menu Acquire then Open Protocol and choose a saved voltage protocol file. For this experiment, the protocol has been created to hold membrane potential at -40mV, deliver a single 50 ms voltage step to -100 mV and ramp to +40 mV over 200 ms. This protocol is executed every 2 seconds. The ramp protocol allows for observation of the reversal potential and inward rectification of GIRK channels. For voltage-gated ion channels, a rectangular voltage step may be more appropriate.</li>
	<li>Approach cell using fine adjustment of the manipulator, release positive pressure, after touching the cell surface apply negative pressure, and monitor increase in resistance in the Seal Test oscilloscope window. Once resistance is in the GΩ range, the electrode has sealed onto membrane (Gigaseal has formed).</li>
	<li>Apply a pulse of negative pressure to break the cell membrane and get access into the cell. Once in the whole-cell configuration, you will register increased capacitive currents.</li>
	<li>Switch to Membrane Test in Clampex and write down the access resistance Ra, membrane resistance Rm, membrane capacitance Cm and verify by eye the exponential fit of the theoretical curve to the real capacitative current. Membrane capacitance is proportional to cell size and will be used later to calculate current density.</li>
	<li>Switch back to Seal Test, set V-Out (membrane potential) to -40 mV and filter at 10 kHz</li>
	<li>Compensate for membrane capacitance and series resistance on the amplifier with the following steps:
	<ol class="lalpha">
 	<li>Switch the WHOLE CELL CAP switch on. Adjust WHOLE CELL CAP and SERIES RESISTANCE dials back and forth, until you get a flat test pulse. You may also need to adjust pipette capacitance FAST CAP knob.</li>
		<li>Turn on % PREDICTION knob to about 60-80%, again re-adjusting WHOLE CELL CAP and the SERIES RESISTANCE dial (you may have to adjust pipette capacitance compensation FAST CAP knob as well).</li>
		<li>Turn on % COMP- to 100% without oscillating the cell, while trying to keep the pulse flat by re-adjusting WHOLE CELL CAP and SERIES RESISTANCE (you may have to adjust pipette capacitance compensation FAST CAP knob as well). Adjust the lag by reducing the time constant.</li>
		<li>Write down values for Cm (membrane capacitance), Rs (series resistance), %COMP, %PRED and Lag time set on the amplifier in the notebook. The Cm and Rs should be approximately the same as the Cm and Rm measured in the Membrane Test above.</li>
 </ol>
 </li>
	<li>Turn on external solution valve to control the bath solution. Set the amplifier 8-pole Bessel filter to 2kHz, open a voltage clamp protocol and begin recording currents.</li>
	<li>We record GIRK currents using a ramp protocol that steps to -100 mV for 50 ms then ramps from 100 mV to +40 mV over 200 ms and is delivered every 2 seconds. The ramp protocol allows for observation of the reversal potential and for inward rectification property of GIRK channels. At room temperature (22-25° C), the GIRK current should reverse near -50 mV (with 20 mM KCl in the bath), which is near the calculated equilibrium potential for K (EK = -50 mV), then remain fairly flat at potentials positive to EK.</li>
	<li>Experimental Protocol: After recording a stable baseline, switch to solution 20K + Carbachol and wait for peak of response, switch back to 20K bath solution, then apply 20K + Ethanol and wait for peak response, then switch to 20K bath solution, and finally to 20K + Ba2+ solution. During application of these drugs you will see changes in the amplitude of GIRK currents (most pronounced at -100 mV). Note the trace number corresponding to the application of different extracellular solutions in the notebook. A single Clampfit file will contain all of the sweeps for the entire experiment.</li>
</ol>
5. Data Analysis
We use Clampfit 9.2 software to analyze data GIRK currents at -100 mV.
<ol>
	<li>Open the file with the recording, set cursor at -100 mV. By default you will see all the traces overlaid on the screen. By pressing "Write cursors" icon you will create Excel-like spreadsheet with numerical data.</li>
	<li>By assigning X to the trace number column and Y to the cursor column and pressing "Create Graph" icon you can quickly draw a time-course plot of the experiment.</li>
	<li>Calculate basal current by subtracting current in presence of Ba2+ from current recorded in external perfusion solution alone ("20K current" minus "20K + Ba2+ current"). Calculate induced currents by subtracting basal current in 20K from the activated currents ("20K + Carbachol current" minus "20K current"; "20K + Ethanol current" minus "20K current"). Calculate current density (pA/pF) by dividing the current by membrane capacitance (read directly from the amplifier). Calculate percent activation by dividing induced currents by basal currents and multiplying by 100.</li>
</ol>
6. Representative Results
You should be able to record current from cells transfected with wild-type channels. For GIRK channels measured in the extracellular solution containing 20 mM K+ (and 145 mM K+ intracellular) they should exhibit strong inward rectification and a reversal potential of ~ -50 mV, which is an essential property of potassium channels. The wild-type current will increase strongly upon carbachol application (3-5 fold increase over basal current) and respond similarly to the 100 mM ethanol. Note: the sign convention for electrophysiology is that inward current is negative and outward current is positive. For the L257W mutation in the alcohol binding pocket of GIRK2, you will see attenuated responses to both carbachol and ethanol. This suggests that L257 is involved in alcohol and G protein activation of GIRK2 channels. For further examples of the results of GIRK2 mutagenesis, see recent publication by Aryal et al.1.
<img src="/files/ftp_upload/2189/2189fig1.jpg" alt="Electrophysiology data analysis, graphs showing GIRK2 channel activity, used in patch-clamp study." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Window of Clampfit 8.0 shows data file for wild-type GIRK2 recording. Upper left panel shows superimposed sweeps recorded in response to a ramp voltage protocol in the presence of different external bah solutions. Cursor 1 is positioned at -100 mV. The cursor values are written to the spreadsheet (Right panel). Lower left panel shows the plot of sweep number vs., current. Note the increase in current (downward deflection) with ethanol (EtOH) and carbachol (Carb) and current inhibition with Ba2+.
<img src="/files/ftp_upload/2189/2189fig2.jpg" alt="Electrophysiology analysis with patch clamp data, GIRK2 L257W; ion current graphs, Clampfit software." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Window of Clampfit 8.0 shows data file for a mutant GIRK2-L257W recording. Descriptions are same as in Figure 1. Note the loss of carbachol (Carb) activation and reduction of EtOH-induced currents in the mutant channels.

<ol><li>Aryal, P., Dvir, H., Choe, S., Slesinger, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+discrete+alcohol+pocket+involved+in+GIRK+channel+activation%5BTitle%5D)+AND+%22Nature+Neurosci%22%5BJournal%5D)">A discrete alcohol pocket involved in GIRK channel activation.</a> Nature Neurosci. 12, 988-995 (2009).</li>
<li>Panchenko, V. A., Glasser, C. R., Mayer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+similarities+between+glutamate+receptor+channels+and+K%2B+channels+examined+by+scanning+mutagenesis%5BTitle%5D)+AND+%22J+Gen+Phys%22%5BJournal%5D)">Structural similarities between glutamate receptor channels and K+ channels examined by scanning mutagenesis.</a> J Gen Phys. 117, 345-360 (2001).</li>
<li>Doyle, D. A., Cabral, M. orais, Pfuetzner, J., Kuo, R. A., Gulbis, A., Cohen, J. M., Chait, S. L., &amp, B. T., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure+of+the+potassium+channel%3A+molecular+basis+of+K%2B+conduction+and+selectivity%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The structure of the potassium channel: molecular basis of K+ conduction and selectivity.</a> Science. 280, 69-77 (1998).</li>
<li>Nishida, M., Cadene, M., Chait, B. T., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystal+structure+of+a+Kir3.1-prokaryotic+Kir+channel+chimera%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Crystal structure of a Kir3.1-prokaryotic Kir channel chimera.</a> EMBO J. 26, 4005-4015 (2007).</li>
<li>Tao, X., Avalos, J. L., Chen, J., MacKinnon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystal+structure+of+the+eukaryotic+strong+inward-rectifier+K%2B+channel+Kir2.2+at+3.1+%26Aring%3B+resolution%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Crystal structure of the eukaryotic strong inward-rectifier K+ channel Kir2.2 at 3.1 &Aring; resolution.</a> Science. 326, 1668-1674 (2009).</li>
</ol>

No conflicts of interest declared.

Site-directed mutagenesis is a classical approach to study structure-function relationships of ion channels. It is based on the premise that by changing a critical residue of the ion channel one should be able to observe pronounced changes in the channel function. In contrast, many mutations in non-critical position can be introduced without major perturbations to the channel function. Typically, the residue in question is first mutated to alanine, a small non-polar amino acid, followed by mutation to tryptophan, the largest of amino acids2. If the residue is important to the channel function, both mutations will change channel activity. If a less essential residue is mutated, the effects of mutation, especially to alanine are often unremarkable. Tryptophan mutations are more likely to cause changes due to steric hindrances associated with the tryptophan size. Often, when the structural information is lacking, systematic mutation to alanine or tryptophan within a fragment of channel (called alanine or tryptophan scan) can be used to locate critical residues. It is important to note that multiple clones with different point mutations can be prepared in parallel allowing for testing multiple residues within relatively short time. Recently, multiple crystal structures of the channels have become available3,4,5. They can be used to propose potential key residues to be tested using the approach described in this protocol.
Measurements on wild-type channel should always accompany the measurement of a mutant channel to serve as a positive control to assure that transfection and electrophysiological experiments were done correctly. Point mutations may lead to lack of a functional current, a current similar to the wild-type or altered regulation of the channel activity. Lack of measurable current may be due to improper folding or altered trafficking. In such case it may be useful to find out whether the channel traffics correctly to the surface using a biotinylation assay or immunofluorescence staining against extracellular tag (without cell permeabilization) to distinguish between non-functional channels on the plasma membrane and channels retained inside the cell. Finally, it is advisable to investigate not only basal activity of the channel but also consequences of mutation on the channel regulation. Different mechanisms of modulation of the channel function could be studied depending of the ion channel of interest including regulation by G proteins, PIP2, ions, ATP concentration, phosphorylation (using tyrosine, serine or threonine mutation), ubiquitination or sumoylation (using lysine mutation) and direct channel openers or blockers.

<table><tbody><tr><td>Chemicals</td><td>Sigma</td><td>Varies</td><td></td></tr><tr><td>Mini Prep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>Radiacwash</td><td>Biodex</td><td>005-100</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>BD Biosciences</td><td>CB40210</td><td>Poly-L-lysine can also be used</td></tr><tr><td>Maxi Prep Kit</td><td>Qiagen</td><td>12263</td><td></td></tr><tr><td>QuickChange XL II Site Directed Mutagenesis Kit</td><td>Stratagene</td><td>200512-5</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-027</td><td></td></tr><tr><td>Cover slips &amp;#710;&amp;#8230; 12 mm</td><td>Warner Instruments</td><td>CS-12R</td><td></td></tr><tr><td>Capillary Glass Tubing Borosilicate-standard wall</td><td>Warner Instruments</td><td>G150-3</td><td></td></tr><tr><td>&lt;a href="http://www.leica-microsystems.com/products/light-microscopes/clinical/inverted-microscopes/details/product/leica-dmi3000-b/" target="blank"&gt;Inverted fluorescence microscope&lt;/a&gt;</td><td><a href="http://www.leica-microsystems.com/products/light-microscopes/clinical/inverted-microscopes/details/product/leica-dmi3000-b/" target="_blank"><img src="/img/new_window.png"> Leica</a></td><td>DMI3000 B</td><td></td></tr><tr><td>Electrophysiology</td><td>Nikon/&lt;br/&gt;Leica/&lt;br/&gt;Zeiss</td><td>Any inverted DIC&lt;br/&gt;(Hoffmann)&lt;br/&gt;microscope with fluorescence</td><td></td></tr><tr><td>Amplifier</td><td>Molecular Devices</td><td>Axon Axopatch 200B amplifier</td><td></td></tr><tr><td>Perfusion system</td><td>Warner Instruments</td><td>VC-6 Perfusion MM-6</td><td></td></tr><tr><td>Digitizer</td><td>Molecular Devices</td><td>Digidata 1320 digitizer</td><td></td></tr><tr><td>Manipulators</td><td>Sutter Instruments</td><td>Manipulator: MP-85</td><td></td></tr><tr><td>Electrode Puller</td><td>Narishige</td><td>PC10 vertical electrode puller</td><td></td></tr><tr><td>Isolation Table</td><td>Technical Manufacturing Corporation</td><td>Air table</td><td></td></tr></tbody></table>

mutagenesis and functional analysis of ion channels heterologously expressed in mammalian cells

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

G-Protein gated inwardly rectifying potassium channels or grk are important for regulating the excitability of neurons. Gks can be activated by both G-protein coupled receptors GPCRs, such as the muscarinic receptor M two R and by alcohol to study the effects of mutations in the gerk alcohol binding site. On its activation, a single point mutation is introduced into the grk CD NA by site directed mutagenesis purified gerk constructs are transfected, along with YFP constructs into mammalian cells, and the transfection efficiency is assessed by fluorescence imaging.Whole cell patch clamp analysis is then performed to compare the alcohol and G-protein dependent activation. This visual experiment demonstration will provide you with key information on how to study the function of LY channels. Today we'll be looking at a question of how alcohol activates group channels.The site directed neogenesis technique shown here can provide insight into ion channel function. This technique can also be applied to other systems where a mutation in a gene was known to cause human disease. These Are demonstration of hot cell patch Climbing technique is critical because there are many steps that require precision, proper electronic compensation and practice for generating usable data.The DNA used for transfection in this protocol is prepared as follows. First, GRK subunit, DNA from PCR is directly cloned into the mammalian expression. Plasmid PC DNA 3.1 point mutations are introduced using appropriately designed primers along with the quick change XL two site directed mutagenesis kit.Using this system, PCR is performed in which the entire plasmid is replicated, and point mutations encoded by the primers are introduced. Following PCR, the replicated plasmids are digested with DPN one restriction enzyme and then transformed by heat shock into XL 10 gold ultra competent e coli. The transformed bacteria are grown on LB agar plates with ampicillin bacteria harboring the plasmid are ampicillin resistant and will produce colonies.The isolated colonies are picked and grown in mini cultures. Mini preps are performed to isolate the plasmid DNA. The DNA is then sequenced to identify mutants successfully transformed.E coli are grown in a larger batch and maxi preps are performed to prepare larger quantities of plasmid DNA plate about one times 10 to the five HEC 2 9 3 T cells into each of two wells. On a 12 well plate, one for the mutant and one for the control. Wild type channel incubate at 37 degrees Celsius.After eight to 24 hours, the cells will adhere to their substrate and are ready for transfection. Transfect the cells with lipo 2000 according to the manufacturer's instructions with 0.2 micrograms of channel CDNA 0.4 micrograms of M two R receptor CDNA and 0.04 micrograms of Y-F-P-C-D-N-A. To identify successfully transfected cells incubate the cells for 24 hours.After the desired incubation time has passed, place the cells on the stage of an inverted fluorescence microscope to determine the transfection efficiency. Compare fluorescent and DIC images to determine the percentage of cells transfected. Return the culture dish back to the incubator.Clean 12 millimeter glass cover slips and store in ethanol until use. Remove the residual ethanol by flaming in the sterile cabinet. Place the cover slips in a 24 well plate and coat them with 0.2 milligrams per milliliter.Poly de lycine 24 hours after transfection trypsin eyes the cells and plate four times 10 to the three cells per well. In four to eight wells of a 24 well dish containing poly de lycine coated glass cover slips incubate for an additional 24 to 48 hours prior to performing wholesale patch clamping. Prepare patch electrodes using a two step vertical puller and boro silicate electrode glass.Ideally, the finished electrode will have three to seven mega ohms resistance and will not require additional manipulation such as heat polishing. For patch clamping, place a cover slip with transfected cells in a 35 millimeter dish containing 20 millimolar potassium or 20 K external solution. Place the dish on the stage of an inverted fluorescence microscope.The patch clamping setup used here employs a perfusion system. This system contains the extracellular solutions that will be applied to the cells which are gravity fed through tubes connected to a manifold. Each solution is controlled remotely with a solenoid pinch valve.Use DIC to focus and find cells. Switch to the YFP filter to locate the YFP positive cells. Once the YFP positive cells have been identified, position the tip of the perfusion system, neuro fluorescent cell, 20 to 30 microns or two to three cell lengths away.Fill an electrode with internal electrode solution. Using a syringe and flexible needle, gently tap the filled electrode to remove air bubbles from the tip. Attach the electrodes to the head stage of an AXO patch 200 B amplifier.Check that the settings on the amplifier again equals one configuration. Whole cell switch is beta equals one, and the lowpass vessel filter is set to 10 kilohertz. Apply positive pressure with the syringe and clamp to keep the electrode tip clean.Then use a micro manipulator to place the tip of the electrode in the bath and move to above the cell. Switch the amplifier to V track mode and the meter knob to V track. Then use the pipette offset potential meter on the amplifier to zero the pipette potential by bringing voltage on the amplifier meter display to 0.00.Alternatively, switch to V clamp mode on the amplifier and select the test pulse button to deliver a test voltage step. The rectangular voltage step will appear in the seal test window of clamp. Adjust the pipette offset on the amplifier to bring the baseline current to zero nano and pairs.The seal test will show the electrode resistance, which should be three to seven mega ohms. Next, close the seal test window. Select acquire open protocol in the dropdown menu.Then select a saved voltage protocol. Here, the protocol selected has been pre-programmed to deliver a single voltage step to minus 100 millivolts and ramped to plus 40 millivolts every two seconds. This ramp protocol allows for observation of the reversal potential and for inward rectification property of GK channels.For voltage gated ion channels, a rectangular voltage step may be more appropriate using the fine adjustment of the micro manipulator approach the cell release the positive pressure, and after touching the cell surface, apply slight negative pressure. Monitor the resulting increase in resistance in seal test. Once resistance is in the giga ome range, the electrode has sealed onto the membrane.This is referred to as a giga seal. Using a syringe, apply a pulse of negative pressure to break the membrane and gain access into the cell. Once in the whole cell configuration, the seal test will register large capacitive currents that are visible as spikes or transient at the beginning and end of the test.Pulse in seal test. Next, click on the membrane test button. In clamp X, record the access resistance membrane resistance.A membrane capacitance in a notebook verify exponential fit by eye. The membrane capacitance is proportional to the cell size and will be used to calculate current density access. Resistance and membrane resistance are used to evaluate whether the recording will be satisfactory for continuing.A good recording will have a low access resistance. Now close the membrane test window and click on the seal test button again in the seal test window set V out to minus 40 millivolts to compensate for membrane capacitance and series resistance on the amplifier, switch the whole cell cap on, adjust the whole cell cap and series resistance back and forth until the transient is minimized and the current is a flat line. If needed, adjust the pipette capacitance with the fast cap knob on the amplifier.Too much or too little compensation will leave a residual capacity transient. Next turn the percentage prediction knob to 60 to 80%readjust the whole cell cap series resistance and fast cap as needed to obtain a flat line in the seal test. Finally, turn percentage comp to about 100%without oscillating the cell, whilst trying to keep the pulse as linear as possible by readjusting whole cell cap series resistance and fast cap potentia meters.Once resistance and capacitance compensation is achieved, write down the value set for the amplifier capacitance series resistance percent, compensation percent prediction and lag time in a notebook, turn on the external solution valve for the control 20 K bath solution. Set lowpass vessel filter on the amplifier to two kilohertz. Then start recording currents by clicking on the record button in the software.Look for the inward rectification property of the ger current. This is seen as a large inward current negative to the potassium reversal potential that is determined by the nert equation EK and a small outward current positive to ek. After recording a stable baseline, determine receptor mediated activation of currents by applying extracellular solution 20 k plus carbahol.Once peak activation is observed and current reaches steady state, switch back to the K bath solution and wait until baseline current level is reached. Next to study alcohol activation of gert currents. Switch to external solution 20 k plus 100 millimolar ethanol and wait for peak response.Then switch back to the 20 K bath solution and wait until baseline current level is reached. Finally, to determine the basal gert currents switch to the 20 K plus 20 K barium solution and wait for the response here. The inhibition of gerk current should be evident being most pronounced at minus 100 millivolts throughout the procedure.Note the trace sweep number corresponding to the application of different extracellular solutions. A single clamp fit file will contain all the sweeps for the entire experiments. To analyze GK data, use the clamp FIT 8.2 software.According to the instructions in the accompanying written protocol, two different wholesale recordings were obtained from HEC 2 9 3 T cells expressing either wild type or mutant GK channels and analyzed according to the instructions in this video as shown here for wild type GK two. The panel on the left contains a window with several superimposed currents that were produced by the voltage ramp protocol in the presence of different solutions. Note the inward rectification.That is the larger inward current at minus 100 millivolts around 50 milliseconds on the current trace. The small outward current between zero and plus 40 millivolts around 200 to 260 milliseconds on the current trace originates from an endogenous voltage gate gator potassium channel. It is not the Gert current cursor.One is placed over the maximal inward current, and the cursor values are written to the spreadsheet. In the spreadsheet. The trace number and current at minus 100 millivolts are seen.The data include basal current, carbahol induced and alcohol evoked currents, as well as a barium inhibited current. Likewise, the voltage ramp currents in the presence of different solutions. 20 k plus 100 millimolar ethanol or 20 k plus five millimolar carbahol or 20 k plus barium are shown for the mutant channel L 2 57 w.Note the significantly smaller alcohol and carbo alcohol-induced currents. These findings suggest that this residue plays an important role in gating of GK channels by alcohol and g beta gamma subunits. After watching this video, you should have a good understanding of how to construct a mutation in an and start and study its effect on channel function using advanced electrophysiologic techniques.Once master, this site directed neogenesis technique can be done in two to three days and scaled up to five or more mutations. The development of these techniques paved the way for ion channel biophysicists to explore basic properties of ion channel function and gating. In addition, they allow researchers to study the human diseases that are caused by a single mutation in an ion channel gene.

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Research

JoVE Journal

Biology

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).

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

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

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.

One-channel Cell-attached Patch-clamp Recording

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Controllable Ion Channel Expression through Inducible Transient Transfection

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

Expression and Purification of Mammalian Bestrophin Ion Channels

This protocol describes techniques used to determine ion channel structures by cryo-electron microscopy, including a baculovirus system used to efficiently express genes in mammalian cells with minimum effort and toxicity, protein extraction, purification, and quality checking, sample grid preparation and screening, as well as data collection and processing.

Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

The pharmacological targeting of ion channels is a promising approach to treating solid tumors. Detailed protocols are provided for characterizing ion channel function in cancer cells and assaying the effects of ion channel modulators on cancer viability.

Screening Ion Channels in Cancer Cells

Learn site-directed mutagenesis and patch clamp analysis to study GRK ion channel function in mammalian cells. This method reveals how point mutations affect alcohol and G-protein activation.

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

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