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.
