The overall goal of this procedure is to record BK channel activation under controlled voltage, while also controlling the intracellular solution. This is accomplished by first expressing BK channels in Xenopus Cytes by injecting them with BK channel mRNA. The second step of the procedure is to prepare the perfusion system.
The third step of the procedure is to form a giga seal between the oocyte membrane and the patch pipette. The final step of the procedure is to excise the membrane patch. Ultimately, results obtained by using patch clamp technology coupled with a profusion system show that BK channels are activated by changes in voltage and calcium concentration.
Hi, I'm Jin Cho Young from Dr.Jamie Zus lab in Washington University. This method can help answer key questions in the neuroscience field, including questions concerning the molecular mechanism for ion channel activation. So let's get started.
The first step in the procedure starts a couple of days before the patch clamping and uses BK channel mRNA that was transcribed in vitro. After harvesting the Xenopus Lavu Cytes stage five to six, inject 0.05 to 50 nanograms of BK channel mRNA into an cyte. Using a nano inject two auto nanoliter injector.
Repeat the injection on a dozen oocytes for each mRNA following the injection. Rinse the oocytes twice using ND 96 solution. Then place the oocytes in a culture plate and incubate them at 18 degrees Celsius for two or more days to allow time for the oocytes to express the BK channel.
Following two to five days of incubation, the oocytes should be expressing BK channels and so are already for patch clamping. Before handling the oocytes, set up everything else you'll need for patch clamping, starting with the perfusion system. The system shown here is the automate valve link.
16 profusion system pressurized nitrogen pushes profusion solutions out of the solution reservoirs through the profusion tubings and into the profusion pencil and tip. The flow of each stream of profusion solution is controlled by one reservoir valve and one electronic valve. In this protocol, one of the reservoirs is not pressurized and functions as a waste collector, as well as a pressure release mechanism.
Perfusion solutions of differing concentrations of calcium or magnesium may be prepared prior to the experiments. Add the perfusion solutions to the reservoirs and label each one to indicate the concentration of calcium or magnesium. To set up the profusion system first, connect the tubings to the profusion pencil.
Then turn on the electronic valve controller and open the electronic valves to assure that the tubings and the profusion pencil are free of clogs and air bubbles. Push deionized water through each tubing using a syringe filled with deionized water. At this point, connect the tubings to the solution reservoirs and open the valve of gas cylinder to apply pressurized to nitrogen.
Open one reservoir valve to fill the tubing with one of the reservoir solutions. If needed, click the reservoir valve to remove bubbles in the solution. Close the reservoir valve after the tubing is filled with solution, fill the perfusion tip with water and screw it onto the perfusion pencil.
Be careful not to trap any bubbles. When connecting the tip, fix the perfusion pencil to the bath stage using modeling clay, make sure the profusion tip is in the bath space. The profusion system is now set up.
At this point, add deionized water to the bath. Make sure the profusion tip is submerged in the water. Test that each perfusion solution comes out of the perfusion tip correctly.
Close the electronic valve connected to the tubing of the waste collector and open the valve for one of the profusion solutions. Under the microscope, observe the jet of profusion fluid coming out of the perfusion tip. Close the valve for that profusion solution and open the valve for the waste collector.
The profusion jet should almost disappear. Repeat the same test for each of the perfusion solutions. When you've verified that all of the perfusion solutions flow correctly, replace the deionized water in the bath with the desired bath solution.
Now it's time to set up the recording apparatus. You should recoat the silver recording electrode with silver chloride before each patch clamping session. First, remove the electrode wire from the pipette holder and submerge the tip half of the electrode wire in a vial containing fresh bleach for at least 15 minutes.
This deposits a layer of silver chloride on the wire. Rinse the electrode wire with deionized water and blot dry. Then install the silver silver chloride electrode back in the pipette holder.
Now turn on your computer. Plug the hardware key into the printer port of your computer. The hardware key must be plugged for you to use the data acquisition software, which is HEA pulse.
Switch on the amplifier and start the data acquisition software. HEA pulse. Load the protocol file and adjust the configuration settings such as the stimulus scale.
The goal for adjusting the configuration settings is to assure that the test potential is exactly equal to the command potential. Now we're ready to prepare the oocytes. Retrieve the oocytes from the incubator.
Submerge the oocyte in stripping solution for five to 10 minutes. The stripping solution detaches the viel membrane from the plasma membrane, which makes it possible to strip the vitel membrane. After five to 10 minutes, gently strip the vitel membrane from the oocyte.
Using two forceps. A divi OC site is extremely fragile and therefore should be moved as little as necessary. Taking care to prevent exposing the Devi oocyte to air or bubbles use a glass pipette filled with enough solution to transfer the oocyte to the bath.
The channels on the oocyte membrane are now exposed and ready for the patch pipette. Pull a patch pipette by inserting a glass capillary tube into the micro pipette puller. Observe the pipettes under a microscope to determine the tip shape and opening diameter.
The opening diameter should be two to four micrometers to reduce capacitive current during recording, and to ensure a smooth tip, coat the tip with wax and then fire. Polish it under a microscope. Fill the pipette tip by placing the tip of the pipette inside the pipette solution and drawing the plunger.
This wets the tip of the pipette. Next, fill the pipette one third full with pipette solution using a syringe. Then place the pipette in the pipette holder with the silver, silver chloride electrode inside.
Make sure the wire electrode is in contact with the pipette solution. The next step is to excise an inside out patch from the prepared oocyte. First, find the clear edge of the oocyte under the microscope.
Using the manipulator, move the patch pipette close to the oocyte, making sure it is submerged. In the bath solution, record the series resistance of the pipette. The ideal series resistance of the patch pipette is between one to 1.5 milli ohms when filled with pipette solution and submerged in bath solution.
Now slowly push the pipette against the oocyte until its resistance approximately doubles. Obtain a giga ohm seal by applying gentle suction by mouth through a suction tube connected to the pipette holder. After confirming that a giga seal was formed, stabilize the patch by holding it at minus 30 millivolts for a couple of minutes.
To obtain an inside out patch, excise the patch by quickly pushing the pipette further into the OO site and then gently pulling it out. The inside out patch is now ready for patch clamping. Move the pipette holding the inside out patch to about 100 microns away from the opening of the perfusion tip.
Close the electronic valve for waste collecting and open the reservoir valve for the desired profusion solution. Begin recording currents across the patch. Change the voltage and profusion solutions as desired.
When you are finished recording, clean the profusion tubings pencil and tip by pushing through deionized water to avoid clogs. The patch clamp recordings obtained here show that the wild type BK channels on an oocyte membrane patch respond to both voltage and calcium. This figure shows a patch recording with nominal zero calcium in the perfusion solution.
The square waves above the current traces indicate the voltage applied to the patch. Four voltages were applied ranging from 50 millivolts to 200 millivolts in increments of 50 millivolts. The maximum current, approximately two nano amps was obtained with the maximum voltage of 200 millivolts.
This increase in current amplitude with increasing voltage indicated that the open probability of BK channels increased with voltage. This figure shows the response of the inside out patch to different voltages. When there was one micromolar calcium in the perfusion solution, voltage steps were applied.
As in the proceeding figure, the response to the 200 millivolt voltage step, the top response increased to approximately three nano amps when there was calcium in the Perfu eight as compared to two nano amps with no calcium. This indicates that the open probability of the BK channels increased in the presence of calcium. In summary, this patch clamping procedure showed that the BK channels were voltage and calcium dependent.
After watching this video, you should have a good understanding of how to record the Iron Channel activation using patch clamp technique coupled with a pro fueling system.Now. Thank you for watching and good luck with your experiments.
