The overall goal of this procedure is to obtain low noise recordings of ionic and gating currents from voltage gated ion channels expressed in zap cytes with high temporal resolution of fast channel kinetics. This is accomplished by first injecting the recorded ionic channel mRNA into the frog oocytes. The second step is to create agar bridges by shaping the capillary tubing and then filling the tubing with a salt containing agar solution.
Next, the cut open rig is prepped for experimentation by placing all the components, including the UO site and agar bridges into designated locations. The final step is to insert the micro electrode into the UO site to allow for voltage clamp recordings. Ultimately, the recording protocol is used to show ionic currents from the ion channels located in the oocyte membrane.
The main advantage of the cut open voltage clamp technique is that allows for stable low noise recordings of a very large population of ion channels. It also allows for rapid resolution of channel kinetics. A visual demonstration of this method is critical as some steps such as placement of the O site, the top chamber, and the agar bridges are difficult to learn.
In this procedure, make at least six agar bridges by heating up one end of a boro silicate capillary tube in a medium flame. For each of them. Make sure that the end of the capillary tubing is in the top portion of the blue flame.
Once the capillary tube has heated up, use the forceps to make a 90 degree bend. The bending site should have a smooth curvature rather than an abrupt corner, or it may significantly reduce the internal diameter of the glass, which makes filling more difficult and increases the resistance of the bridge. Subsequently, heat the capillary tube approximately 25 millimeters from the un bent end.
Make a second 90 degree bend at this location in the same direction as the first bend. After the capillary tubes have cooled down, use a diamond tipped glass cutter to trim the legs of the bridge to approximately five millimeters. Next, insert platinum wires into the capillary tubes of the three current supplying bridges.
Cut off any excess platinum wire so that there is no wire exposed outside of the tube. Push the platinum wire further into the capillary tube with a fine tipped implement so that the wire is one millimeter shorter than the glass on both ends of the capillary tube. Then add the capillary bridges one at a time to a pre-made agar solution with the legs facing up.
Once there are no air bubbles or pockets in the bridges, retrieve the bridges from the Agar solution and place the bridges on a paper towel to dry. Alternatively, fill the bridges by pushing Agar solution through a syringe attached to a small pipette tip. Afterward, prepare the cut open rig under a dissecting microscope by applying a small amount of Vaseline around the edge of the hole on the top side of the middle chamber and the bottom side of the upper chamber with a very fine tipped object.
Then at three molar potassium chloride solution into the manifold slots holding silver, silver chloride pellets. In this step, install the top chamber without an UO site. Slide the top chamber off center so that the holes in the two chambers do not overlap.
Then fill all the chambers with external solution and place all the bridges in their respective chambers. After that, make sure the external command and both clamps are turned off. Check the current reading on the amplifier and adjust the P set on the back of the bath guard head stage to zero current with a small screwdriver.
Subsequently, switch the bath guard switch on the amplifier to active. Then adjust the GS offset on the bath guard head stage to obtain zero current with a small screwdriver, repeat between active and passive until both are reasonably close to zero. Next, remove the agar bridge ends and top chamber.
Transfer an UO site into the middle bath chamber using a pipette pump, make sure the UO site is positioned over the hole in the center of the bath. Then remove excess external solution from the bottom bath using a pipette pump to create a seal between the UO site and bath surface. Now, place the top bath chamber over the UO site so that the hole in the chamber is centered on top of the UO site.
Using a thumb middle finger and tweezers slowly apply pressure down on the chamber until it is pressed tightly against the oocyte in order to expose only a small portion of the membrane to the upper bath through the hole afterward, add external solution to the top and bottom baths until they're almost full. Then place the free legs of the agar bridges into the external solution of each bath. Make sure that each bridge is resting in its correct bath location.
Start a test protocol in the recording software check to make sure the vertical displacement of the horizontal section between the two peaks of the test pulse is less than or equal to 100 nano amps. If the horizontal section demonstrates greater than 100 nano amps vertical displacement as shown, then increase the tightness of the top chamber on the cyte. Subsequently, switch the bath guard switch on the amplifier to active.
Next, remove the external solution in the bottom bath and replace with saponin solution. After the saponin solution has been added, observe the repeating test pulse on the computer monitor. If the peak of the test protocol reduces or disappears, it indicates that there is a bubble below.
The cyte in this case completely replaced the saponin solution. With new saponin solution, the cyte has been perme. When the downward slope of the current spike in the test protocol decreases.
Once the cell is remove the saponin solution and fill the bath with internal solution. Then stop the test protocol. Next, check for any possible contact between the solutions in different baths and crystallize potassium chloride between the wells of the manifold as these can cause short circuits and erratic behavior.
If there is solution, contact between the baths aspirate the excess solutions out of the two baths. For crystallized potassium chloride. Use the squirt bottle to wash off the crystallized potassium chloride and then aspirate the leftover water.
Then use a modified syringe to inject three molar potassium chloride into a micro electrode. Flick the micro electrode several times. Now, mount the potassium chloride filled electrode on the micro manipulator arm by inserting the wire filament into the open micro electrode end.
Push the end of the micro electrode into the filament holder and make sure the electrode is not loose. After that, tighten the electrode fastener. Then swing the micro manipulator arm into position over the cyte baths and tighten the clamps to prevent further arm movement.
Lower the electrode until the electrode tip just breaks the surface of the liquid in the bath. Press the V one button on the cyte voltage clamp and then adjust the V one offset knob to reduce the V one voltage to zero. Perform the same adjustment for V two.
The potential difference V one minus V two should read zero millivolts on the voltage clamp display. After that, continue to lower the electrode down towards the visible patch of the UO site in the top bath while looking through the microscope. Once the micro electrode is very close to the UO site, watch the V one minus V two reading to see when the electrode enters the uoc site.
The V one minus V two voltage will become negative when the micro electrode enters the cell. At this time, open the data collection protocol in the recording software. Flip the clamp switch on the uoc site voltage clamp amplifier to the on setting.
Next, adjust the potential to match the command by adjusting the knob located on the I head stage. Then start recording currents by commencing the data recording protocol. Here are the selected test pulse traces.
During oocyte permeable, the increase of the time constant of decay seen in the traces demonstrates an increase of oocyte permeable. This figure shows the wild type sodium channel results from cut open voltage clamping. Here are the traces of recorded current from different voltage stimuli.
The current voltage curve shown here represents the voltage dependence of peak current. While attempting this technique, it's important to remember to have the voltage clamp turned off before you enter the cell and to turn the voltage clamp off before exiting the cell. Failure to do this can cause damage to the agri bridges and they will likely need to be replaced.
