Hello, I'm Dr.Ola Anderson in the Department of Physiology and Biophysics at Weill Cornell Medical College in New York City. My name is Olson. I'm a computational biomedicine student in the lab of Olive Anderson.
And I'm Shail Collingwood, a research technician also from the Anderson lab. Today we'll show you how to form a plane, a biolayer, how to isolate a small membrane using the Biolayer Punch electrode, how to record single channel activity and how to analyze the results and thereby use Gramin channels as probes of lipid B layer properties. In a previous video, we showed you how to get ready for these experiments.
We showed you how to prepare the chamber that we used, and we showed you how to prepare the Bilayer punch electrode that we use to isolate the small layer of membrane where we record the channel activity. So let's punch some bilayers To begin setting up for bilayer experiments at 2.5 mils of the temperature equilibrated electrolyte solution to each compartment of the pre-painted chamber. The solution we usually use is one molar sodium chloride plus 10 millimolar.
He piece buffered to pH seven, adjust the microscope and the light source so the hole in the Teflon is centered in your field of view and brightly illuminated. Insert the glass micro pipette into a micro electrode holder. Connect a syringe to the holder with a piece of tigon tubing.
Fill the pipette in electrode holder by suction. Be careful to avoid bubbles as they may break the electrical connection between the silver pellet in the electrode holder and the pipette tip. Apply a small amount of petroleum jelly to the screw, then place it in the micro electrode holder.
The petroleum jelly helps to make an airtight seal. Plug the electrode holder into the patch clamp head stage in the Faraday cage. Position the micropipet tip centered and right behind the hole in the Teflon partition.
Place the coiled up silver electrode into the front chamber. Fix it in place with modeling clay and connect it to the voltage command circuit. Finally, balance the electrodes.
This is critical because the two electrodes, the one in the front and the one in the micro electrode holder may have slightly different half cell potentials. The half cell potential is the difference between the silver metal and the electrode and the electrolyte solution adjacent to the silver chloride coating. Balance the electrodes by adjusting the manual offset so that the current flowing between the two compartments fluctuates between negative 200 and positive 200 picoamps on the patch clamp.
The offset potential will change with time, so you balance the electrodes at regular intervals throughout the experiment to form the lipid bilayer membrane. Clean a fire polished past pipette with fch solution as described in the accompanying video. Dip the cleaned pipette into the lipid decking solution and let it rise about one millimeter up into the pipette.
Place the pipette tip just in front of the hole in the Teflon partition and gently paint a bubble of the lipid solution onto the hole. When successful, a brightly colored lipid bubble forms across the hole with a Taurus or donut shape of lipid attaching it to the Teflon partition. Let to the membrane thin, which can be facilitated by applying a 100 to 200 millivolt potential across the membrane.
Place the glass micro pipette tip in the Taurus and let it sit for a few minutes to provide for a better seal between the pipette and the bilayer. At this point, you are ready to isolate a membrane patch using a glass micro pipette. Now dope, the membrane with gramin.
This gramin solution was made from a one micromolar stock that was diluted to one to 10 nanomolar. In dimethyl sulf oxide, usually one to 10 microliters of the dilution is added to each side of the membrane so that the final concentration is in the picaMolar range. Use the magnetic stir barss to stir the solution for about two minutes and wait another 10 minutes before forming a new small membrane to record single channel activity.
This time weight allows for gramin to be incorporated into the membrane. Check the channel activity by applying voltage. Generally, we want about one channel event per second.
The channels are visible as discrete up and downward changes in the membrane current. You are now ready to begin the actual experiment. First, record the control current traces.
Balance the electrode as demonstrated earlier, form a small membrane or patch as described in the accompanying video. Apply positive 200 millivolts and record the single channel activity. You should collect 100 to 200 single channel current transitions.
Repeat this step with negative 200 millivolts. Break the patch by expelling electrolyte through the pipette tip. Using the nylon screw, rebalance the electrodes retreat pipette to the rear chamber, blow out excess lipid and form a new patch.
You may also repeat these steps to require recordings at other potentials. Now you're ready to record the experimental current traces at the bilayer modifier. In this case, the antidepressant fluoxetine to both sides of the membrane.
The fluoxetine stock solution should be diluted to 25 millimolar in DMSO. Add five microliters of this solution to achieve 50 micromolar concentration in the chamber DMSO may affect the bilayer properties. Therefore, it is important to limit the amount of DMSO in the solution to 25 microliters 1%or 150 millimolar.
Stir for five minutes and wait 10 minutes to allow the modifier to equilibrate between the aqueous solution and the membrane. Now, record the channel activity as described previously in the presence of the modifier. Repeat these steps to record channel activity at increasing concentrations of the modifier.
We learn a lot about changes in the bilayer by quantifying channel lifetimes and appearance rates. To do such an analysis, we need to detect channel formation and disappearance. Here we use a transition based algorithm where the computer calculates the first derivative to determine rapid changes in the current and marks them as channel events.
The events are stored as a time series to help us determine channel lifetimes pre and post transition. Current levels are also stored and help us determine channel amplitudes as the experiment is in progress. The computer constructs a current transition amplitude histogram, which enables you to follow the acquisition of current transitions.
It also enables you to evaluate the quality of the experiment. If the current transition amplitudes scatter widely, you have problems. If the histogram is tight, you are doing well.
When you have accumulated more than 200 transition events, calculate the average current and standard deviation for the transitions in the main peak. You can estimate the channel appearance rate simply by dividing the number of transitions in the current transition amplitude histogram by the observation time. Remember to also divide by two because a channel is counted twice in the histogram once when it appears and once when it disappears.
Analyze the time series of transition events to get the channel lifetime distributions. Determining the lifetimes is straightforward in current traces where you have no more than one conducting channel at any time. In this case, the computer detects whether each transition is a channel appearance or a disappearance, and a disappearance event can only belong to the preceding appearance event.
The time between the two transitions is the channel lifetime. In instances where the channel transitions occur too close to be distinguished, you have to manually assign appearances and disappearances. The assignment is more complex in case you have two or more simultaneously conducting channels because now you do not know which channel disappearance belongs to which channel appearance.
For this reason, it is preferable to have a low channel activity. You may solve the assignment problem by using a random number generator to assign appearances to disappearances. Thus, you end up with a series of channel lifetimes.
This is converted into a lifetime histogram, which is in turn converted into a survivor plot, determine the average lifetime by fitting a single exponential distribution to the survivor plot using a non-linear leased squares curve fitting and experiments where the large membrane does not break. When you add the bilayer modifier, one can compare the effect on the channel appearance rates. Such comparison is not warranted if the large membrane breaks membrane protein function is regulated by the cell membrane lipid composition.
This regulation is due to a combination of specific lipid protein interactions and more general interactions between the lipid bilayer and the membrane proteins. These interactions are particularly important in pharmacological research. As many current pharmaceuticals on the market are amphi that can alter lipid bilayer properties, which can in turn lead to altered membrane protein function.
Gramin channels can be used as molecular force transducers to report if amphiphiles can alter lipid bilayer properties. Plotting the changes in lifetime and appearance rate as a function of modifier. Concentration gives insight into changes in bilayer properties.
Increased frequency means that the bilayer is more easily deformed to accommodate Gram site and dimerization. Increased lifetime means that the bilayer deformation associated with channel formation is not as energetically costly. A modifier may increase frequency and lifetime by increasing membrane elasticity or affecting curvature.
We have now shown how to use chromo channels to check whether small molecules alter elastic bilayer properties. And that's it. Thanks for watching.
Good luck with your experiments.
