Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

The overall goal of this procedure is to functionally reconstitute mezcal channels into a droplet interface, bilayer system, and to examine the response of these mechano sensitive channels to harmonic mechanical stimuli. This method enables the study of the mechano electrical activity of the V 23 T mescal mutant in response to harmonic mechanical excitation after reconstitution into a droplet interface by layer system. The main advantage of this technique is that it utilizes the technical advantages of both the patch clamp technique and the droplet interface by layer methods with the ability to be miniaturized as well as the ability to control the chemical composition of each side of the membrane.

To formulate, liposomes, prepare 10 milliliters of a two milligram per milliliter lipid solution by adding 10 milliliters of potassium chloride buffer to 20 milligrams of D-P-H-P-C synthetic lipids purchased as lyophilized powder. Make sure both lipid vehicles and buffer solution are thoroughly mixed. The mixture should look homogenous and hazy when everything is dissolved.

Freeze the mixture at minus 20 degrees Celsius now completely thaw the new lipid mixture, letting the mixture thaw at room temperature and not in a heated environment. After a total of six freeze thaw cycles, use a commercially available extruder to extrude the lipids by forcing the whole lipid suspension first through a 0.4 micron polycarbonate membrane filter, and then six times through a 0.1 micron membrane filter. This process yields particles with diameters near 100 nanometers.

To manufacture the oil reservoir, drill two opposing holes through the wall of a two inch diameter, 1.5 centimeter long acrylic cylinder. The holes should be one millimeter in diameter and one centimeter from the bottom. Then drill two four millimeter holes concentric to the previously drilled one millimeter holes.

The depths of the holes should be one millimeter each. Take care not to drill them all the way through. These holes are made to fit the rubber gaskets.

Place and glue two rubber gaskets having one millimeter inner diameters in the bigger holes in order to prevent oil from leaking. Then glue the machine to cylinder to a 10 centimeter by 10 centimeter thin acrylic sheet using any multipurpose epoxy on the day of the experiment, cut a seven centimeter length of 2 250 micron diameter silver wires, and then immerse their tips in bleach for two hours. To form a silver chloride coating a gray color indicates that a silver chloride coating has been formed.

Using a glass cutter split a 10 centimeter long borrow silicate glass capillary of one millimeter outer diameter and point 58 millimeter inner diameter into two five centimeter capillaries. Using a one milliliter syringe with an 18 gauge blunt tip needle, fill the capillaries with the P-E-G-D-M-A hydrogel to prevent the hydrated hydrogel from swelling out of the capillary. Keep a three millimeter clearance at the tips and make sure there are no air bubbles in the capillaries.

Insert the silver, silver chloride electrodes into the hydrogel filled capillaries. Cure the P-E-G-D-M-A hydrogel through free radical photo polymerization upon exposure to ultraviolet light for two minutes at one watt using an ultraviolet spot, the experiment is set up under a faraday cage grounded to a ground connection on the patch amplifier. Attach one of the micro pipettes to a straight micro electrode holder that has a male connector.

Then connect the micro electrode holder to the head stage of the patch amplifier. To connect the head stage to the ground solder an 18 gauge insulated copper wire to an appropriate connector for the head stage mount the head stage on a three axis manual micro manipulator by first attaching the head stage mounting plate to the micro manipulator, and then connecting the head stage to the mounting plate. Using the appropriate screws, attach the second micro pipette to the linear actuator through a lab made connector and then mount both on a second microm manipulator.

The micro pipettes should be opposing each other aligned and horizontally leveled. Next in a glass vial mix 0.1 milliliter of the D-P-H-P-C liposomes with 0.01 milliliter of the V 23 T mescal protea liposome solution. Using a 34 gauge microfill needle, fill the tips of both micro pipettes with a proteosome solution.

Place the reservoir on the top of an upright microscope and feed the micro pipettes through the opposing one millimeter holes. Then fill the reservoir to the surface with hexa decane to form the spherical droplets at the tip of the micropipets. Use a third 10 micron diameter bo silicate glass micro pipette mounted on a third microm manipulator to dispense diluted V 23 T mescal protea liposome solution at the tips of the micro pipettes and form the droplets.

Control the size of the droplets by decreasing or increasing the volume as desired, and let them rest for 10 minutes for the monolayers to form completely. Then bring the droplets into contact by layer. Formation will occur within one to two minutes.

After setting up the software and equipment as described in the text protocol, use A BNC cable to connect the output of a waveform generator to the external command input front switch. Send a 10 hertz, 500 millivolt peak tope triangular waveform to the head stage. Using the microm manipulator.

Move the glass micro pipettes horizontally to bring droplets into contact until they slightly touch and wait for bilayer thinning to occur. Progression of the bilayer formation process can be seen visually through the microscope and can be monitored by current measurement. Adjust the bilayer size to about 250 microns in diameter by controlling the position of the droplet mounted on the actuator using the manipulator, the bilayer size can be estimated visually through the microscope.

Once the bilayer has formed and is stable, stimulate the droplets by sending a sinusoidal signal using a function generator to stimulate the mescal protein incorporate in the bilayer. Send a sinusoidal wave form with a 175 micron peak to peak amplitude, 0.2 hertz frequency, and 50%duty cycle to the Pieto electric server controller. Atypical real-time current recording is shown here.

The measured capacitance of the bilayer increases suddenly indicating bilayered thinning and formation before it reaches a steady state. The bilayer could be controlled by changing the droplets position when the DIB is mechanically stimulated while maintaining a constant DC potential across the membrane. The V 23 T mutant of mezcal generates reliable activities including mainly sub conductive states and occasionally full opening events.

Gating occurs mainly at peak compression indicating that it is related to an increase in bilayer tension as it is shown in the polar plot. Here, the ability of this technique to be used to test other types of biomolecules is highlighted here. Ethin, which is a voltage gated ion channel, increases the membrane permeability upon the application of a DC transmembrane potential One master.

This technique can be done in two hours if it's performed properly. While attempting this procedure, it's important to remember not to accidentally break the glass capillaries or otherwise. You have to restart the setup and make sure all wires are connected after its development.

This technique paved the way for researchers in the field of stretch activated channels to explore new methods through which external forces can be conveyed to the gate of DIB embedded mechanism sensitive channels. After watching this video, you should have a good understanding of how to form droplet interface by layers, incorporate mechano sensitive channels in the artificial membranes and stimulate these channels mechanically.