Lipid-Protein Membrane Structure-Function Characterization using Droplet Interface Bilayers

In this work, we study how physical and electrical properties of lipid membranes and their environment impact ion conduction in droplet interface bilayer model systems. Traditional patch clamp electrophysiology doesn't probe larger scale membrane biophysics, but this single platform captures both single-channel activity and membrane areas larger in size on the millimeter scale to more extensively study membrane biophysics. To begin, prepare the chlorinated agarose-coated silver wire electrodes and lipid vesicle solutions for electrophysiological experiments.

For droplet interface bilayer formation, place a clear bottom Petri dish on the stage of an inverted microscope. Fill the dish with alkane oil such as 100%hexadecane to a depth of approximately five millimeters. After attaching the electrodes to the patch clamp headstage and ground electrode holder, use the micromanipulator controls to lower both agarose-coated ball ends into the oil to a depth of approximately 2.5 millimeters below the oil surface, avoiding rapid motion to prevent collisions with the Petri dish.

Adjust the microscope focus until both ball ends and their agarose coatings are in sharp focus. Position the electrodes near the edge of the field of view, so that both can be observed simultaneously. Using a calibrated two-microliter pipette, aspirate the extruded DPhPC lipid vesicles doped with gramicidin A.Slowly dispense 250 nanoliters of the suspension directly onto each agarose-coated ball end using a separate pipette tip for each without touching the agarose shell.

Allow the droplets to spread over the agarose surface and visibly sag downward due to gravity and reduced surface tension, forming a pendant droplet shape. Observe sagging from the side through the Petri dish wall if it is clear. After five minutes, gently nudge the anti-vibration table to assess the droplet.

Confirm that the sagging droplets move with a slight delay relative to electrode motion, indicating the formation of fully-coated lipid monolayer droplets. If this behavior is not observed, wait an additional two to three minutes and repeat the assessment. Ensure the microscope, anti-vibration table, Faraday cage, amplifier, digitizer, function generator, and computer are connected to a common ground.

Arrange the instrument connections according to the manufacturer's setup guide and configure the essential electrical and optical setup, following the detailed instructions for droplet interface bilayer experiments. Configure an external function generator or the acquisition software output channel to generate a 10-Hertz, 10-millivolt triangle voltage wave form. Confirm the amplitude and frequency on a connected oscilloscope and in the acquisition software.

After droplets have sagged for approximately 10 minutes, use the micromanipulators to bring the two droplets into gentle contact. Turn on the 10-Hertz, 10-millivolt triangle wave form and monitor the capacitive current response in the acquisition software. Adjust the droplet contact area by slightly moving the electrodes until the peak-to-peak capacitive current response to the waveform is between approximately 100 and 200 picoamperes.

Or the contact area is no more than approximately 1/4 of the droplet diameter. Wait for spontaneous bilayer formation indicated electrically by an expanding peak-to-peak capacitive current response and optically by an increasing internal reflection with an oval appearance at the contact region. Confirm that pure lipid bilayers display rectangular current plateaus in response to the triangular voltage stimulus, while gramicidin A-doped bilayers exhibit sloped plateaus, reflecting ensemble ionic conduction.

Once stable-capacitive or conductive responses are confirmed, turn off the triangle wave form and allow the droplet interface bilayer to equilibrate for at least 15 minutes before applying stimulation protocols. In the video capture software, set the frame rate to at least 20 frames per second. Select the appropriate camera and objective lens settings.

Next, click Acquire and select New Protocol. In the Acquisition Mode tab, select Episodic Stimulation. Set runs per trial and sweeps per run to one.

Adjust the sweep duration to four seconds longer than the total experimental duration, allowing a two-second pre-and post-stimulus baseline. Set the sampling rate to between five to 10 kilohertz. In the Inputs tab, assign the recording channel to the current input.

And then, in the Outputs tab, assign the stimulation channel to the voltage output controlling the electrodes. Open the Waveform tab. In column A, set type to Step, first level and delta level to zero millivolts, and first duration to 2, 000 milliseconds to define a pre-stimulation baseline.

In column B, set type Pulse for paired pulse facilitation. Specify the values for first duration and first level voltage amplitude and set the train rate to define the interpulse interval corresponding to the target duty cycle. Adjust the pulse width to 100 milliseconds.

In column C, for paired pulse depression, set type to Pulse with the same amplitude and pulse duration. Set the train rate to achieve the desired lower duty cycle and set the pulse width. Next, in column D, configure the post-stimulation parameters identical to the pre-stimulation baseline.

Review the protocol and save it with a descriptive name. Start video recording and immediately start electrical acquisition and stimulation by clicking the Record button in each software. During the first 60 seconds of stimulation, observe that the current increases and may reach a plateau.

During the last 60 seconds, observe that the current decreases. At the end of the protocol, stop electrical acquisition and video recording simultaneously. Finally, perform post-processing to extract membrane area and compute normalized current, area, and flux from imaging and electrical data.

Sequential images captured spontaneous bilayer zipping and area expansion when two sagging droplets were brought into contact. The bright inner oval-shaped reflections at the droplet contact were used to estimate bilayer diameter and membrane area over time. Paired pulse facilitation from zero seconds to 60 seconds and paired pulse depression from 60 seconds to 120 seconds were delivered using 100-millisecond pulses.

The representative current responses during on periods and the stimulation patterns were recorded. Normalized current for gramocydin A-doped DPhPC bilayers in hexadecane and dodecane/hexadecane oils is shown during paired pulse facilitation and paired pulse depression. Normalized membrane area measured at 30 time points showed similar area evolution in both oil conditions despite differing currents.

Normalized flux separated area-independent changes in conductance and was consistent with changes in membrane conduction beyond simple area expansion. Extended paired pulse depression stimulation for up to 30 minutes produced long-term depression-like behavior in hexadecane membranes, but maintained elevated flux in dodecane/hexadecane membranes consistent with long-term potentiation-like behavior. This protocol allows us to measure how membrane structure and ion conduction change through electrically-driven restructuring of droplet interface bilayers.

This platform can support many different stimulation protocols in dibs with ion channels, without ion channels, and even photoisomerizable channels to better probe membrane dynamics and electrical properties. Because this tunable platform probes both macroscale and microscale membrane biophysics, future experimentation would greatly improve from using fluorescence spectroscopy to more confidently separate protein channel activity from membrane pore conduction.