Monitoring Leucine-Rich Repeat Containing 8 Channel (LRRC8/VRAC) Activity Using Sensitized-Emission Förster Resonance Energy Transfer (SE-FRET)

Our research focuses on the properties and function of membrane transport, specifically ion transporters and channels such as LRRC8-formed anion and osmolyte channels. We investigate their biophysical mechanisms, regulatory processes, and cell physiological functions. In the end, we also aim at elucidating how their dysfunction may lead to disorders in humans.

Many labs have advanced our understanding of LRRC channels, resolving structures of LRRC8 complexes, elucidating electrophysiological properties, and uncovering diverse physiological roles. However, a crucial question remains:how are these channels biochemically and physiologically regulated and how does the subunit composition influence their activation and roles within the cell? This method allows observing LRRC8 channel activity in compartments typically inaccessible for electrophysiology such as intracellular compartments without disturbing cytosolic composition like whole-cell patch-clamp would.

Its subcellular resolutions enables monitoring of differentially activated LRRC8 channels within a single cell and allows for continuous monitoring during physiological processes. With this FRET-based sensor, we will investigate the regulation and function of LRRC8 VRAC channels under different physiological conditions. Key questions are:how the subunits rearrange their confirmation;which signal transduction processes are involved with different stimuli, and whether there are differences amongst the different paralogs of LRRC8;and whether there are subcellular differences in the physiological processes.

To begin, prepare the isotonic, hypotonic, and hypertonic buffers. Using an osmometer, measure the osmolarity of the buffer. The day before transfection, seed one times ten to the power of five HeLa cells in two milliliters of cell culture medium on a 35-millimeter dish.

Culture the cells overnight at 37 degrees Celsius and 5%carbon dioxide. The following day, dropwise, add transfection solution in a spiral motion to the dish. Move the dish five times horizontally and vertically on the surface of the bench to mix the solution.

Culture the cells overnight at 37 degrees Celsius and 5%carbon dioxide. The next day, check for transfection under a fluorescent microscope. Take the cells expressing both the donor and acceptor constructs and aspirate the cell culture medium.

Wash the cells three times with two milliliters of isotonic buffer. After aspirating isotonic buffer, add three milliliters of isotonic buffer to the cells and place the sample dish on the microscope stage. Load microscopy FRET settings or set up the necessary channels for a FRET experiment:donor excitation-donor emission, DD;donor-acceptor, DA;and acceptor-acceptor, AA.Find a field of view with at least one cell expressing both the donor and acceptor constructs and adjust the channel settings if necessary.

Remove the plate from the microscope stage and place it back into the incubator until the experiment. Take the cells expressing only the donor construct and aspirate the culture medium. Wash the cells three times with two milliliters of isotonic buffer.

After the last wash, add three milliliters of isotonic buffer to the cells and place the sample dish on the microscope stage. Find a field of view with at least one cell expressing the donor construct. Image all the donor-donor, acceptor-acceptor, and donor-acceptor channels.

Draw a region of interest or ROI around the cells and measure the mean intensity of donor-acceptor and donor-donor channels. For background subtraction, draw an ROI in the donor-acceptor and donor-donor channels where only the background signal is found and measure the mean intensity. After imaging the cells, draw an ROI around the cells and measure the mean intensity of the donor-acceptor and acceptor-acceptor channel.

For background subtraction, draw an ROI in the donor-acceptor and acceptor-acceptor channels where only the background signal is found and measure the mean intensities. Finally, calculate the correction factors, beta and gamma, using the values determined for background-corrected IDA, background-corrected IDD, and background corrected IAA. To begin, aspirate the buffer from the previously prepared HeLa cells expressing the donor and acceptor.

Wash the cells with three milliliters of isotonic buffer and place the sample dish on the microscope stage. For aspiration of the isotonic buffer, fix and adjust a hose cannula so its tip reaches the bottom of the dish. For adding buffers, fix and adjust the tubing to allow gravity-driven buffer flow to drop into the dish.

Find a field of view with at least one cell expressing the donor and acceptor construct simultaneously and acquire an image. For background subtraction for the FRET signal, draw an ROI in the donor-acceptor channel where only the background signal is found and measure the mean intensity. For SE-FRET quantification, draw an ROI around the cell and measure the mean intensity in the donor-donor, donor-acceptor, and acceptor-acceptor channels for all images in the time series.

Set up a time-lapse experiment for the channels donor-donor, donor-acceptor, and acceptor-acceptor with an interval of 10 seconds and a duration to cover all conditions of the stimulation sequence. Start acquisition of the time-lapse imaging and plot the SE-FRET traces. After baseline measurement, replace the buffer with gravity flow.

In the live experiment, aspirate the isotonic buffer via the hose cannula, applying a vacuum with a syringe. Add three milliliters of the buffer of the next condition by gravity flow while continuing the time-lapse imaging. To measure another condition, wash the sample in the next buffer as shown previously while continuing the time-lapse imaging.

Using the FRET-based method, the activity of the volume-regulated LRRC8 anion channel was monitored during osmotic stimulation and SE-FRET reduction correlated with extracellular hypotonicity. In other experiments, LRRC8 VRAC activation by isosmotic stimuli such as diacylglycerol signaling or myocyte activation was also detected. LRRC8 VRAC activity was monitored upon apoptosis induction in HeLa cells expressing LRRC8A mCerulean3 and LRRC8E mVenus.

Tumor necrosis factor alpha and cycloheximide caused a robust SE-FRET decrease, which recovered in a hypertonic medium. DMSO treatment did not reduce SE-FRET, confirming specificity for LRRC8 VRAC.