Patch-Clamp Techniques for Single Endolysosomal Vesicle Analysis

Endolysosomal compartments are the smallest organelles in cells. Traditional electrophysiologic techniques are unable to directly measure their voltage and current changes. The endolysosome patch-clamp technique enables direct recordings, allowing us to study previously in the ion channels.

The major experimental challenge is that not all compounds effectively enlarge lysosomes. Identifying suitable cells is difficult, and even with ideal lysosomes, successful ion channel recording are not guaranteed. We developed the endolysosome-patch clamp technique in enabling direct electrophysiologic recordings from endolysosomal membranes.

This breakthrough allowed us to characterize ion channels, such as TRPMLs and the TPCs revealing their roles in pathogen defense, neuron degeneration, and metabolic disorders. Our method enables direct lysosomal ion channel recordings, overcoming whole-cell patch-clamp size limitations, and improving vesicle selection while integrating fluorescence-based techniques. To begin, install the capillary tube into the polar.

Press the green pull button on the keypad. Loosen the clamping knob and remove the pulled pipettes from the polar. Next, place the pulled-patch pipettes into the Microforge holder.

Inspect the pipette tips using a 35x objective lens, combined with a 15x eyepiece for a total magnification of 525x. Use the micro manipulator to bring the patch pipettes close to the filament. Set the temperature dial to 80.

Once the heater is turned on, press and hold the foot switch for one to two seconds to apply a brief heat pulse. After polishing, place the polished pipettes into a sealed box to prevent dust contamination. Add one milliliter of the bath solution to the chamber.

Remove a cover slip with treated HEK 293 cells from the 24 well plate. Transfer the cover slip to the microscope chamber. Use a homemade plastic filling needle to fill the isolation pipette with pipette solution.

Install the filled pipette at the front end of the patch-clamp setup. Under a microscope with a 40x objective and 10x eyepiece, move the isolation pipette close to sufficiently enlarged endosomes or lysosomes. Lower the isolation pipette using the micro manipulator until it touches the plasma membrane edge.

Quickly move the pipette to tear off a small piece of the membrane. Using the same pipette, press the cell from the opposite side to squeeze out endosomes/lysosomes approximately two micrometers from the cell. Fill a freshly polished patch pipette with the appropriate pipette solution.

Install the pipette at the front end of the amplifier. Apply a positive pressure of 20 to 50 millibar to the pipette and maintain it by locking the valve. Next, move the pipette tip into the bath solution and position it in the center of the field of view.

Apply repeated current pulses to determine the pipette resistance, seal resistance, and series resistance. Monitor the pipette tip size, seal formation, and the establishment of the whole endolysosomal configuration. Quickly move the pipette close to the top of the target vesicle.

Observe the vesicle moving or rolling due to fluid flow from the pipette. Adjust the offset voltage of the pipette to zero millivolts. Immediately release the positive pressure to draw the vesicle toward the pipette, forming a gigaseal within one second.

For whole vesicle recording, use a high-voltage short pulse of two to five milliseconds to disrupt the membrane at the contact point between the pipette and the organelle. Set the voltage from 500 to 1000 millivolts. To record current, conduct endolysosomal voltage clamp experiments using a wide range of input voltages to assess the membrane properties.

During gigaseal formation, the current response decreased rapidly, indicating the successful establishment of the lysosome attached mode. Repeated application of continuous input voltage recorded currents across the endolysosomal membrane, showing basal, agonist-activated, and leakage currents.