Patch-Clamp Techniques for Single Endolysosomal Vesicle Analysis

Summary

This protocol details a method for directly measuring ion channel activity on intracellular vesicles using a manual endolysosomal patch-clamp system. We illustrate the method that involves enlarging endolysosomes and manually isolating these vesicles. This approach ensures that researchers can accurately replicate and apply the procedure.

Abstract

Endolysosomal ion channels are critical for endolysosomal ion and pH homeostasis, membrane potential regulation, and vesicle trafficking. However, electrophysiologically accessing these channels within small intracellular vesicles has been a challenge. The development of endolysosomal patch-clamp techniques has been instrumental in overcoming this barrier, allowing for the direct measurement of ion channel activity in endolysosomal membranes.

Compared to existing planar patch-clamp techniques, endolysosomal patch-clamp can simultaneously record multiple cells and easily combine with other measurement methods. Manual operation offers the advantage of visualizing targeted vesicles. It also addresses the limitation of the indispensable presence of Ca²⁺ on one side of the endolysosomal membrane, increasing the flexibility of experimental design. Utilizing endolysosomal patch-clamp techniques enables the direct measurement and analysis of ion channel activity within endolysosomes.

Given the close link between aberrant endolysosomal ion channel function and diseases such as neurodegenerative diseases and metabolic disorders, investigating and modulating these channels may unveil new drug targets. By restoring intracellular ion balance, we may alleviate or cure related diseases. Therefore, this technique is pivotal for discovering new drug targets and developing relevant medications.

Introduction

Ion channels play a crucial role in numerous physiological processes. While surface ion channels have received significant attention, the importance of intracellular channels, particularly those within endolysosomes, is gradually being acknowledged. The endolysosomal system is composed of multifunctional, membrane-bound organelles specialized for fundamental cellular functions, including recycling endosomes (RE), early endosomes (EE), late endosomes (LE), lysosomes (LY), and hybrid organelles with both endolysosomal and other compartment characteristics, such as phagosomes and autophagosomes.

EE, also known as sorting endosomes (SE), are one of the earlier destinations for materials internalized from the plasma membrane (PM). EEs are critical compartments responsible for sorting cargo into various endocytic pathways, such as the maturation pathway to LE/LY for degradation, the rapid recycling pathway back to the PM, and the slow recycling pathway involving the recycling compartment or peripheral RE. Multivesicular bodies (MVBs) derived from endosomes are spherical compartments surrounded by a limiting membrane, which can be filled with intraluminal vesicles (ILVs)¹. To maintain the normal function of these organelles, they require membrane ion channels to regulate vesicular pH, osmolarity, and signal transduction. However, measuring the activity of these channels is not straightforward.

For ion channels located on the plasma membrane, the patch-clamp technique developed in the 1970s has long been the gold standard method². However, accessing channels electrophysiologically within small intracellular vesicles has remained a challenge. Applying the gold standard for measuring ion channels on the plasma membrane to that on intracellular organelles faces three main challenges. First, the size of endolysosomes is typically very small (less than 1 µm in diameter), making them difficult to observe and isolate under a microscope and smaller than the opening diameter of typical glass micropipettes, rendering the experiment inoperable. Second, isolating endolysosomes directly from target cells while maintaining organelle integrity requires special skills. Third, due to the absence of a cytoskeleton in intracellular organelles, forming a seal on the endolysosomal membrane within the patch pipette and then rupturing it to achieve a whole-endolysosome configuration can be challenging, as it compromises the structural integrity of the organelle³.

Several methods have been developed to overcome these issues, including lipid bilayer recording, modifying lysosomal targeting sequences, and solid-supported membrane-based electrophysiology (SSM or SSME) techniques. The lipid bilayer recording method involves reconstructing synthetic phospholipid membranes with purified ion channels, enabling detailed electrophysiological study of membrane protein function under controlled conditions^4,5. Modifying lysosomal targeting sequences on ion channels involves the redirection of endolysosomal ion channels to the plasma membrane for measurement using conventional patch-clamp methods⁶. Solid-supported membrane-based electrophysiology (SSM or SSME) techniques, also known as the endolysosomal planar patch-clamp method, use solid substrate planar glass chips with small apertures (<1 µm in diameter) in microstructured planar borosilicate chips. These small aperture glass chips allow for the analysis of small, even native, endolysosomes using a pressure-suction control system (Nanion). However, in the first two methods, the ion channels are not in their natural physiological environment. Attempts to record lysosomal channels expressed on the plasma membrane or reconstituted into lipid bilayers have largely produced uncertain and contradictory results.

Although planar patch-clamp techniques have effectively addressed the issue of artificial interference and offer the advantage of high-throughput measurements, the solutions used are also limited by this method. The endolysosomal patch-clamp technique introduced in this article can simultaneously record multiple cells and easily combine with other measurement methods. Manual operation provides the advantage of visualizing target vesicles. It also overcomes the unavoidable limitation of Ca²⁺ in the solution on one side of the endolysosomal membrane, increasing the freedom of experimental design³. Recently, endolysosomal patch-clamp techniques have played a key role in drug development research. For example, in neurodegenerative diseases, this technique has helped identify new drugs targeting endolysosomal ion channels associated with Alzheimer's and Parkinson's diseases^7,8. Researchers can also use this technique to explore the role of endolysosomal ion channels in tumor cells⁹, thereby controlling tumor growth and proliferation. Regarding metabolic diseases, endolysosomal patch-clamp studies are revealing compounds that regulate endolysosomal ion channels, offering new treatment approaches for diabetes and obesity. The endolysosomal patch-clamp technique aids in understanding endolysosomal dysfunction and finding potential therapies⁶, significantly enhancing our understanding of endolysosomal ion channel functions and promoting the discovery of new drug targets.

Protocol

1. Instrument setup

1. Hardware
   NOTE: See Figure 1 for a standard electrophysiology rig setup.
   1. Shield the setup from external interference using a table and a Faraday cage.
   2. Use an inverted microscope with a micromanipulator for stably positioning the microelectrode.
   3. Set up an amplifier to collect and amplify the acquired signals.
   4. Use a digitizer to convert the analog signals into digital signals.
   5. Use data acquisition and analysis software to set up experimental protocols and extract meaningful, analyzable results from the collected data.
2. Electrodes
   1. Use two electrodes: a bath electrode and a pipette electrode.
      NOTE: Platinum and silver chloride have the best polarization properties. Silver chloride electrodes consist of silver wire or pellets that have been chlorided, thus having an AgCl layer on the external surface of the wire or pellet (Figure 2).
3. Pipette preparation
   1. Pulling
      1. Install the capillary tube into the puller.
         NOTE: The parameter used for pipette fabrication below are instrument-specific. The instrument we used in this paper is mentioned in Table of Materials.
      2. Under experimental conditions (room temperature 22 ˚C, through-shape heater filament, borosilicate glass with filament) that do not burn out the heater filament, run a RAMP test on the puller to determine the heat value (HEAT) required to melt the glass capillary.
      3. Design a new pulling program with six separate pull cycles using the HEAT, velocity (VEL), and time (TIME) parameters (Table 1).
         NOTE: HEAT refers to the parameter that heats the heater's filament, allowing the glass filament to melt. Velocity indicates the speed at which the puller pulls the filament in both directions. Time represents the duration of the interval between each cycle.
      4. Press the green Pull button on the keypad.
      5. Loosen the clamping knob and remove the pipettes from the puller.
      6. Inspect the pipette tips under the microforge's eyepiece to determine the tip diameter, sharpness, and geometry. Ensure the tip diameter is 0.5-0.9 µm.
         NOTE: As the filament is used more frequently, the heat value required to melt the glass capillary will change.
      7. If the tip size does not meet expectations, adjust the HEAT and VEL parameters. Higher temperatures and faster speeds will produce finer, longer tips, and vice versa. Adjust the HEAT by increments of 5 degrees and the speed by increments of 3.
   2. Polishing
      1. Place the pulled patch pipettes into the microforge holder.
      2. Inspect the pipette tips using a 35x objective lens (combined with a 15x eyepiece, resulting in 525x magnification).
      3. Use the micromanipulator to bring the patch pipettes close to the filament.
      4. Set the temperature dial to 80.
      5. Use the footswitch to turn on the heater and apply a brief heat pulse (1 - 2 s), monitoring the polishing process through the microforge eyepiece.
      6. Repeat the process until all pipettes are polished.
      7. Place the completed pipettes in a sealed box to prevent dust from entering.
         NOTE: The goal is to quickly melt the tip so that the glass reforms the final tip geometry without making it too sharp. This prevents the pipette from penetrating the vesicle membrane it is pressed against and effectively promotes seal formation. Following the final polish, the inner path of the pipette tip should be very narrow, straight, and linear.
         The best recording pipettes typically have a resistance of 5 - 8 MΩ after fire polishing. The tip opening diameter should be 2 µm. A perfectly polished patch pipette is essential for successful gigaseal formation, as this is one of the key techniques crucial to the procedure's success.

2. Sample preparation

1. Cell Culture (using HEK293 as an example)
   1. Culture the cells in a standard humidified incubator at 37 °C with 5% CO₂.
   2. Culture the HEK293 cells in low-glucose DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin.
   3. Place poly-L-lysine-coated 12 mm coverslips in a 24-well plate.
2. Organelle enlargement
   1. Add 1 µM vacuolin-1 to HEK293 cells in the 24-well plate (from step 2.1) and incubate at 37 °C with 5% CO₂ overnight until enlarged vesicles begin to form.
      NOTE: The size of the enlarged endolysosomes can reach 1-10 µm, depending on the tool, compound, incubation time, and cell type (Figure 3 and Table 2).
3. Solution preparation
   NOTE: The standard solution preparation aims to simulate the ionic concentrations inside the cell and organelles. For example, the standard solutions for measuring ion channels on lysosomes are as follows:
   1. Prepare bath solution (in mM):140 K-methanesulfonate (MSA), 5 KOH, 4 NaCl, 0.39 CaCl₂, 1 EGTA, 10 HEPES. Adjust the pH to 7.2 with KOH. Adjust the osmolarity to 300 mosm/L with glucose.
   2. Prepare pipette solution (in mM):140 Na-MSA, 5 K-MSA, 2 Ca-MSA, 1 CaCl₂, 10 HEPES, 10 MES. Adjust the pH to 4.6 with MSA. Adjust the osmolarity to 310 mosm/L with glucose.
      NOTE: Pass the solution through a 0.2 µm filter, prepare 45 mL aliquots in 50 mL conical tubes, and store them at 4 °C for up to 2-4 weeks.

3. Organelle isolation

1. Remove a coverslip with treated cells (see section 2) from the 24-well plate, transfer it to the microscope chamber, and add 1 mL of bath solution.
   NOTE: Cells must not be stored in the bath solution for >1 h for whole-endolysosomal patch-clamp experiments that involve endolysosome isolation, sealing, and recording. After 1 h, the enlarged endolysosomes will shrink, adhere to the pipette, and become difficult to isolate.
2. Use a homemade plastic filling needle to fill the isolation pipette with pipette solution and install the pipette at the front end of the patch-clamp setup.
3. Set the diagonal movement angle of the micromanipulator to be close to 30° (factory default value).
4. Under the microscope (40x objective and 10x eyepiece), examine the cells for sufficiently enlarged endosomes or lysosomes located near the cell membrane edge for isolation. Move the isolation pipette close to the selected cell.
5. Lower the isolation pipette using the micromanipulator until it touches the edge of the plasma membrane (Figure 4). Quickly move the isolation pipette horizontally to tear off a small piece of the plasma membrane.
6. Using the same pipette, press the cell from the opposite side to squeeze out the endosome/lysosomes to a distance of ~2 µm from the cell.
   NOTE: If there is residual cell membrane on the organelle, it may prevent the formation of a gigaseal.
7. Examine the isolated endolysosome under the microscope.

4. Gigaseal formation

1. Fill a freshly polished patch pipette with the appropriate pipette solution and install the pipette at the front end of the amplifier.
2. Apply 20 - 50 mbar of positive pressure to the pipette (or use 0.03-0.05 mL from a 1 mL syringe for pressure control) and maintain this pressure (lock the valve).
   NOTE: 1 mbar (millibar) = 0.001 bar = 0.1 kPa (kilopascal) = 1 hPa (hectopascal) = 1,000 dyn/cm2.
3. Moving the pipette tip into the bath solution, in the center of the field of view, confirm that the pressure is high enough to see the liquid flowing into the bath solution.
4. Apply repeated current pulses (+5 mV; 5 ms) 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.
5. Quickly move the pipette close to the top of the target vesicle. Bring the pipette near the vesicle until the vesicle moves or rolls due to the fluid flow from the pipette (Figure 5).
6. Adjust the offset voltage of the pipette to 0 mV. Immediately release the positive pressure. Ideally, the vesicle will be drawn toward the pipette and connect to the membrane, forming a gigaseal (1-20 GΩ) within 1 s.Look for a decrease to <10 pA of the current amplitude evoked by the voltage pulse of 5 mV, which indicates successful gigaseal formation.
   NOTE: Stable positive pressure is essential for gigaseal formation. Sometimes, leaks in the tubing can prevent gigaseal formation. It is recommended to use a pressure gauge to ensure there are no leaks. If a leak is detected, reattach the tubing or apply a small amount of petroleum jelly to the seams to improve the seal.

5. Current measurement

1. Membrane breakage
   NOTE: Depending on the experimental needs, the electrode-to-membrane contact can be categorized into four modes: cell-attached mode, whole-cell mode, inside-out mode, and outside-out mode (Figure 6).
   1. For whole vesicle recording, use a high-voltage short pulse (ZAP pulse) of 200 µs or 500 µs to break the membrane at the contact point between the pipette and the organelle. Set the voltage from -500 mV to -1,200 mV, decreasing by 100 mV each time until the membrane is disrupted.
   2. For inside-out (single channel recording) mode, after forming the gigaseal, pull the pipette away from the organelle, tearing off a small piece of the membrane, exposing the side that was originally facing the vesicle lumen.
   3. For the outside-out mode, after forming the gigaseal and transitioning to the whole vesicle mode, pull the pipette away from the organelle, tearing off a small piece of the membrane while maintaining a small vesicular structure.
2. Current recording
   1. Record the current across the endolysosomal membrane as the input voltage varies in endolysosomal voltage-clamp patch-clamp experiments.
      1. Control these voltages by voltage steps or voltage ramps; use a wide range of input voltages (e.g., -100 mV to +100 mV) in endolysosomal voltage-clamp experiments.

Results

The following describes the current shapes observed during endolysosomal patch-clamp experiments. If the current shape is not as expected, it could be due to poor contact or leakage. Poor contact may occur if the reference electrode is not fully in contact with the bath solution or if the pipette electrode is about to break. Leakage can happen if there is a gap between the chamber and the coverslip allowing fluid to flow onto the objective lens or the stage; having too much or too little pipette solution could also resul...

Discussion

Electrophysiological experimental setups have four main laboratory requirements: i) environment: methods to keep the sample healthy; ii) optics: methods to visualize the sample; iii) mechanics: methods to stably position the microelectrode; and iv) electronics: methods to amplify and record the signal.

To successfully perform endolysosomal patch-clamp experiments, several key steps are crucial. First, the condition of the cells-the cells must be tightly adhered to the coverslip so that when th...

Materials

Name	Company	Catalog Number	Comments
BOROSILICATE GLASS	SUTTER INSTRUMENT	BF150-75-10	O.D.:1.5 mm, I.D. 0.75 mm 10 cm length, with filament
Digidata 1140A	Axon Instruments
Inverted microscope IX73	OLYMPUS
MODEL P-97 micropipette puller	SUTTER INSTRUMENT
MPC-200	SUTTER INSTRUMENT
MultiClamp 700B	Axon Instruments
POLISHER
Quick Release Chamber	Warner instruments	641943	QR-40LP, for 25 mm Coverslips