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 on

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.

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