The endo-lysosomal system is a series of intracellular organelles responsible for recycling and degradation of macromolecules. Endo-lysosomes express several functionally diverse ion channels, crucial for regulating organelle trafficking and intracellular signalling as well as maintaining the acidic luminal pH for optimal enzymatic activity. Dysfunctions within the endo-lysosomal system are associated with multiple disorders including lysosomal storage disorders and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Lysosomal ion channels have therefore gained significant attention as potential targets for novel therapeutics.
Due to their localisation to small intracellular compartments, endo-lysosomal ion-channels have been historically challenging to study using conventional patch-clamp techniques. However, recent advances in lysosomal biology make it possible to enlarge and extract endo-lysosomes for subsequent recording using manual patch-clamp electrophysiology. This approach allows for the characterisation of channel function within the native environment, facilitating better physiological understanding.
Materials and Methods
Endo-lysosomal extraction technique
Cells are incubated for at least 2 h with vacuolin-1 to enlarge endo-lysosomes.
Prior to recordings, cells are briefly incubated with neutral red to visualise acidic intracellular compartments.
A small diameter glass pipette is used to rupture the cell and to excise enlarged endo-lysosomes.
Isolated endo-lysosomes are subsequently patched using a fresh, fire polished glass pipette.
Transient receptor potential mucolipin 1 (TRPML1), is Ca2+ permeable, nonselective cation channel, expressed in late endosomes and lysosomes. TRPML1 is a key regulator for multiple cellular functions including lysosomal signalling, autophagy, and membrane trafficking. Mutations in MCOLN1, encoding TRPML1, cause mucolipidosis type IV, a severe autosomal recessive lysosomal storage disorder, characterised by diverse symptoms including delayed psychomotor development and ocular aberrations. TRPML1 has further been implicated in the pathogenesis of Alzheimer’s disease and other neurodegenerative disorders, making it an attractive target for drug discovery.
TRPML1 is regulated by pH, with the highest activity under acidic conditions as found in the lysosomal lumen (Figure 2). In this example, experiments were performed in whole cell configuration on the manual patch clamp platform using a variant lacking the organelle targeting motif (TRPML1-4A), that can be expressed at the plasma membrane and would, therefore, also be suitable for high throughput screening.
Results
pH dependence of TRPML1-4A activation – whole cell recordings
Figure 2a. TRPML1-4A current traces at decreasing extracellular pH valuesFigure 2b. ML-SA1 induced TRPML1-4A activation increases with acidic pH
To investigate these channels in their native environment, we established a refined manual patch clamp technique to directly record from isolated endo-lysosomes, suitable for investigating potential therapeutic agents as exemplified in the example shown in Figure 3.
The cardiac late Na+ current (late INa) generates persistent inward currents throughout the plateau phase of the ventricular action potential and is an important determinant of repolarisation rate, EADs and arrythmia risk¹. As inhibition of late INa can offset drug effects on hERG and other repolarising K⁺conductances, it is one of the key cardiac channels in the Comprehensive in vitro Pro-arrythmia Assay (CiPA) panel being developed by the FDA to improve human clinical arrythmia risk assessment²̛ ³.
Cardiac toxicity remains the leading cause of new drug safety side-effects. Current preclinical cardiac safety assays rely on in vitro cell-based ion channel assays and ex vivo and in vivo animal models⁽¹⁾. These assays provide an indication of acute risk but they do not always predict the effect of chronic compound exposure, as recently seen with oncology drugs. Therefore, new assays are required to characterise chronic structural and functional effects in human cells earlier in drug discovery. Impedance-based technology can provide more accurate chronic cardiotoxicity measurements in an efficient manner using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).
