Lysosomal diseases

Ion channels and transporters as key players.

Unlocking the Secrets of Lysosomes: Ion Channels and Transporters in Lysosomal Diseases

Lysosomes are intracellular organelles which serve as the digestive system of the cell. Lysosomes degrade material taken up from outside the cell, as well as obsolete components of the cell itself. The inside of the lysosome is highly acidic, which is essential for the function of the lysosomal hydrolases. The acidic pH is maintained by a proton pump in the lysosomal membrane which actively transports H+ ions into the lysosome. Other ion channels and transporters, such as TMEM175 and TRPML, are also present in the lysosomal membrane and mutations in these proteins have been linked to various diseases including neurodegenerative diseases like Parkinson’s and Alzheimer’s disease, and cancer.

The rapidly evolving field of lysosomal

ion channel and transporter research

Parkinson´s disease risk protein TMEM175

Recent developments in neurodegenerative disease research very clearly show that no detail is too small to miss. Focusing even on tiny structures in the cellular function, such as the cellular organelles (lysosomes and mitochondria) can bring significant advancement in helping affected patients. The involvement of impaired lysosomal and mitochondrial function in Parkinson´s disease (PD) is not new. However, TMEM175 a novel, constitutively active ion channel involved in regulating lysosomal pH and autophagy, emerged as a highly relevant potential target for the treatment of PD. Patients carrying a mutation in the TMEM175 gene, carry significant risk for Parkinson’s disease development and earlier age-of-onset, presumably due to compromised lysosomal and mitochondrial function, leading to increasing aggregation of insoluble proteins such as phosphorylated α-synuclein. The lack of specific experimental tools has hindered further investigation into the exact role of TMEM175 in normal lysosomal function and pathological processes. However, successful recordings of TMEM175 have been performed using automated patch clamp and solid-supported membrane- (SSM-) based electrophysiology, opening new avenues for deciphering organelle function.

DCPIB dose response curve for K+ flux through TMEM175 in lysosomes

Figure shows DCPIB dose response curve (left) and individual current traces (right) for K+ flux through TMEM175 in lysosomes purified from TMEM175 overexpressing HEK293 cell culture. From the dose dependent peak currents we established EC50 and Imax values for the modulation of K+ flux by DCPIB.

TRPML-1 and lysosomal storage disorders

TRPML1 (Transient Receptor Potential Mucolipin 1) is an important cation channel that has gained attention from the medical and scientific communities due to its vital role in cellular physiology and its potential as a therapeutic target.

TRPML1 is primarily found within the lysosomes, the recycling centers of our cells. It plays a crucial role in physiology as a sensor for reactive oxygen species and is an essential player of lysosomal adaption in situations of nutrient starvation. TRPML1 exhibits a unique ion selectivity, mediating calcium efflux, but also the passage of sodium, and potassium ions. Understanding the intricate gating mechanisms and ion conductance properties of TRPML1 holds the key to unraveling its physiological functions and potentially developing targeted therapies for diseases associated with its malfunction. Dysfunctions in TRPML1 have been linked to lysosomal storage disorders such as Mucolipidosis Type IV, which affect multiple organ systems and manifest as developmental delays, motor impairments, and visual problems.

Lysosomal ion channels and transporters in disease

TMEM175*
Parkinson’s disease
TRPML1*
Mucolipidosis Type IV leading to hepatosplenomegaly, dysmorphic features and neurological disorders
ClC-7*
Osteopetrosis, neuronal ceroid lipofuscinoses (LSD), neurodegeneration
Cystinosin*
Cystinosis (LSD)
SLC15a4*
Hypotrichosis 4, Systemic Lupus Erythematosus
Sialin
Sialic Acid Storage Disease (LSD), muscular, cerebellar and CNS disorders
Presenilin-1 dependent v-ATPase
Alzheimer disease
TPC1/2
Parkinson's disease, non-alcoholic fatty liver disease, Ebola infection, cancer, cardiac dysfunction and diabetes
ATP13A2
Parkinson’s disease

*Listed targets have been validated on SURFE2R instruments. (LSD=lysosomal storage disease)

Techniques for organelle research

Recording lysosomes

Technologies such as automated patch clamp (APC) or solid-supported membrane- (SSM-) based electrophysiology offer options for recording activity of ion channels and transporters located even in small cellular structures, such as lysosomes at high throughput, relevant for drug discovery and validation.

Automated patch clamp platforms can be used to study lysosomal ion channels. TPC1/2 ion channels in lysosomes have been recorded using the Port-a-Patch. Upscaling the throughput to Patchliner and SyncroPatch 384 allows for faster ion channel mutation screening as done by recording plasma membrane TMEM175 in wild type and mutated cell.

The SURFE2R N1 and SURFE2R 96SE , both applying SSM-based electrophysiology, can measure ion and substrate flow by transporters and ion channels directly from intact lysosomes. Such measurements have been successfully performed for TMEM175, TRPML1 and ClC-7. The SSM-based approach allows for stable and robust recordings from proteins residing in organellar membranes with a throughput of up to 10,000 data points per day.

 

 

 

 

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