patch-clamp-techniques-for-single-endolysosomal-vesicle-analysis

Patch-Clamp Techniques for Single Endolysosomal Vesicle Analysis

Endolysosomal ion channels are critical for endolysosomal ion and pH homeostasis, membrane potential regulation, and vesicle trafficking. However, ...

National Taiwan University

National Taiwan University Hospital

Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly and can be classified either as dry or as neovascular (or wet). Neovascular AMD is characterized by a strong immune response and the inadequate release of cytokines triggering angiogenesis and induction of photoreceptor death. The pathomechanisms of AMD are only partly understood. Here, we identify the endolysosomal two-pore cation channel TPC2 as a key factor of neovascularization and immune activation in the laser-induced choroidal neovascularization (CNV) mouse model of AMD. Block of TPC2 reduced retinal VEGFA and IL-1β levels and diminished neovascularization and immune activation. Mechanistically, TPC2 mediates cationic currents in endolysosomal organelles of immune cells and lack of TPC2 leads to reduced IL-1β levels in areas of choroidal neovascularization due to endolysosomal trapping. Taken together, our study identifies TPC2 as a promising novel therapeutic target for the treatment of AMD.

TPC2 promotes choroidal angiogenesis and inflammation in a mouse model of neovascular age-related macular degeneration.

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3 mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3 reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.

The role of two-pore channel 2 (TPC2), one of the few cation channels localized on endolysosomal membranes, in cancer remains poorly understood. Here, we report that TPC2 knockout reduces proliferation of cancer cells in vitro, affects their energy metabolism, and successfully abrogates tumor growth in vivo. Concurrently, we have developed simplified analogs of the alkaloid tetrandrine as potent TPC2 inhibitors by screening a library of synthesized benzyltetrahydroisoquinoline derivatives. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against cancer cells and impairs proangiogenic signaling of endothelial cells to a greater extent than tetrandrine. Simultaneously, toxic effects on non-cancerous cells are reduced, allowing in vivo administration and revealing a TPC2 inhibitor with antitumor efficacy in mice. Hence, our study unveils TPC2 as valid target for cancer therapy and provides easily accessible tetrandrine analogs as a promising option for effective pharmacological interference.

Gene editing and synthetically accessible inhibitors reveal role for TPC2 in HCC cell proliferation and tumor growth.

This work reports the color-tunable mixed photoluminescence (PL) emission from an Alq3 organic layer in an Au-Alq3-Au plasmonic structure through the combination of organic fluorescence emission and another form of emission that is enabled by the surface plasmons in the plasmonic structure. The emission wavelength of the latter depends on the Alq3 thickness and can be tuned within the Alq3 fluorescent spectra. Therefore, a two-color broadband, color-tunable mixed PL structure was obtained. Obvious changes in the Commission Internationale d'Eclairage (CIE) coordinates and the corresponding emission colors of Au-Alq3-Au samples clearly varied with the Alq3 thickness (90, 130, and 156 nm).

Color-tunable mixed photoluminescence emission from Alq3 organic layer in metal-Alq3-metal surface plasmon structure.

Two pore channels (TPCs) and mucolipins (TRPML) are the most prominent cation channels expressed in endolysosomes. Recently, roles of TPCs and TRPML2 have been revealed in regulating and detecting osmotically-driven changes in the surface-to-volume ratio of endolysosomes to promote endocytic and recycling traffic. TPCs and TRPML2 are highly expressed in macrophages and contribute to immune cell function. Here, we provide an overview of the emerging roles of these channels in innate immune cells, in particular macrophages, and highlight two models for osmo-mechanical regulation of intracellular organelle volume, trafficking, and cell homeostasis involving either TPCs or TRPML2.

Two-pore and TRP cation channels in endolysosomal osmo-/mechanosensation and volume regulation.

Infectious diseases and cancer are among the key medical challenges that humankind is facing today. A growing amount of evidence suggests that ion channels in the endolysosomal system play a crucial role in the pathology of both groups of diseases. The development of advanced patch-clamp technologies has allowed us to directly characterize ion fluxes through endolysosomal ion channels in their native environments. Endolysosomes are essential organelles for intracellular transport, digestion and metabolism, and maintenance of homeostasis. The endolysosomal ion channels regulate the function of the endolysosomal system through four basic mechanisms: calcium release, control of membrane potential, pH change, and osmolarity regulation. In this review, we put particular emphasis on the endolysosomal cation channels, including TPC2 and TRPML2, which are particularly important in monocyte function. We discuss existing endogenous and synthetic ligands of these channels and summarize current knowledge of their impact on channel activity and function in different cell types. Moreover, we summarize recent findings on the importance of TPC2 and TRPML2 channels as potential drug targets for the prevention and treatment of the emerging infectious diseases and cancer.

Endolysosomal cation channels point the way towards precision medicine of cancer and infectious diseases.

Two-pore channels TPC1 and TPC2 are ubiquitously expressed pathophysiologically relevant proteins that reside on endolysosomal vesicles. Here, we review the electrophysiology of these channels. Direct macroscopic recordings of recombinant TPCs expressed in enlarged lysosomes in mammalian cells or vacuoles in plants and yeast demonstrate gating by the Ca-mobilizing messenger NAADP and/or the lipid PI(3,5)P. TPC currents are regulated by H, Ca, and Mg (luminal and/or cytosolic), as well as protein kinases, and they are impacted by single-nucleotide polymorphisms linked to pigmentation. Bisbenzylisoquinoline alkaloids, flavonoids, and several approved drugs demonstrably block channel activity. Endogenous TPC currents have been recorded from a number of primary cell types and cell lines. Many of the properties of endolysosomal TPCs are recapitulated upon rerouting channels to the cell surface, allowing more facile recording through conventional electrophysiological means. Single-channel analyses have provided high-resolution insight into both monovalent and divalent permeability. The discovery of small-molecule activators of TPC2 that toggle the ion selectivity from a Ca-permeable (NAADP-like) state to a Na-selective (PI(3,5)P-like) state explains discrepancies in the literature relating to the permeability of TPCs. Identification of binding proteins that confer NAADP-sensitive currents confirm that indirect, remote gating likely underpins the inconsistent observations of channel activation by NAADP.

Electrophysiology of Endolysosomal Two-Pore Channels: A Current Account.

Endolysosomal ion channels are a group of ion channel proteins that are functionally expressed on the membrane of endolysosomal vesicles. The electrophysiological properties of these ion channels in the intracellular organelle membrane cannot be observed using conventional electrophysiological techniques. This section compiles the different electrophysiological techniques utilized in recent years to study endolysosomal ion channels and describes their methodological characteristics, emphasizing the most widely used technique for whole endolysosome recordings to date. This includes the use of different pharmacological tools and genetic tools for the application of patch-clamping techniques for specific stages of endolysosomes, allowing the recording of ion channel activity in different organelles, such as recycling endosomes, early endosomes, late endosomes, and lysosomes. These electrophysiological techniques are not only cutting-edge technologies that help to investigate the biophysical properties of known and unknown intracellular ion channels but also help us to investigate the physiopathological role of these ion channels in the distribution of dynamic vesicles and to identify new therapeutic targets for precision medicine and drug screening.

Electrophysiological Techniques on the Study of Endolysosomal Ion Channels.

The glycoprotein CD44 is a key regulator of malignant behaviors in breast cancer cells. To date, hyaluronic acid (HA)-CD44 signaling pathway has been widely documented in the context of metastatic bone diseases. Core 1 β1,3-galactosyltransferase (C1GALT1) is a critical enzyme responsible for the elongation of O-glycosylation. Aberrant O-glycans is recognized as a hallmark in cancers. However, the effects of C1GALT1 on CD44 signaling and bone metastasis remain unclear. In this study, IHC analysis indicated that C1GALT1 expression positively correlates with CD44 in breast cancer. Silencing C1GALT1 accumulates the Tn antigen on CD44, which decreases CD44 levels and osteoclastogenic signaling. Mutations in the O-glycosites on the stem region of CD44 impair its surface localization as well as suppress cell-HA adhesion and osteoclastogenic effects of breast cancer cells. Furthermore, in vivo experiments demonstrated the inhibitory effect of silencing C1GALT1 on breast cancer bone metastasis and bone loss. In conclusion, our study highlights the importance of O-glycans in promoting CD44-mediated tumorigenic signals and indicates a novel function of C1GALT1 in driving breast cancer bone metastasis.

Truncation of GalNAc-type O-glycans Suppresses CD44-mediated Osteoclastogenesis and Bone Metastasis in Breast Cancer.

Human genetic diversity can reveal critical factors in host-pathogen interactions. This is especially useful for human-restricted pathogens like  serovar Typhi ( Typhi), the cause of typhoid fever. One key defense during bacterial infection is nutritional immunity: host cells attempt to restrict bacterial replication by denying bacteria access to key nutrients or supplying toxic metabolites. Here, a cellular genome-wide association study of intracellular replication by . Typhi in nearly a thousand cell lines from around the world-and extensive follow-up using intracellular  Typhi transcriptomics and manipulation of magnesium availability-demonstrates that the divalent cation channel mucolipin-2 (MCOLN2 or TRPML2) restricts  Typhi intracellular replication through magnesium deprivation. Mg currents, conducted through MCOLN2 and out of endolysosomes, were measured directly using patch-clamping of the endolysosomal membrane. Our results reveal Mg limitation as a key component of nutritional immunity against  Typhi and as a source of variable host resistance.

Human variation impacting  restricts  Typhi replication by magnesium deprivation.

Melanoma arising from pigment-producing melanocytes is the deadliest form of skin cancer. Extensive ultraviolet light exposure is a major cause of melanoma and individuals with low levels of melanin are at particular risk. Humans carrying gain-of-function polymorphisms in the melanosomal/endolysosomal two-pore cation channel TPC2 present with hypopigmentation, blond hair, and albinism. Loss of TPC2 is associated with decreased cancer/melanoma proliferation, migration, invasion, tumor growth and metastasis formation, and TPC2 depleted melanoma cells show increased levels of melanin. How TPC2 activity is controlled in melanoma and the downstream molecular effects of TPC2 activation on melanoma development remain largely elusive. Here we show that the small GTPase Rab7a strongly enhances the activity of TPC2 and that effects of TPC2 on melanoma hallmarks, in vitro and in vivo strongly depend on the presence of Rab7a, which controls TPC2 activity to modulate GSK3β, β-Catenin, and MITF, a major regulator of melanoma development and progression.

Rab7a is an enhancer of TPC2 activity regulating melanoma progression through modulation of the GSK3β/β-Catenin/MITF-axis.

Osteosarcoma is an aggressive bone malignancy with a high propensity for drug resistance and metastasis, leading to poor clinical outcomes. This study investigates the role of core 1 β1,3-galactosyltransferase 1 (C1GALT1) in osteosarcoma, focusing on its implications in chemoresistance. Our findings reveal that high expression of C1GALT1 is associated with advanced stages, adverse overall survival, and increased recurrence rates. Elevated levels of C1GALT1 were observed in doxorubicin-selected osteosarcoma cells, where its suppression significantly promoted doxorubicin-induced apoptosis and reduced drug efflux. Pharmacological inhibition of C1GALT1 using itraconazole replicated these effects, suggesting a potential therapeutic strategy to overcome chemoresistance. Additionally, we identified the involvement of the ATP-binding cassette (ABC) transporter ABCC1 in the drug-resistance phenotype mediated by C1GALT1. C1GALT1-mediated O-glycan changes were found to influence the cell-surface targeting and lysosomal degradation of ABCC1, thereby modulating its efflux capacity. In vitro and in vivo studies confirmed that C1GALT1 impacts ABCC1 expression and function, further supporting its role in osteosarcoma chemoresistance. These results highlight the clinical relevance of C1GALT1 as a biomarker for prognosis and a potential therapeutic target for osteosarcoma. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Article	Year
TPC2 promotes choroidal angiogenesis and inflammation in a mouse model of neovascular age-related macular degeneration. Life science alliance\| PubMed ID: 34183443	2021
Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice. Nature communications\| PubMed ID: 35031603	2022
Gene editing and synthetically accessible inhibitors reveal role for TPC2 in HCC cell proliferation and tumor growth. Cell chemical biology\| PubMed ID: 33626324	2021
Color-tunable mixed photoluminescence emission from Alq3 organic layer in metal-Alq3-metal surface plasmon structure. Nanoscale research letters\| PubMed ID: 25328506	2014
Two-pore and TRP cation channels in endolysosomal osmo-/mechanosensation and volume regulation. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 33279607	2021
Endolysosomal cation channels point the way towards precision medicine of cancer and infectious diseases. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie\| PubMed ID: 35240524	2022
Electrophysiology of Endolysosomal Two-Pore Channels: A Current Account. Cells\| PubMed ID: 35954212	2022
Electrophysiological Techniques on the Study of Endolysosomal Ion Channels. Handbook of experimental pharmacology\| PubMed ID: 36871125	2023
Truncation of GalNAc-type O-glycans Suppresses CD44-mediated Osteoclastogenesis and Bone Metastasis in Breast Cancer. Molecular cancer research : MCR\| PubMed ID: 37040171	2023
Human variation impacting restricts Typhi replication by magnesium deprivation. Cell genomics\| PubMed ID: 37228749	2023
Rab7a is an enhancer of TPC2 activity regulating melanoma progression through modulation of the GSK3β/β-Catenin/MITF-axis. Nature communications\| PubMed ID: 39562548	2024
C1GALT1 expression predicts poor survival in osteosarcoma and is crucial for ABCC1 transporter-mediated doxorubicin resistance. The Journal of pathology\| PubMed ID: 39844613	2025

Cheng-Chang Chen

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