Lysosomal dysfunction and impaired clearance of pathological α-synuclein aggregates are recognized as central mechanisms in the pathogenesis of Parkinson’s disease (PD). Genome-wide association studies have identified variants in TMEM175 as significant risk factors. TMEM175 encodes a lysosomal K + /H + channel that plays a pivotal role in maintaining lysosomal pH stability, regulating autophagic flux, and supporting mitochondrial respiration. While complete loss of TMEM175 activity may paradoxically confer resilience in toxin-induced PD models, partial dysfunction, commonly observed in human risk variants, appears to drive disease progression and accelerate neurodegeneration by compromising lysosomal enzyme function, promoting α-synuclein accumulation, and increasing neuronal susceptibility to oxidative stress. Pharmacological activation of TMEM175 thus represents a promising therapeutic strategy, offering an integrated approach to target converging pathogenic mechanisms in PD. To this end, we established a comprehensive experimental pipeline integrating high-throughput screening with detailed mechanistic assays to identify and validate TMEM175-positive modulators. A cornerstone of this work is the development of a physiologically relevant lysosomal electrophysiology platform. Using automated patch-clamp (SyncroPatch 384, Nanion), we implemented a scalable system capable of compound testing across a 384-chip array in the whole-lysosome configuration. As breakthrough, we advanced manual patch-clamp recordings on lysosomes isolated from iPS-derived dopaminergic neurons carrying either wild-type or disease-associated TMEM175 variants. These recordings reached the single-channel level, providing direct resolution of TMEM175 activity. Strikingly, application of DCPIB and 4-AP induced pronounced alterations in both the amplitude and duration of individual channel events, underscoring the unique mechanistic insights accessible only through single-channel analysis. This unprecedented capability establishes a versatile framework that unites high-throughput discovery with single-channel resolution, enabling precise pharmacological characterization of TMEM175. These advances illuminate TMEM175’s role in PD pathogenesis and pave the way for rational development of novel modulators to combat neurodegeneration.
Sorbo et al. (Sun,) studied this question.