A Comparative Study on the Lysosomal Cation Channel TMEM175 Using Automated Whole-Cell Patch-Clamp, Lysosomal Patch-Clamp, and Solid Supported Membrane-Based Electrophysiology: Functional Characterization and High-Throughput Screening Assay Development

The lysosomal cation channel TMEM175 is a Parkinson's disease-related protein and a promising drug target. Unlike whole-cell automated patch-clamp (APC), lysosomal patch-clamp (LPC) facilitates physiological conditions, but is not yet suitable for high-throughput screening (HTS) applications. Here, we apply solid supported membrane-based electrophysiology (SSME), which enables both direct access to lysosomes and high-throughput electrophysiological recordings. In SSME, ion translocation mediated by TMEM175 is stimulated using a concentration gradient at a resting potential of 0 mV. The concentration-dependent K+ response exhibited an I/c curve with two distinct slopes, indicating the existence of two conducting states. We measured H+ fluxes with a permeability ratio of PH/PK = 48,500, which matches literature findings from patch-clamp studies, validating the SSME approach. Additionally, TMEM175 displayed a high pH dependence. Decreasing cytosolic pH inhibited both K+ and H+ conductivity of TMEM175. Conversely, lysosomal pH and pH gradients did not have major effects on TMEM175. Finally, we developed HTS assays for drug screening and evaluated tool compounds (4-AP, Zn as inhibitors; DCPIB, arachidonic acid, SC-79 as enhancers) using SSME and APC. Additionally, we recorded EC50 data for eight blinded TMEM175 enhancers and compared the results across all three assay technologies, including LPC, discussing their advantages and disadvantages.

Automated summary

This study investigated the function of TMEM175 channel in lysosomes using solid supported membrane-based electrophysiology (SSME). The results showed that TMEM175 is a pH-dependent ion channel, and decreasing cytosolic pH inhibits its function. Additionally, the effects of different compounds on TMEM175 were evaluated and the advantages and disadvantages of different experimental techniques were compared.