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- Title
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.
- Authors
Bazzone, Andre; Barthmes, Maria; George, Cecilia; Brinkwirth, Nina; Zerlotti, Rocco; Prinz, Valentin; Cole, Kim; Friis, Søren; Dickson, Alexander; Rice, Simon; Lim, Jongwon; Fern Toh, May; Mohammadi, Milad; Pau, Davide; Stone, David J.; Renger, John J.; Fertig, Niels
- Abstract
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.
- Subjects
HIGH throughput screening (Drug development); ELECTROPHYSIOLOGY; PARKINSON'S disease; MEMBRANE potential; TRP channels; CONCENTRATION gradient
- Publication
International Journal of Molecular Sciences, 2023, Vol 24, Issue 16, p12788
- ISSN
1661-6596
- Publication type
Article
- DOI
10.3390/ijms241612788