Bazzone, Andre and Barthmes, Maria and George, Cecilia and Brinkwirth, Nina and Zerlotti, Rocco and Prinz, Valentin and Cole, Kim and Friis, Soren and Dickson, Alexander and Rice, Simon and Lim, Jongwon and Toh, May Fern and Mohammadi, Milad and Pau, Davide and Stone, David J. and Renger, John J. and Fertig, Niels (2023) 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. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 24 (16): 12788. ISSN 1661-6596, 1422-0067
Full text not available from this repository. (Request a copy)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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | ION CHANNELS; POTASSIUM; PROTEINS; BINDING; ASSOCIATION; VALIDATION; TMEM175; ion channels; Parkinson's disease (PD); screening technologies; drug development; lysosomes; solid supported membrane-based electrophysiology (SSME); (SURFER)-R-2; automated patch-clamp (APC); lysosomal patch-clamp |
| Subjects: | 500 Science > 570 Life sciences |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 12 Mar 2024 14:30 |
| Last Modified: | 12 Mar 2024 14:30 |
| URI: | https://pred.uni-regensburg.de/id/eprint/59815 |
Actions (login required)
 |
View Item |
