Membrane Transport 2023
When: 24.08. -25.08.2023
Venue: University of St Andrews, UK.
Go to the Conference website here.
Meet Dr. Ali Obergrussberger
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Nanion is proud sponsor of this conference.
Nanion sponsored Talk in Session 3: The Transport Physiologist´s Toolbox in 2023
Friday, 25.08.2023 at 12:00 SCT.
Talk: Identification of novel TMEM175 modulators using high throughput automated patch clamp and solid supported membrane- (SSM-) based electrophysiology platforms
Presented by Ali Obergrussberger, Nanion Technologies
TMEM175 is a novel, constitutively active ion channel involved in regulating lysosomal pH and autophagy. Mutations in this gene impair normal lysosomal and mitochondrial function, thereby increasing aggregation of insoluble proteins such as phosphorylated α-synuclein, leading to symptoms typical of Parkinson’s Disease (PD). Consequently, TMEM175 demonstrates significant potential as a key player in the treatment of PD. The lack of specific pharmacological tools has hampered further investigation into the exact role of TMEM175 in normal lysosomal function and pathological processes.
Advancements in high-throughput screening technologies have allowed rapid assessment of large numbers of compounds against ion channel drug targets using automated patch-clamp. We have successfully developed recombinant cell lines expressing wild-type (WT) TMEM175, a gain of function (Q65P), and loss of function (M393T) mutants using stably transfected HEK cells. Here, we report the characterization of the WT TMEM175 cell line performed using high-throughput automated patch-clamp electrophysiology and show reproducible concentration-response curves with the potassium channel inhibitor 4-aminopyridine (4-AP). A rapid and robust, automated high-throughput electrophysiology screening assay was subsequently developed to enable the identification of both activators and inhibitors of TMEM175.
Additionally, we have used solid-supported membrane- (SSM-) based electrophysiology (SSM-E) for TMEM175 recordings from lysosomal membranes purified and prepared from the WT TMEM175 cell line. Dose-dependent signal enhancement and inhibition of TMEM175 currents were successfully characterized. The SSM-E approach allows for stable and robust recordings from proteins residing in organellar membranes with a throughput of up to 10,000 data points per day.
In summary, the TMEM175 stable cell line was characterized using an automated patch-clamp and SSM-based electrophysiology. We developed and executed robust, high throughput, and high content direct electrophysiological intracellular screening assays, with unusually high success rates. The successful development of TMEM175 electrophysiology assays capable of identifying novel pharmacological tools will enable investigation of the role of this exciting target in normal physiology and in disease.