High-throughput electrophysiology and fluorescence-based screening platforms for identification of activators of the lysosomal ion channel TMEM175
Journal
ICMS 2024 UK
Author(s)
Year
2024
TMEM175 is a cation permeable ion channel located on the lysosomal and endosomal membrane which functions as a proton-activated proton channel and a potassium ion leak channel. This channel maintains lysosomal luminal pH through direct efflux of protons and as a counter-ion flux of potassium ions to balance influx of cytosolic protons by the V-ATPase (Cang et al., 2015). This has a significant role in maintaining lysosomal function that becomes dysregulated in disease leading to compromised proteolytic processing.
Loss of function variants, such as the M393T point mutation, are risk factors for earlier onset and increased severity of neurodegenerative diseases including Parkinson’s disease (Wie et al., 2021; Nalls et al., 2015). Unstable lysosomal pH leads to decreased lysosomal catalytic activity, decreased glucocerebrosidase activity, impaired autophagosome clearance, e.g. of accumulated α-synuclein, by the lysosome and decreased mitochondrial respiration.
Small molecule activators of TMEM175 may have therapeutic potential to normalise luminal pH and restore lysosomal activity to patients suffering neurodegeneration or those who are at risk of an early onset. Development of screening capabilities are therefore the first step in identifying novel chemical starting points for drug discovery.
Here we show assay data from Charles River Laboratories comparing cell lines constitutively expressing the WT and M393T variants of human TMEM175 and WT mouse TMEM175 channels. Activation of TMEM175 was observed in automated patch-clamp (Sophion Qube) for both outward Cs+ and inward H+ currents, and FLIPR Thallium flux assay after addition of the tool activator DCPIB.