TMEM175: Apparent drug potencies are different in lysosomes and plasma membrane—comparative study employing a novel screening technology

SSM-based electrophysiology (SSME) has emerged as a powerful technique for studying transport proteins of intracellular localization, which are challenging to assess using conventional methods. Here, we present a recent study on the lysosomal H+ and K+ channel TMEM175 involved in the onset of Parkinson’s disease, including its functional characterization and medium to high throughput drug screening. We have purified lysosomes from a HEK cell line overexpressing TMEM175 and employed H+ and K+ concentration gradients to stimulate ion flux through TMEM175 at 0 mV. Based on the dose response curves we confirmed a PH/PK permeability ratio of 48,500, aligning with existing literature. We also investigated the pH dependence of K+ flux. Cytosolic protons lead to a reduction of TMEM175 activity, indicating a mechanism that prevents oversaturation of the cytosol under acidic stress. In contrast, TMEM175 function seems to be independent of the natural pH gradient across the lysosomal membrane. Next, we established a high-throughput method based on SSME to assess the drug potency of tool compounds, 4-AP and Zn as inhibitors; DCPIB, arachidonic acid and SC-79 as enhancers. To validate our approach, we compared the results obtained from SSME with those from whole-cell automated patch-clamp, where TMEM175 resides in the plasma membrane, as well as lysosomal patch-clamp. Our comparative analysis revealed that the presence of the lysosomal membrane environment is crucial for accurately estimating drug potencies for TMEM175. In conclusion, our research raises SSME as a versatile technology capable of efficiently characterizing electrogenic target proteins and facilitating high-throughput drug development. By unlocking the potential of SSME, we gain valuable insights into the function of transporters and channels and pave the way for the discovery of novel therapeutic interventions.