A Deep Dive into TMEM175

In recent years, TMEM175 has emerged as a promising therapeutic target for the treatment of Parkinson’s disease. This lysosomal ion channel has been shown to play an essential role in maintaining lysosomal pH and function. Although originally identified as a potassium leak channel, recent studies suggest that TMEM175 is, in fact, a proton-activated, proton-selective channel.

The very fact that TMEM175 is located on the lysosomal membrane makes its functional analysis quite challenging. Early studies were mostly carried out on cell lines overexpressing TMEM175 on the plasma membrane, thus making it possible to use the traditional manual patch clamp and automated patch clamp technique to study the channel’s function. The obvious drawback of this approach is that the overexpressed channel is located in an unnatural lipid and protein environment of the plasma membrane, which could affect its function.

Therefore, to enable the measurement of lysosomal ion channels in a more natural environment, the lysosomal patch clamp technique has been introduced. Nevertheless, to make the lysosomes accessible to patch-clamp recordings, they have to be enlarged, for example, using vacouline-1 treatment, resulting in lysosomal pH alkalinization and potential effects on TMEM175. Furthermore, patching enlarged lysosomes is quite tedious and of very low throughput.

So, it’s clear that, while useful, these approaches aren’t without their limitations when it comes to functional characterization of intracellular ion channels.

In a recent comparative study, researchers from Nanion Technologies, SB Drug Discovery, Cerevel Therapeutics, Assay Works, and the University of Regensburg delved into the characterization of TMEM175 using three different techniques: automated whole-cell patch clamp (APC), lysosomal patch clamp (LPC), as well as solid supported membrane-based electrophysiology (SSME). The authors demonstrated that while every approach has its specific uses, the functional characterization of TMEM175 using SSME broadened our understanding, revealing the kinetic properties of K+ and H+ translocation and the effect of cytosolic and lysosomal pH on TMEM175 conductivity. Interestingly, the authors also showed that compounds act differently on TMEM175 located in the plasma membrane compared to lysosomal TMEM175, emphasizing the importance of conducting experiments under conditions closely mirroring native environments. In this regard, SSME stands out as it uses non-treated lysosomes of natural size, providing an environment most similar to the native one.

Does it mean that SSME is better suited for studying intracellular ion channels than the patch clamp? Perhaps, but it’s not that straightforward. Each method – whether it’s APC, LPC, or the promising SSME – has its own strengths and weaknesses. For instance, while lysosomal patch clamp and SSME offer a more natural environment for TMEM175, automated patch clamp offers higher throughput, greatly surpassing that of LPC and maintaining a slight edge over SSME. Indeed, in contrast to LPC, both APC and SSME offer automated preparations, measurement workflows, and data analysis capabilities. The only difference is that APC systems enable up to 384 parallel recordings, while SSME currently supports up to 96.

In conclusion, all three techniques complement one another due to their unique capabilities, with SSME filling a gap, enabling electrophysiological investigation with decent throughput on non-treated lysosomes without the requirement of a running cell culture.

Good to see how SSME, with its great potential, has positioned itself as a formidable tool in early drug discovery.

You can learn more about the SSME approach here.

Need more details related to the study? Find the full article here.