Introduction

Solid supported membrane-based electrophysiology (SSM-E) offers novel approaches for electrophysiological recordings from lysosomal membrane proteins such as transporters, ligand-gated and leak ion channels in their native membrane environment.

Method

The method of SSM-E relies on the adsorption of any membrane, native, cell culture-derived or organellar, to a lipid coated electrode, i.e the solid supported membrane, and the direct current read-out caused by the capacitive charging of the membranes. The 3 mm diameter electrode entails a >1000-fold amplification of the currents compared with conventional patch clamp, allowing for the measurements of low-conducting membrane proteins, such as transporters. The fact that also intracellular membranes can be accessed by SSM-E, their accessibility for investigation and characterization drastically improves.

Results & Conclusions

Here, we present a study on TMEM175 channels residing in lysosomes using SSM-E. We found an average permeability ratio between protons and potassium of P_H/P_K = 48.500 and similar conductivities for K⁺, Rb⁺, and Cs⁺. We also found that TMEM175 activity is downregulated to 30% of I_max upon cytosolic acidification with a pK=7.0, while TMEM175 is resistant to lysosomal acidification. We also investigated dose-dependent effects on TMEM175 (n=8 sensors) exerted by blockers, i.e. Zn²⁺ (IC₅₀ = 1.5 ± 0.2 mM) and 4-AP (IC₅₀ = 1.7 ± 0.3 mM), and enhancers, i.e. DCPIB (EC₅₀ = 10 ± 5 µM; E_max = 275 ± 37 %) and arachidonic acid (EC₅₀ = 2 ± 0.4 µM; E_max = 168 ± 5 %). As expected, the enhancer SC79 which acts via PKA has only little effects on TMEM175 activity (E_max = 109 ± 5 %) in our in vitro assay.