EC50 and IC50 Measurements of TRPM8 in Lipid Bilayers

Abstract

Ensemble measurements of ion channel currents, made in the presence of pharmaceutical compounds, enable determination of compound activity and potency in the form of IC50 and EC50 values, and are traditionally performed with Patch Clamp. An alternative platform for ion channel study involves the formation of artificial bilayers from the contact of lipid monolayers at aqueous/oil interfaces. This technique has the potential for automation and parallel measurements. Previous work with this platform has demonstrated measurements of physiologically relevant ion channels at the single molecule level. Here we present measurements of the cold and menthol receptor TRPM8 at the single channel and ensemble level. We studied the effect of menthol and temperature on the open probability of TRPM8 and found a single-channel conductance of 64 ± 6 pS, comparable to previous work. In addition, we measured ensemble activity of TRPM8 as a function of temperature and varying PI(4,5)P2 concentration. Our apparatus enables introduction of pharmaceutical compounds to the solution surrounding the bilayer during measurement. We introduced solutions containing menthol and 2-aminoethoxydiphenyl borate (2-APB) at variable concentration to bilayers during ensemble TRPM8 measurements, enabling us to efficiently and repeatedly measure an IC50 value of 4.9 μM for 2-APB and an EC50 value of 111.8 μM for menthol, both in agreement with literature. These developments indicate that artificial bilayers may play an expanded role in future ion channel studies, including drug discovery and safety screening.