Miniaturized and High-Throughput Approaches for Measuring Ion Channel Function and Ligand Activity

Ion channels are integral membrane proteins known to regulate various physiological processes, including cell signaling, proliferation, secretion and membrane potential, by actively mediating ion transport across the membrane. Over years of ion channel research, numerous methodologies have been established to evaluate their functional characteristics or their modulation by ligands. These approaches can be classified into two main categories, i.e., electrophysiological and non-electrophysiological methods. Electrophysiological techniques have been extensively used to assess the biophysical properties of ion channels, as they represent the only method that can directly measure current flow through the channel. Among the available electrophysiological methods, the Planar Lipid Bilayer (PLB) technique excels for analyzing ion channel behavior at the single-molecule level enabling experiments under highly controlled conditions. On the other hand, non-electrophysiological methods, which include cell-based high-throughput screening (HTS) technologies, measure the channel activity indirectly. Fluorescence-based methods are widely used and are based on ion-specific fluorescent probes sensitive to intracellular ion concentrations. Thus, changes in fluorescence signals caused by ion concentration modifications due to ionic flux reflect how the absence or presence of specific compounds can modulate ion channel activity. In this work, both approaches have been evaluated and optimized. The PLB technique was used for the single-channel characterization of a novel synthetic two-pore domain potassium (K2P) channel, namely ancestral TREK1, or ancTREK1. This protein represents the inferred ancestral form of the modern TREK1 (TWIK-related K+ channel 1), reconstructed through the phylogenetic comparison of TREK-family sequences across a broad range of vertebrate species. Measurements were performed after protein reconstitution into Black Lipid Membranes (BLMs) formed via Montal-Mueller (folding) method using a miniaturized electrophysiological device developed by Elements S.r.l. By contrast, a fluorescence-based proton-flux assay was used as a high-throughput screening (HTS) platform to perform preliminary experiments on ligand-dependent activity modulation of hHV1 (human voltage-gated proton channel 1). The main characteristic of this channel is to allow protons (H+) flux across the membrane in a perfectly selective way. This distinctive property makes this channel suitable for the use of a pH-sensitive fluorescent probe which can easily detect pH modifications due to proton flux-mediated by hHV1 across the cell membrane. This could potentially enable large-scale screening for putative compounds targeting this proton channel. Overall, this work shows how both miniaturized approaches can be valuable resources to evaluate ion channel activity. Specifically, fluorescence-based methods as a helpful platform to speed up the screening process of large libraries of molecules; and PLB, combined with advanced miniaturized systems, like the one presented in this work, as an advantageous tool for ion channel single-molecule characterization.