Single-Channel Electrophysiology Using a Miniaturized Montal-Mueller Planar Lipid Bilayer System for Recording a Synthetic K2P Channel.

The functional reconstitution of ion channels into planar lipid bilayers provides a strategy to investigate channel activity at the single-molecule level. However, conventional Montal-Mueller bilayer systems typically require bulky experimental setups, manual chamber assembly, and relatively large recording volumes, which increase reagent consumption and may introduce additional sources of electrical noise due to extended wiring and external headstages. Here, we present a miniaturized, ready-to-use bilayer platform (BLMchipMM) integrated with a compact amplifier that enables stable single-channel recordings with reduced reagent consumption and simplified operation. This protocol describes the use of this system for performing single-channel electrophysiology measurements. Using this system, we reconstituted a synthetic K2P channel (ancTREK1) and used it as a proof-of-principle test protein to validate reconstitution and single-channel recording. Single-channel recordings show a current-voltage relationship in Mg2+-free symmetric K+ solutions, with conductance values consistent with those reported for TREK1 channels. The successful reconstitution of this K2P-like channel demonstrates the utility of this system for single-channel analysis in artificial membranes.