Genetically engineered rapamycin responsive K2P channels

Establishment of electrical potentials across biological membranes is a universal feature of all cells. Tandem pore domain (K2P) potassium ion channels play pivotal roles in maintaining cellular membrane potentials, shaping physiological responses across a diverse range of cell types. With only a limited repertoire of high-aAinity and subtype-selective K2P modulators available for experimental or therapeutic use, we devised a strategy to genetically engineer K2P channels that are potently activated by rapamycin or non-immunomodulatory rapamycin analogs. Insertion of the FRB domain of mTOR into a short flexible cytoplasmic loop between the second and third transmembrane (TM) domains of the TREK1 K2P channel yielded fusion channels that are activated by nanomolar concentrations of rapamycin. Rapamycin-induced potentiation requires recruitment of an FKBP binding partner, from either the endogenous pool of FKBP within the cell or through fusion of FKBP to the C-terminus of TREK1. Formation of an FRB/rapamycin/FKBP ternary complex within the core of the TREK1 channel leads to an increase in TREK1 single-channel open probability and unitary current, mimicking positive modulatory eAects of conventional TREK1 activating cues. Cryo-EM structures demonstrate that rapamycin-induced ternary complex formation rigidifies the position of the FRB and stabilizes the TM2/TM3 loop in an active channel conformation. We demonstrate that FRB fusion can be employed to successfully activate several K2P channel isoforms, providing chemogenetically targetable tools for direct manipulation of cellular membrane potential.