K2P potassium channels play critical roles in maintaining resting membrane potential, conducting background potassium leak currents across a range of cell types. Modulation of K2P channel activity alters cellular excitability by dictating action potential firing thresholds, contributing to the fine-tuning of physiological responses. As the repertoire of high affinity and subtype selective K2P modulators is limited, we created a set of genetically engineered K2P channels that respond to rapamycin or non-immunomodulatory rapamycin analogs. To accomplish this, we inserted the FRB domain of mTOR into a short flexible cytoplasmic loop that links the TM2 and TM3 helices of the TREK1 K2P channel. This TREK1/FRB fusion was functionally indistinguishable from TREK1 WT channels but exhibited an 8-fold activation after administration of nanomolar concentrations of rapamycin. We demonstrate that rapamycin-induced potentiation of the TREK1/FRB channel requires recruitment of an FKBP binding partner, either from the endogenous pool of FKBP within the cell or via fusion of FKBP to the c-terminus of TREK1. Electrophysiological properties of the TREK1/FRB fusion indicate that formation of the FRB/rapamycin/FKBP ternary complex within the TREK1 TM2/TM3 loop leads to a conformational movement of the TREK1 TM4 helix that mimics conventional TREK1 activation gating. Cryo-EM structural studies demonstrate that the positioning of the FRB domain within the TREK1 TM2/TM3 loop is flexible in the absence of rapamycin but becomes rigidified after formation of the FRB/rapamycin/FKBP ternary complex, suggesting a mechanism by which rapamycin binding results in conformational changes that gate the TREK1 channel. We show that this FRB fusion approach can be generalized to successfully activate multiple additional K2Ps, providing chemo-genetically targetable tools for the study of K2P physiology in vivo and a new class of DREADDs that can be utilized to manipulate cellular membrane potential.
Khajoueinejad et al. (2026) studied this question.