Down- to up-state transition is the default pathway in TREK K2P channel activation and does not involve a lipid occluded pore

Two crystallographic states of mechanosensitive TREK/TRAAK K2P channels - a low-activity down-state and a high-activity up-state - have been proposed to underlie gating, but the origin of the low activity remains debated. Competing models suggest either lipid-mediated pore block or selectivity filter (SF) inactivation. Using systematic mutagenesis of M2/M4 helices, we identified 16 highly active mutants and assessed their activation mechanisms via free-energy calculations, molecular dynamics simulations, and a state-dependent pharmacological probe. The computational approaches reliably predicted mutation-induced shifts in the down-up equilibrium. We further show that intracellular acidification and regulatory lipids primarily stabilize the up-state, consistent with stretch, temperature, and dephosphorylation. These findings support the down-up transition as the principal physiological activation pathway and suggest that mechanosensitivity arises from the larger membrane footprint of the up-state. Our data argue against a physiological role of a lipid-blocked pore and instead support gating via conformational control of the SF in TREK/TRAAK channels.