Cross-talk of filter gate and lower helices drives polymodal regulation and disease in TREK channels.

The TREK two-pore domain potassium channels regulate membrane excitability and respond to diverse physiological stimuli. Single-site mutations of TREK are associated with neurodevelopmental disorders and can cause pathological channel hyperactivation. Here, using the OneOPES enhanced-sampling molecular dynamics framework, we characterize the energetics of conformational transition along a predefined pathway connecting the up and down states of TREK-2, together with initial conformational rearrangements of the selectivity filter under physiological stimuli and disease-mimicking conditions. The simulations reveal a shared energetic axis coupling the lower helices, proximal C-terminus, and selectivity filter to govern channel gating. Predicted conformational effects of a disease-mimicking mutation were validated electrophysiologically using a conformation-sensitive TREK-2 inhibitor. Together with our previous unbiased MD simulations, these results establish an energetic framework for TREK-2 regulation and dysfunction, complementing prior structural and mechanistic studies while highlighting the potential and methodological challenges of advanced enhanced-sampling approaches for resolving complex conformational landscapes of ion channels.