A chemical perturbation scan on a K2P channel modulator pocket identifies affinity hotspots

Ion permeation in the two-pore-domain K+ (K2P) channel TREK1 is gated largely at the selectivity filter (SF), by a C-type-like inactivation mechanism involving distortions of the SF structure; and by a carbonyl flip, which disrupts K+ coordination. Both are prevented by a small-molecule activator bound adjacent to the SF, in a site termed the modulator pocket; yet which pocket regions carry the binding signal, and whether they couple to gating, is unclear. We use 46 chemical substitutions on the activator ML335, evaluated by non-equilibrium alchemical free-energy calculations, as an affinity perturbation scan on the lower- and upper pocket. The response is strongly asymmetric: Lower-ring substitutions impair binding, which we trace to a {pi}-{pi} stacking network connecting the M3, P2, P1 helices, and the SF. Conversely, upper-ring substitutions are tolerated and reshape contacts with the Cap-P1 and SF2-M4 loops; and with the upper SF. The identified affinity hotspots overlap with residues independently implicated in TREK gating by external cues such as pH, temperature, pressure, and cytosolic-sensor transduction, suggesting that high-affinity pocket sites might also shape the gating landscape.