A chemical perturbation scan on a K2P channel modulator pocket identifies affinity hotspots
- 1Max Planck Institute for multidisciplinary science
Abstract
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.
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BibTeX
@misc{mendezotalvaro2026chemical,
title = {{A chemical perturbation scan on a K2P channel modulator pocket identifies affinity hotspots}},
author = {E. Mendez-Otalvaro and B. de Groot},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.13.758835},
url = {https://www.biorxiv.org/content/10.64898/2026.10.13.758835v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A chemical perturbation scan on a K2P channel modulator pocket identifies affinity hotspots
AU - E. Mendez-Otalvaro
AU - B. de Groot
PY - 2026
DA - 2026/10/13
PB - bioRxiv
DO - 10.64898/2026.10.13.758835
UR - https://www.biorxiv.org/content/10.64898/2026.10.13.758835v1
AB - 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.
ER -