BPS2026 – MD simulation reveals ion-specific modulation of ligand-induced TRPA1 activation by PIP2

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal ion channel that senses noxious stimuli and contributes to pain, inflammation, and disease pathology. Although phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is known to modulate multiple TRP channels, its binding pocket on TRPA1 remains unknown and its functional role is controversial, with reports of both inhibition and activation. Using a systematic search docking and induced-fit docking (IFD), we identified a putative PIP2 binding pocket at the interface of S3, the S4-S5 linker, and the interfacial helix (IFH) of the adjacent subunit. This site enables the negatively charged phosphate headgroups to form salt bridges and hydrogen bonds with basic residues, while the acyl tails extend into the transmembrane domain. We then employed large-scale all-atom molecular dynamics simulations to investigate how PIP 2 influences ligand-induced TRPA1 activation under conditions that either Na + or Ca 2+ is the permeating ion. Our simulations revealed ion-specific dual effects—in Na + , PIP 2 -enhanced, ligand-induced activation by stabilizing the S4-S5 linker and promoting Na + permeation; In contrast, in Ca 2+ , PIP 2 -suppressed activation by disrupting the D915-R919 salt bridge and restricting Ca 2+ flux. PIP 2 binding was stabilized by distinct residue networks in Na + vs. Ca 2+ conditions, suggesting allosteric mechanisms dependent on ionic context. Moreover, we observed an alternative ion permeation pathway between S5 and S6, associated with helix packing stability. Blue light-activated CRY2-pseudojanin/ CIBN PIP 2 depletion experiments further validated our simulations—in Na + environments, PIP 2 was required for both activation and sustained opening of TRPA1 by GNE551, while in Ca 2+ environments, PIP 2 promoted agonist-induced desensitization. These findings strongly support our model of dual, context-dependent modulation of TRPA1 gating by PIP 2 . Together, this work advances mechanistic understanding of TRPA1 regulation and highlights PIP 2 as a dynamic lipid cofactor shaping ligand-dependent channel activity.