Identification of a Modulatory Site of Action for the Volatile Anesthetic Isoflurane in TREK1 Tandem Pore Potassium Channels

Many members of the K2P family of potassium channels are modulated by volatile anesthetic (VA) agents. While individual residues within a subset of K2P channels are known to be important for anesthetic modulation, those actually contributing to the VA binding site have not yet been revealed. Using a validated photoaffinity analog of the haloether VA isoflurane (azi-isoflurane), we identified a VA binding site within the TREK1 K2P channel. Azi-isoflurane was reacted with recombinantly expressed, purified and reconstituted TREK1 protein, and modified residues were detected by liquid chromatography-tandem mass spectrometry. Two TREK1 residues are modified by Azi-isoflurane: a glycine in the inner helix bundle crossing region of TM2 and a membrane exposed residue in the TM2/TM3 loop. Protection from azi-isoflurane modification by photolabeling in the presence of 100-fold excess isoflurane was only observed for the inner helix bundle glycine, suggesting that the parent VA isoflurane specifically binds in this region. Two electrode voltage clamp studies on TREK1-expressing Xenopus leavis oocytes demonstrate that mutagenesis of the identified inner helix glycine to tryptophan causes a marked increase in channel activity and eliminates TREK1 anesthetic sensitivity. In molecular dynamics simulations, an azi-isoflurane molecule placed in the TREK1 inner helix bundle region makes stable contacts with this glycine as well as a TM3 residue known to modulate anesthetic responsiveness of TASK K2P channels. Mutating the glycine to tryptophan substantially decreases azi-isoflurane binding affinity, as calculated using free energy perturbation. Future work combining photoaffinity labeling, functional characterization, and MD simulation will aim to understand the mechanism by which anesthetic binding at this region leads to channel activation, whether all VAs share this modulatory site, and whether the identified modulatory site is shared by all anesthetic sensitive K2P channels.