Pharmacological and Biophysical Characterization of the Modulation and Pore Binding-Relevant Features of the Human Twik-Related Spinal Cord K+ (hTRESK) Channels

The human TWIK-related spinal cord K⁺ channel (hTRESK; K_2P18.1) is a member of the two-pore-domain potassium (K_2P) channel family, which regulates cellular excitability by generating background K⁺ currents and stabilizing the resting membrane potential. Despite its physiological and therapeutic relevance, mechanistic understanding of hTRESK modulation has been limited by the absence of a high-resolution structure and the lack of selective pharmacological tools.

In this study, I systematically characterized the pharmacology of hTRESK and identified multiple high-affinity inhibitors as well as a previously unrecognized direct activator, PD-307243. Screening of structurally diverse small molecules and endogenous lipids, including anandamide (AEA), using excised patch recordings from Xenopus laevis oocytes revealed several potent inhibitors, including C101248 and AEA, which inhibited hTRESK with nanomolar affinity. The same approach identified PD-307243 as a novel activator of hTRESK.

To define the molecular basis of ligand interaction, I combined pharmacological competition assays, mutagenesis, and substituted cysteine accessibility method (SCAM)-based protection experiments. Quaternary ammonium (QA⁺) ions, established pore-binding probes in other K_2P channels, competed with all characterized hTRESK modulators, demonstrating that these compounds access a shared region within the channel pore. Notably, PD-307243 also competed with chemically diverse inhibitors, indicating that activators and inhibitors converge on an overlapping interaction site. Furthermore, all tested modulators exhibited altered affinity upon mutation of the pore-lining residue F352, implicating this region as a key determinant of ligand recognition.

SCAM analysis identified three MTSET-accessible pore residues, F145, L148 and F352. Protection experiments demonstrated that representatives of distinct inhibitor classes selectively protected L148 from covalent modification, identifying this residue as being in close spatial proximity to the common modulator interaction region. These findings support the existence of an unselective drug interaction site (UDIS) within the hTRESK pore.