Uncovering Structural Determinants of SK2 and SK3 Channel Blockade for Selective Inhibitor Development

Small-conductance calcium-activated potassium (SK/KCa2) channels selectively conduct K+ ions and are activated by Ca2+ via calmodulin molecules. The three known isoforms (SK1–3), predominantly expressed in the central nervous system, are key regulators of neuronal excitability and represent attractive therapeutic targets for neurological disorders such as schizophrenia and depression. Although blockers such as apamin and UCL-1684 are available, their potential is constrained by limited subtype selectivity and a narrow therapeutic window. Therefore, the development of new non-peptidic blockers with improved affinity and selectivity for SK2 or SK3 channels is crucial. This requires a better knowledge of the structural elements that influence ligand interactions.

Recent cryo-EM structures of SK2 revealed a particular conformation of the extracellular S3–S4 loop and identified a phenylalanine residue at its tip as a critical determinant of blocker binding. Building on these findings, we investigated the role of this residue in SK2 and SK3 channels using site-directed mutagenesis. A combination of in vitro patch clamp and binding assays showed that substituting phenylalanine with alanine markedly reduces sensitivity to apamin and UCL-1684, whereas replacement with tyrosine largely preserves channel inhibition. AlphaFold3 structural modeling of the tyrosine mutants, based on the SK2 cryo-EM structure, supports these results, suggesting that tyrosine maintains and even stabilizes the S3–S4 loop conformation. Finally, our phylogenetic analysis revealed that this tyrosine variant appeared naturally in few species and today’s occurrences mainly form a monophyletic group within the Actinopterygii class, suggesting a potential tuning of the channel function in this group.

Overall, our findings support previous structural data on SK2 and extend these insights to SK3, offering valuable guidance for the rational design of subtype-selective SK channel blockers.