New insight into the modulatory role of the S3-S4 loop in SK2/3 potassium channels

Small-conductance calcium-activated potassium (SK/KCa2) channels selectively allow the passage of K+ ions and are activated by Ca2+ through interactions with calmodulin. There are three known isoforms (SK1-3 ; encoded by the KCNN1-3 genes), which have distinct but overlapping patterns of tissue distribution, particularly in the heart and central nervous system. SK channels play a crucial role in regulating neuronal excitability by controlling the firing rate and patterns of neurons. Due to their physiological roles and distribution, they are potential targets for treating various disorders, including schizophrenia, depression, Alzheimer’s and Parkinson’s diseases, as well as atrial fibrillation. Although blockers like apamin and UCL-1684 already exist, their use in therapy is limited by a narrow therapeutic window and poor selectivity towards SK subtypes. Consequently, developing new non-peptidic blockers with both high affinity and selectivity for SK2 or SK3 channels is essential. This requires a better knowledge of the structural elements that influence ligand interactions.
In this project, our goal is to gain a deeper understanding of how SK channels interact with the well-known blocker apamin by exploring their 3D structures. Using models generated with AlphaFold2, we identified a unique conformation of the S3S4 loop in SK1, SK2, and SK3, with a conserved phenylalanine pointing toward the exit of the channel, which may play a critical role in the interaction with several blockers, including apamin. To explore this hypothesis, we created various mutants of this phenylalanine in SK2 and SK3 channels and examined their activity and sensitivity to apamin and UCL-1684. Through a combination of in vitro patch-clamp and binding experiments, we showed that the mutation of phenylalanine to alanine disrupts the interaction with both compounds while the mutation to tyrosine has no major impact. Molecular docking experiments allowed us to generate highly confident models of the interactions of SK1-3 with these two blockers. The results obtained in this study may be useful in the research and development of new pharmacophores.