Investigating the structure and pharmacology of SK channels through molecular docking and in vitro patch clamp

Small conductance calcium-activated potassium (SK) channels are gated by intracellular Ca2+ via calmodulin molecules. Three isoforms (SK1-3) have been identified, showing different but overlapping tissue expression in the central nervous system. SK1 and SK2 proteins display considerable overlap in cortex and hippocampus while SK3 expression is higher in the monoaminergic cell regions. SK channels play an important role in neuronal excitability by modulating the firing rate and firing pattern of neurons, and represent potential targets for the treatment of various CNS disorders. Therefore, the development of new nonpeptidic blockers combining high affinity and selectivity towards SK2 or SK3 channels (and possibly versus one another) is crucial and requires a better knowledge of the structural features essential to the affinity of these ligands. In this project, we aimed to better understand the interaction between SK channels and the archetypical blocker apamin by studying the 3D structures of SK proteins and their activity. From models obtained using AlphaFold, we observed a particular conformation of the S3-S4 loop in SK1-3, which does not seem to be present in IK. Furthermore, in the first three subtypes, we observed the presence of a phenylalanine residue in this loop that appears to be located just outside the channel pore and could play a key role in the interaction with apamin. To validate this hypothesis, we generated different mutants of this phenylalanine in SK2 and SK3 subunits and studied their sensitivity to apamin and UCL1684 by coupling molecular docking to in vitro patch-clamp experiments.