Phylogeny of SK channels and functional characterization of the conserved Phe in the S3-S4 loop

Small-conductance calcium-activated potassium channels (K_Ca2.x or SK) are critical regulators of neuronal excitability, and dysregulation of their function is linked to central nervous system disorders, including schizophrenia and depression. Structural analyses of the SK2 channel identified a phenylalanine at the tip of the S3-S4 extracellular loop as a key determinant for channel blockade by apamin and UCL1684. To investigate the functional significance of this residue, we used site-directed mutagenesis to substitute phenylalanine with either alanine or tyrosine in both SK2 and SK3 subtypes. Electrophysiological recordings and ligand-binding assays revealed that the alanine substitution renders both channels insensitive to the two compounds. The phenylalanine-to-tyrosine substitution has no significant impact on the sensitivity of SK channels to UCL1684, indicating a conserved conformation of the S3-S4 loop and the specific low conductance structure of the selectivity filter. The SK3 tyrosine mutant does, however, display a 5-fold reduced sensitivity to apamin, possibly linked to an additional stabilizing hydrogen bond (H-bond) between the tyrosine and the selectivity filter. The tyrosine mutation is not expected to significantly affect K⁺ current according to molecular dynamic simulations. The phylogeny of SK channels (SK1-4) is consistent with a model of vertebrate gene evolution driven by a whole-genome tetraploidization, with the first duplication occurring prior to the last common ancestor of vertebrates, and the second occurring separately after the split between jawless fishes and jawed vertebrates lineages. SK4 channels form a divergent group of paralogs presenting an early loss of the SK characteristic S3-S4 loop. A tyrosine residue represents the most frequent natural substitution of the conserved S3-S4 phenylalanine, and most occurrences are found in a predominantly monophyletic group within the SK2 channel branch. Natural selection may have promoted this substitution by conferring reduced sensitivity to peptidic toxins produced by competing species, as illustrated by the human SK3-apamin interaction.