Abstract

Small-conductance (KCa2) and intermediate-conductance (KCa3.1) calcium-activated K+ channels are voltage-independent and share a common calcium/calmodulin mediated gating mechanism. Most existing positive gating modulators like EBIO, NS309 or SKA-31 activate both channels with similar potencies. We previously performed a structure activity relationship study with the aim of optimizing the benzothiazole pharmacophore of SKA-31 towards KCa3.1 selectivity and identified SKA-121 (5-methylnaphtho[2,1-d]oxazol-2-amine), which displays 40-fold selectivity for KCa3.1 over KCa2.3. In order to understand why introduction of a single CH3 group in 5-position of the benzothiazole/oxazol system could achieve such a gain in selectivity for KCa3.1 over KCa2.3 we first localized the binding site of the benzothiazoles/oxazoles to the calmodulin binding domain (CaM-BD)/CaM interface and then used the RosettaLigand computational modeling software to generate models of the KCa3.1 and KCa2.3 CaM-BD/CaM complexes with SKA-121. Based on a combination of mutagenesis and structural modeling we suggest that all benzothiazoles/oxazoles-type KCa activators bind relatively "deep" in the CaM-BD/CaM interface and hydrogen-bond with E54 on CaM. In KCa3.1 SKA-121 forms an additional hydrogen-bond network with R362. NS309 in contrast sits more "forward" and directly hydrogen-bonds with R362 in KCa3.1. Mutating R362 to serine, the corresponding residue in KCa2.3 significantly reduces the potency of SKA-121 suggesting that R362 is responsible for the generally greater potency of KCa activators on KCa3.1. The increase in SKA-121's KCa3.1 selectivity compared with its parent SKA-31 seems to be due to better overall shape complementarity and hydrophobic interactions with S372, M368 on KCa3.1 and M72 on CaM at the KCa3.1-CaM-BD/CaM interface.