UC Davis

Intermediate-conductance (K_Ca3.1) and small-conductance (K_Ca2) calcium-activated K⁺ channels are gated by calcium binding to calmodulin (CaM) molecules associated with the calmodulin-binding domain (CaM-BD) of these channels. The existing K_Ca activators, such as naphtho[1,2-d]thiazol-2-ylamine (SKA-31), 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309), and 1-ethylbenzimidazolin-2-one (EBIO), activate both channel types with similar potencies. In a previous chemistry effort, we optimized the benzothiazole pharmacophore of SKA-31 toward K_Ca3.1 selectivity and identified 5-methylnaphtho[2,1-d]oxazol-2-amine (SKA-121), which exhibits 40-fold selectivity for K_Ca3.1 over K_Ca2.3. To understand why introduction of a single CH₃ group in five-position of the benzothiazole/oxazole system could achieve such a gain in selectivity for K_Ca3.1 over K_Ca2.3, we first localized the binding site of the benzothiazoles/oxazoles to the CaM-BD/CaM interface and then used computational modeling software to generate models of the K_Ca3.1 and K_Ca2.3 CaM-BD/CaM complexes with SKA-121. Based on a combination of mutagenesis and structural modeling, we suggest that all benzothiazole/oxazole-type K_Ca activators bind relatively "deep" in the CaM-BD/CaM interface and hydrogen bond with E54 on CaM. In K_Ca3.1, SKA-121 forms an additional hydrogen bond network with R362. In contrast, NS309 sits more "forward" and directly hydrogen bonds with R362 in K_Ca3.1. Mutating R362 to serine, the corresponding residue in K_Ca2.3 reduces the potency of SKA-121 by 7-fold, suggesting that R362 is responsible for the generally greater potency of K_Ca activators on K_Ca3.1. The increase in SKA-121's K_Ca3.1 selectivity compared with its parent, SKA-31, seems to be due to better overall shape complementarity and hydrophobic interactions with S372 and M368 on K_Ca3.1 and M72 on CaM at the K_Ca3.1-CaM-BD/CaM interface.