Structural Determinants For Selectivity Of The Positive Kca Channel Gating Modulator, Ska-121

Small-conductance (KCa2) and intermediate-conductance (KCa3.1) calcium-activated K+ channels are voltage-independent and share a common calcium/calmodulin mediated gating mechanism. Existing positive gating modulators like EBIO, NS309 or SKA-31 activate both KCa2 and KCa3.1 channels with similar potency. We recently performed a structure activity relationship (SAR) study with the aim of optimizing the benzothiazole pharmacophore of SKA-31 towards KCa3.1 selectivity and have identified SKA-121 (5-methylnaphtho[2,1-d]oxazol-2-amine), which displays 41-fold selectivity for KCa3.1 (EC50 109 nM ± 14 nM) over KCa2.3 (EC50 4.4 ± 1.6 μM). SKA-121 is a typical positive-gating modulator, which shift the calcium-concentration response curves of KCa3.1 and KCa2.3 to the left but also increases open probability at saturating Ca2+ concentrations. In order to understand why introduction of a “simple” CH3 group in 5-position of the benzothiazole/oxazol system could achieve such a significant gain in selectivity for KCa3.1 over KCa2.3 we are currently performing site-directed mutagenesis of the calmodulin binding domain (CaMBD) guided by the crystal structure of the KCa2.2 CAMBD and structural modeling. We used the RosettaLigand method to generate models of SKA-121 binding to both the KCa3.1 and KCa2.3 C-terminal CaMBD in complex with calmodulin and then mutated channel residues suggested to be within 4A of SKA-121. Based on the results of the mutagenesis and the model we hypothesize that an extensive hydrogen bond network is responsible for the selectivity of SKA-121 for KCa3.1 over KCa2.3. The NH2 group of SKA-121 forms hydrogen bonds with M51 and E54 on calmodulin. E54 is part of a hydrogen bond network including R362, E295 and N300 of the KCa3.1 channel. Mutation of R362 to S breaks this hydrogen bond network and significantly reduces SKA-121 activity on KCa3.1.Supported by U54NS079202 and T32-HL086350 from NIH.