A new negative allosteric modulator, AP14145, for the study of small conductance calcium-activated potassium (K_Ca 2)...

Small conductance calcium-activated potassium (K_Ca 2) channels represent a promising atrial-selective target for treatment of atrial fibrillation. Here, we establish the mechanism of K_Ca 2 channel inhibition by the new compound AP14145.

Using site-directed mutagenesis, binding determinants for AP14145 inhibition were explored. AP14145 selectivity and mechanism of action were investigated by patch-clamp recordings of heterologously expressed K_Ca 2 channels. The biological efficacy of AP14145 was assessed by measuring atrial effective refractory period (AERP) prolongation in anaesthetized rats, and a beam walk test was performed in mice to determine acute CNS-related effects of the drug.

AP14145 was found to be an equipotent negative allosteric modulator of K_Ca 2.2 and K_Ca 2.3 channels (IC₅₀  = 1.1 ± 0.3 μM). The presence of AP14145 (10 μM) increased the EC₅₀ of Ca²⁺ on K_Ca 2.3 channels from 0.36 ± 0.02 to 1.2 ± 0.1 μM. The inhibitory effect strongly depended on two amino acids, S508 and A533 in the channel. AP14145 concentration-dependently prolonged AERP in rats. Moreover, AP14145 (10 mg·kg^-1 ) did not trigger any apparent CNS effects in mice.

AP14145 is a negative allosteric modulator of K_Ca 2.2 and K_Ca 2.3 channels that shifted the calcium dependence of channel activation, an effect strongly dependent on two identified amino acids. AP14145 prolonged AERP in rats and did not trigger any acute CNS effects in mice. The understanding of how K_Ca 2 channels are inhibited, at the molecular level, will help further development of drugs targeting K_Ca 2 channels.