Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K ⁺ channels for atrial fibrillation treatment

Abstract

The Ca 2+ -activated SK4 K + channel is gated by Ca 2+ –calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K + channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N -lobe interact with the proximal carboxyl terminus and the linker S4–S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM–PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4–S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4–S5, not conserved in SK1–SK3 subunits, thereby conferring selectivity and preventing the Ca 2+ –CaM N -lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K + channels by targeting drugs to the CaM–PIP2-binding domain provides a promising anti-arrhythmic therapy.

Characterization and Chemical Synthesis of Cm39 (α-KTx 4.8): A Scorpion Toxin That Inhibits Voltage-Gated K+ Channel KV1.2 and Small- and Intermediate-Conductance Ca2+-Activated K+ Channels KCa2.2 and KCa3.1

“…Inhibition of K Ca 3.1, especially in combination with targeting K Ca 2.2, can be interesting for influencing atrial fibrillation (AF). It was shown recently that inhibition if K Ca 3.1 or K Ca 2.2 in isolation may reduce AF [ 56 , 57 , 58 ]. It may be an interesting scenario to target both ion channels simultaneously, and based on our results Cm39 would be a suitable candidate for a dual-action drug candidate.…”

KCa3.1 Promotes Proinflammatory Exosome Secretion by Activating AKT/Rab27a in Atrial Myocytes during Rapid Pacing

“…Our previous study showed that blocking KCa3.1 overexpression in canine atrial tissue macrophages using TRAM-34 reduced susceptibility to AF [ 9 ]. Recent studies have shown that the KCa3.1 channel allogenic blocker BA6b9 significantly prolonged the atrial and effective atrioventricular refractory periods in the isolated hearts of rats and reduced AF induction ex vivo [ 17 ]. This evidence supports the proarrhythmic role of KCa3.1.…”

The SK4 channel allosteric blocker, BA6b9, reduces atrial fibrillation substrate in rats with reduced ejection fraction

“…These SK4 K + channels are well known to be expressed by immune cells such as T cells, B cells, and macrophages, as well as by endothelial cells, fibroblasts, and proliferating smooth muscle cells of the vascular system ( 30–33 ). Recently, we demonstrated that the SK4 channel protein is widely expressed in the atria of rat and human hearts and to a lesser extent in the ventricles ( 23 ). A novel allosteric blocker, BA6b9, was designed to interact with the calmodulin-PIP2 binding domain, a previously untargeted region of these SK4 channels, at the specific interface of the proximal C-terminus and the linker S4–S5 domain ( 23 ).…”