The unique turret region of Kv3 channels governs the mechanism of action of highly specific positive allosteric modulators.

Abstract Small-molecule modulators of diverse voltage-gated K+ (Kv) channels may help treat severe neurological disorders. However, the development of selective modulators requires an understanding of their mechanism-of-action (MoA). We applied an orthogonal approach to elucidate the MoA of an imidazolidinedione derivative (AUT5), which is a highly specific positive allosteric modulator (PAM) of Kv3.1 and Kv3.2 channels. AUT5 modulation involves positive cooperativity and preferential stabilization of the open state. Critically, we found that the unique and highly conserved extracellular turret region of Kv3.1 and Kv3.2 essentially governs AUT5 modulation. Furthermore, leveraging on the cryo-EM structure of Kv3.1a, atomistic blind docking calculations revealed four equivalent AUT5 binding sites near the turrets and between the voltage-sensing and pore domains of the channel’s tetrameric assembly. Therefore, the unique Kv3 turret emerges as a novel structural correlate of the selective MoA of a new class of Kv3 channel PAMs with a therapeutic potential.