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.

Motor cortex analogue neurons in songbirds utilize Kv3 channels to generate ultranarrow spikes

“…Whereas pharmacological inhibitors provided strong evidence of a major role for Kv3.x channels in RAPNs compared to AId neurons ( Figures 4 — 6 ), they notably lack the specificity for Kv3.x subunits. In contrast, the specific ( Covarrubias et al, 2023 ), novel positive Kv3.1/3.2 modulator, AUT5, potentiates Kv3.1 currents by speeding up activation kinetics ( Taskin et al, 2015 ) and leftward shifting the voltage dependence of activation ( Taskin et al, 2015 ; Covarrubias et al, 2023 ). To examine AUT5 effects, we recorded both spontaneous ( Figure 8A and C , left) and evoked ( Figure 8B and D , left) APs from RAPNs and AId neurons in the whole-cell current-clamp configuration, before and during exposure to 1 µM AUT5 (EC 50 = 1.3 µM, Taskin et al, 2015 ).…”

“…Using the Kv3.1/3.2 positive modulator AUT5, we were able to further correlate the differential expression of Kv3.1 subunits with excitable properties in RAPNs and AId neurons. AUT5 is known to alter gating kinetics, and leftward shift the voltage dependence of activation of Kv3.1 channels ( Boddum et al, 2017 ; Covarrubias et al, 2023 ; Taskin et al, 2015 ). The narrowing of the AP waveform and increase in steady-state firing provide support for a stronger contribution of Kv3.1 in the repolarization of APs and in increasing the availability of Nav channels during high-frequency firing in RAPNs versus AId neurons.…”