Structural basis for heteromeric assembly and subthreshold activation of human M-channel

The M-channel, a heterotetrameric voltage-gated potassium channel formed by KCNQ2 and KCNQ3 subunits, critically regulates neuronal excitability, with dysfunction linked to epilepsy and developmental encephalopathies. Despite its physiological importance, structural mechanisms governing its unique heteromeric assembly and subthreshold gating have remained unresolved. We present cryo-EM structures of human M-channels revealing unprecedented stoichiometric plasticity, with all possible KCNQ2:KCNQ3 configurations (1:3 to 3:1) observed. Electrophysiology of engineered concatemers shows these assemblies recapitulate native function. Structural analyses uncover that KCNQ3’s voltage-sensing domain (VSD) adopts a more depolarized conformation than KCNQ2, explaining its signature subthreshold activation. Leveraging these insights, we developed CLM142, a structure-guided activator with 10-fold greater potency and specificity than withdrawn retigabine. CLM142 enabled open-state structure determination, revealing how PIP2 binding couples VSD movement to pore opening. Our work provides an atomic-resolution framework for understanding M-channel’s unique assembly, physiology, disease mechanisms, and targeted therapeutic design.