K(2P) channel C-type gating involves asymmetric selectivity filter order-disorder transitions

K(2P) potassium channels regulate cellular excitability using their selectivity filter (C-type) gate. C-type gating mechanisms, best characterized in homotetrameric potassium channels, remain controversial and are attributed to selectivity filter pinching, dilation, or subtle structural changes. The extent to which such mechanisms control C-type gating of innately heterodimeric K(2P)s is unknown. Here, combining K(2P)2.1 (TREK-1) x-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and electrophysiology, we uncover unprecedented, asymmetric, potassium-dependent conformational changes that underlie K(2P) C-type gating. These asymmetric order-disorder transitions, enabled by the K(2P) heterodimeric architecture, encompass pinching and dilation, disrupt the S1 and S2 ion binding sites, require the uniquely long K(2P) SF2-M4 loop and conserved “M3 glutamate network,” and are suppressed by the K(2P) C-type gate activator ML335. These findings demonstrate that two distinct C-type gating mechanisms can operate in one channel and underscore the SF2-M4 loop as a target for K(2P) channel modulator development.