Insights into the structure and modulation of human TWIK-2

The<underline>T</underline>andem of pore domain in a<underline>W</underline>eak Inward<underline>R</underline>ectifying<underline>K</underline>⁺channel 2(TWIK-2; KCNK6) is a member of the Two-Pore Domain K⁺(K_2P) channel family, which is associated with pulmonary hypertension, lung injury, and inflammation. The structure and regulatory mechanisms of TWIK-2 remain largely unknown. Here, we present the cryo-electron microscopy (cryo-EM) structure of human TWIK-2 at ∼3.7 Å and highlight its conserved and unique features. Using automated whole-cell patch clamp recordings, we demonstrate that gating in TWIK-2 is voltage-dependent and insensitive to changes in the extracellular pH. We identify key residues that influence TWIK-2 activity by employing structure and sequence-guided site-directed mutagenesis and provide insights into the possible mechanism of TWIK-2 regulation. Additionally, we demonstrate the application of high-throughput automated whole-cell patch clamp platforms to screen small molecule modulators of TWIK-2. Our work serves as a foundation for designing high-throughput small molecule screening campaigns to identify specific high-affinity TWIK-2 modulators, including promising new anti-inflammatory therapeutics.