Insights into the structure and modulation of human TWIK-2

The Tandem of pore domain in a Weak Inward Rectifying K⁺ 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. Despite its physiological relevance, the structure, regulatory mechanisms, and selective modulators of TWIK-2 remain largely unknown. Here, we present a 3.7 Å single particle cryo-electron microscopy structure of human TWIK-2 and highlight its conserved and distinctive 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 site-directed mutagenesis and provide insights into the possible lipid-mediated 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- anti-inflammatory therapeutics.