Mechanisms of intracellular pH gating and local anesthetic inhibition of the two-pore domain K+ channel TASK-2.

TWIK-related acid-sensitive K+ channel 2 (TASK-2) is a pH sensing two-pore K+ (K2P) channel that regulates respiration in brainstem neurons and systemic pH homeostasis in kidney. Despite its physiological importance and implication in disease, the molecular basis for intracellular gating and pharmacological inhibition of TASK-2 is incompletely understood. Here, we combine cryo-EM and single-channel electrophysiology to resolve the mechanisms of TASK-2 gating by intracellular protons and inhibition by the anesthetic bupivacaine. We show protonation of intracellular lysines triggers a unique stacked gating mechanism distinct from other K+ channels. Inner helices unwind and domain swap to form a β-zipper, which we call a Z-gate, that seals the cytoplasmic channel entrance. These conformational changes open lateral fenestrations to the membrane that permit lipids to invade the channel cavity and block the pore. Furthermore, we find bupivacaine inhibits the channel by competing with lipids in the cavity site in a state-dependent manner. These results define a paradigm for intracellular gating of K2Ps and provide a structural foundation for designing more potent and selective channel modulators.