N-type voltage-gated calcium (Ca_V) channels mediate Ca²⁺ influx at the presynaptic terminals in response to action potential and play vital roles in synaptogenesis, neurotransmitter releasing, and nociceptive transmission. Ziconotide is a N-type calcium channel blocker and was regarded as an analgesic agent for the amelioration of chronic pain. Here we elucidate a cryo-electron microscopy (cryo-EM) structure of the apo and ziconotide-bound human Ca_V2.2 complex at near-atomic resolution. This complex structure reveals how the Ca_V2.2, β1, and α2δ1 subunits are assembled, and unraveled the novel binding fashion of ziconotide to Ca_V2.2. In our structures, the second voltage-sensing domain (VSD) is trapped by a PIP₂ molecule and stabilized at a resting-state conformation, which is distinct from the other three VSDs of Ca_V2.2 as well as activated VSDs observed in previous structures of Ca_V channels. The structures also shows that the intracellular gate formed by S6 helices is closed, and a W-helix from the DII-III linker is determined to act as a blocking-ball that causes closed-state inactivation in Ca_V2.2. The ziconotide is docked above the selectivity filter of Ca_V2.2, harbored by the negative-charged extracellular doom fabricated by α2δ1, and thus blocks the entrance of calcium ions. Collectively, our structure provides previously unseen structural insights into fundamental gating mechanisms of Ca_V channels and demonstrated the molecular basis of the analgesic effect of ziconotide.

N-type CaV channel;Gating mechanism;Pore-blocker;Ziconotide;Closed-state inactivation