AbstractThe R-type voltage-gated Ca²⁺ (Ca_v) channels Ca_v2.3, widely expressed in neuronal and neuroendocrine cells, represent potential drug targets for pain, seizures, epilepsy, and Parkinson’s disease. Despite their physiological importance, there have lacked selective small-molecule inhibitors targeting these channels. High-resolution structures may aid rational drug design. Here, we report the cryo-EM structure of human Ca_v2.3 in complex with α2δ−1 and β3 subunits at an overall resolution of 3.1 Å. The structure is nearly identical to that of Ca_v2.2, with VSD_II in the down state and the other three VSDs up. A phosphatidylinositol 4,5-bisphosphate (PIP2) molecule binds to the interface of VSD_II and the tightly closed pore domain. We also determined the cryo-EM structure of a Ca_v2.3 mutant in which a Ca_v2-unique cytosolic helix in repeat II (designated the CH2_II helix) is deleted. This mutant, named ΔCH2, still reserves a down VSD_II, but PIP2 is invisible and the juxtamembrane region on the cytosolic side is barely discernible. Our structural and electrophysiological characterizations of the wild type and ΔCH2 Ca_v2.3 show that the CH2_II helix stabilizes the inactivated conformation of the channel by tightening the cytosolic juxtamembrane segments, while CH2_II helix is not necessary for locking the down state of VSD_II.