The 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.

R 型电压门控 Ca ²⁺ (Ca _v ) 通道 Ca _v 2.3 在神经元和神经内分泌细胞中广泛表达，是治疗疼痛、癫痫、癫痫和帕金森病的潜在药物靶点。尽管它们具有生理重要性，但缺乏针对这些通道的选择性小分子抑制剂。高分辨率结构可能有助于合理的药物设计。_{在这里，我们报告了人类 Ca v} 2.3 与 α2δ−1 和 β3 亚基复合物的低温电子显微镜结构，总分辨率为 3.1 Å。该结构与 Ca _v 2.2 的结构几乎相同，具有 VSD _II处于关闭状态，其他三个 VSD 处于关闭状态。磷脂酰肌醇 4,5-二磷酸 (PIP2) 分子结合到 VSD _II的界面和紧密闭合的孔域。_{我们还确定了 Ca v} 2.3 突变体的冷冻电镜结构，其中重复 II 中的 Ca _v 2 独特胞质螺旋（指定为 CH2 _II螺旋）被删除。这个突变体，命名为 ΔCH2，仍然保留下 VSD _II，但 PIP2 是不可见的，胞质侧的近膜区域几乎看不到。我们对野生型和 ΔCH2 Ca _v 2.3 的结构和电生理特征表明，CH2 _II螺旋通过收紧胞质近膜区段来稳定通道的失活构象，而 CH2 _{II螺旋对于锁定 VSD II}的向下状态不是必需的。