The Ca_v3.2 subtype of T-type calcium channels has been targeted for developing analgesics and anti-epileptics for its role in pain and epilepsy. Here we present the cryo-EM structures of Ca_v3.2 alone and in complex with four T-type calcium channel selective antagonists with overall resolutions ranging from 2.8 Å to 3.2 Å. The four compounds display two binding poses. ACT-709478 and TTA-A2 both place their cyclopropylphenyl-containing ends in the central cavity to directly obstruct ion flow, meanwhile extending their polar tails into the IV-I fenestration. TTA-P2 and ML218 project their 3,5-dichlorobenzamide groups into the II-III fenestration and place their hydrophobic tails in the cavity to impede ion permeation. The fenestration-penetrating mode immediately affords an explanation for the state-dependent activities of these antagonists. Structure-guided mutational analysis identifies several key residues that determine the T-type preference of these drugs. The structures also suggest the role of an endogenous lipid in stabilizing drug binding in the central cavity.

T 型钙通道的Ca _v 3.2 亚型因其在疼痛和癫痫方面的作用而成为开发镇痛药和抗癫痫药的目标。在这里，我们展示了 Ca _v 3.2 单独以及与四种 T 型钙通道选择性拮抗剂复合的冷冻电镜结构，总体分辨率范围为 2.8 Å 至 3.2 Å。这四种化合物显示出两种结合姿势。 ACT-709478和TTA-A2都将其含有环丙基苯基的末端放置在中央空腔中以直接阻碍离子流，同时将其极性尾部延伸到IV-I开窗中。 TTA-P2 和 ML218 将其 3,5-二氯苯甲酰胺基团投射到 II-III 开窗中，并将其疏水尾部置于空腔中以阻止离子渗透。开窗穿透模式立即为这些拮抗剂的状态依赖性活动提供了解释。结构引导突变分析确定了决定这些药物 T 型偏好的几个关键残基。这些结构还表明内源性脂质在稳定中央腔内药物结合方面的作用。