电压门控钙离子通道（Cav）属于电压门控离子通道超家族，分布于细胞膜上，通过响应膜电势的变化控制钙离子的跨膜运输，其功能异常往往导致癫痫、心律不齐等疾病，是重要的制药靶点。Cav配体分子中最为经典的可分为1,4-dihydropyridine （DHP）、benzothiazepine（BTZ）和phenylalkylamine（PAA）三类，其中大量分子已作为药物应用于临床，或被广泛用于Cav性质研究。尽管配体分子的应用已有数十年历史，但它们与Cav间的具体作用机理尚待阐述。本研究对兔源Cav1.1进行了内源提取，通过分别添加DHP类药物分子nifedipine，DHP类激活剂分子Bay K 8644，BTZ类药物分子diltiazem或PAA类药物分子verapamil获得了复合物样品，并利用单颗粒冷冻电镜技术分别解析了这些复合物的结构，整体分辨率分别为2.9 ?，2.8 ?，3.0 ?和2.7 ?。 通过结构分析与比对可以发现，DHP类拮抗剂分子与激活剂分子结合在相同的位点，位于同源结构域III和IV之间的窗口处，且在本研究工作中均与失活状态的离子通道相结合，两个结构中的离子通道呈现出相同的构象。二者间的不同点表现为对应复合物的稳定性不同。拮抗剂分子nifedipin的结合较激活剂分子Bay K 8644更加稳定，可以通过将Cav稳定在失活状态而抑制其通道活性；而激活剂分子Bay K 8644将其亲水基团伸至离子通道的疏水口袋中，使复合物处于一种半稳定状态 (metastable)。本研究中使用的高浓度配体分子使这种半稳定状态的复合物可以保持完整而不会发生解离，从而能在结构解析当中被捕获，这为DHP类激活剂分子在高浓度下表现出拮抗剂的性质提供了结构方面的解释。通过综合分析序列比对结果以及相应氨基酸在结构中的位置与作用，DHP类分子与L-型电压门控钙离子通道间特异性结合的分子机制也得到了阐述。BTZ类分子diltiazem和PAA类分子verapamil均结合在离子透过路径当中，通过阻断离子通道介导的钙离子流来抑制离子通道的活性。Verapamil分子在本研究中呈现出两种不同的结合方式，其中一种的结合位点与diltiazem结合位点有重合。本研究使用结构生物学的手段分析并阐释了三类应用于临床的药物分子和一类经典的DHP类激活剂分子对真核生物L-型电压门控钙离子通道进行功能调控的分子机理，为后续的药物研发提供了重要的理论支持。
Voltage-gated calcium channels (Cav) belong to the voltage-gated ion channel superfamily. Cav channels are located on plasma membrane, responding to changes in the membrane potential and regulating calcium transportation across plasma membrane. Dysfunction of Cav channels can lead to diseases including epilepsy, cardiac arrhythmia, etc. Considering the important physiological functions Cav channels take, they represent important drug targets. Cav channels could be regulated by several ligands, of which the most archetyple ones being classified into three families, namely 1,4-dihydropyridine (DHP), benzothiazepine (BTZ) and phenylalkylamine (PAA), and several members within the three families have been widely applied for treatments for diseases and Cav channels’ function studies. Although the ligand molecules have been used for decades, the precise molecular mechanism of their modulation on L-type Cav channels remains to be elucidatedIn this study, we purified endogenous Cav1.1 complex and reconstituted the protein-drug complex in vitro. Cav1.1 was purified from New Zealand white rabbit (Oryctolagus cuniculus). By separately incubating the purified protein complex with a widely used DHP drug molecule nifedipine, an archetypal DHP agonist Bay K 8644, a BTZ drug molecule diltiazem, and a PAA drug molecule verapamil, we successfully obtained samples of Cav1.1 in complex with the respective ligands, and solved their cryo-EM structures using single particle analysis to overall resolutions of 2.9 ?, 2.8 ?, 3.0 ?, and 2.7 ?, respectively.Detailed analyses and comparison of these structures reveal molecular determinants of ligand binding, and unveil the molecular mechanism underlying ligand regulation.The DHP antagonist and agonist molecules share the same binding site located in the fenestration site between repeats III and IV. Cav1.1 in both structures show nearly identical conformation and are both in the inactivated state. The major difference between the two structures emerges from the different stability of the two complexes. The binding between nifedipine and Cav1.1 is relatively stable compared to its agonist counterpart, and the channel activity is inhibited owing to the stabilization of the inactivated state, while the DHP agonist Bay K 8644 protrudes an electron withdrawing group into the hydrophobic pocket. This kind of hydrophobicity mismatch renders the complex to be in a metastable state despite other interactions between the ligand and the protein largely unaltered. As the concentration of Bay K 8644 increases, the association-disassociation equilibrium of the metastable complex is shifted towards association. The high concentration used in this study makes it possible for the complex to be kept without dissociation. This also explains the observation that with high concentration, DHP agonists convert into antagonists. Diltiazem and verapamil are both pore blockers and bind in the central cavity of the pore domain, directly blocking the ion permeation pathway. It is noteworthy that two binding sites of verapamil are identified, one of which partially overlaps with that of diltiazem. This study elucidates high-resolution structures of Cav1.1 bound to three drug molecules and one archetypal DHP agonist, revealing molecular basis for the modulation of L-type voltage-gated calcium channels by these ligands. These analyses shed light on future structure-guided drug developments.
