Docking of μ-conotoxin GIIIA in the sodium channel outer vestibule
Docking of μ-conotoxin GIIIA in the sodium channel outer vestibule
复制标题
DOI：
10.4161/chan.5112
复制
发表时间：
2007-09-01
期刊：
影响因子：
3.3
通讯作者：
Dudley, Samuel C., Jr.
中科院分区：
文献类型：
--
作者：
Choudhary, Gaurav;Aliste, Marcela P.;Dudley, Samuel C., Jr.
mu-芋螺毒素 GIIIA (mu-CTX) 是电压门控 Na+ 通道选定亚型外前庭的高亲和力配体。该毒素的高亲和力结合和异构体选择性的详细基础尚不清楚。外前庭由四个成孔 (P) 环排列，每个环在前庭口附近都有酸性残基。 mu-CTX 有七个带正电荷的残基，可能与这些酸性 P 环残基相互作用。使用带电毒素和通道残基的成对丙氨酸替换，结合双突变体循环分析，我们确定了每个 P 环酸性残基和选定毒素残基之间特定相互作用的耦合能，以系统地建立对外前庭毒素方向的定量限制。将突变型或天然 Na+ 通道 mRNA 注射到非洲爪蟾卵母细胞中，并通过两电极电压钳测量电流。突变体循环分析揭示了 K9/E403、K11/D1241、K11/D1532 和 R19/D1532 之间新颖、强的毒素通道相互作用。实验确定的相互作用残基对的耦合能为 mu-CTX 对接的分子动力学模拟提供了限制。我们的模拟表明毒素在孔中的精确定位，毒素碱性侧链在高亲和力结合中发挥关键作用。该模型还提供了一组迄今为止尚未描述的毒素通道相互作用的可测试预测，这可能有助于高亲和力结合和通道亚型选择性。
mu-Conotoxin GIIIA (mu-CTX) is a high - affinity ligand for the outer vestibule of selected isoforms of the voltage-gated Na+ channel. The detailed bases for the toxin's high affinity binding and isoform selectivity are unclear. The outer vestibule is lined by four pore forming ( P) loops, each with an acidic residue near the mouth of the vestibule. mu-CTX has seven positively charged residues that may interact with these acidic P-loop residues. Using pair-wise alanine replacement of charged toxin and channel residues, in conjunction with double mutant cycle analysis, we determined coupling energies for specific interactions between each P-loop acidic residue and selected toxin residues to systematically establish quantitative restraints on the toxin orientation in the outer vestibule. Xenopus oocytes were injected with the mutant or native Na+ channel mRNA, and currents measured by two-electrode voltage clamp. Mutant cycle analysis revealed novel, strong, toxin-channel interactions between K9/E403, K11/D1241, K11/D1532, and R19/D1532. Experimentally determined coupling energies for interacting residue pairs provided restraints for molecular dynamics simulations of mu-CTX docking. Our simulations suggest a refined orientation of the toxin in the pore, with toxin basic side-chains playing key roles in high-affinity binding. This modeling also provides a set of testable predictions for toxin-channel interactions, hitherto not described, that may contribute to high-affinity binding and channel isoform selectivity.