Key Laboratory of Tropical Biological Resources, Ministry of Education, Ocean College, College of Materials and Chemical Engineering, Center for Experimental Biotechnology, Hainan University, Haikou Hainan 570228, China.
J Biol Chem. 2010 Apr 16;285(16):12355-66. doi: 10.1074/jbc.M109.079012. Epub 2010 Feb 9.
Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nm IC(50) value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 versus alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC(50) value on alpha3beta2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.
不同的烟碱型乙酰胆碱受体（nAChR）亚型与学习、疼痛感知和疾病状态有关，包括帕金森病和尼古丁成瘾。α-芋螺毒素是最具选择性的 nAChR 配体之一。对 α-芋螺毒素的结构、功能和受体相互作用的机制研究可以为开发新的治疗方法提供平台。以前表征的 α-芋螺毒素具有高度保守的 Ser-Xaa-Pro 基序，这对于有效的 nAChR 相互作用至关重要。本研究对缺乏该高度保守基序但能以 9.8nmIC50 值有效阻断 α3β2nAChR 的新型 α-芋螺毒素 LtIA 进行了表征。与含有 Ser-Xaa-Pro 的 α-芋螺毒素 MII 相比，LtIA 的脱靶速率较快。然而，LtIA 预先阻断 α3β2nAChR 可防止与 MII 相关的缓慢可逆阻断，表明其结合位点重叠。使用 nAChRβ亚基配体结合界面突变来研究 LtIA 对 α3β2 与 α3β4nAChR 的 >1000 倍选择性差异。与 MII 不同，LtIA 对 α3β2(F119Q) 与野生型 nAChR 的 IC50 值增加了 >900 倍，而 T59K 和 V111Iβ2 突变体影响较小。分子对接模拟表明，LtIA 在α3β2nAChR 上具有出乎意料的浅结合位点，包括β2Lys-79。K79A 突变体破坏了 LtIA 的结合，但对存在 Ser-Xaa-Pro 基序的 LtIA 类似物没有影响，这与不同的结合模式一致。
