Atypical α-Conotoxin LtIA from Conus litteratus Targets a Novel Microsite of the α3β2 Nicotinic Receptor
Atypical α-Conotoxin LtIA from Conus litteratus Targets a Novel Microsite of the α3β2 Nicotinic Receptor
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DOI：
10.1074/jbc.m109.079012
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发表时间：
2010-04-16
影响因子：
4.8
通讯作者：
McIntosh, J. Michael
中科院分区：
文献类型：
--
作者：
Luo, Sulan;Akondi, Kalyana Bharati;McIntosh, J. Michael
不同的烟碱乙酰胆碱受体（nAChR）亚型与学习、疼痛感觉和疾病状态（包括帕金森病和尼古丁成瘾）有关。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见解可以作为开发新疗法的平台。先前表征的α-芋螺毒素具有高度保守的Ser-Xaa-Pro基序，其对于有效的nAChR相互作用至关重要。这项研究表征了新型α-芋螺毒素LtIA，其缺乏这种高度保守的基序，但有效地阻断了α 3 β 2 nAChR，IC 50值为9.8 nM。相对于含Ser-Xaa-Pro的α-芋螺毒素MII，LtIA的解离速率较快。然而，用LtIA预阻断α 3 β 2 nAChR阻止了与MII相关的缓慢可逆阻断，表明它们的结合位点重叠。使用nAChR β亚基配体结合界面突变来检查LtIA对α 3 β 2与α 3 β 4 nAChR的> 1000倍选择性差异。与MII不同，与野生型nAChR相比，LtIA对α 3 β 2（F119 Q）的IC 50值增加> 900倍，而T59 K和V111 I β 2突变体几乎没有影响。分子对接模拟表明，LtIA在包括β 2 Lys-79的α 3 β 2 nAChR上具有令人惊讶的浅结合位点。K79 A突变体破坏了LtIA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。
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 alpha 3 beta 2 nAChRs with a 9.8 nM IC50 value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-blockof alpha 3 beta 2 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 alpha 3 beta 2 versus alpha 3 beta 4 nAChRs. Unlike MII, LtIA had a > 900-fold increased IC50 value on alpha 3 beta 2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta 2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha 3 beta 2 nAChR that includes beta 2 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.