Christensen Sean B, Bandyopadhyay Pradip K, Olivera Baldomero M, McIntosh J Michael
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA; George E. Wahlen Veterans Affairs Medical Center, Salt Lake City, UT 84108, USA; Department of Psychiatry, University of Utah, Salt Lake City, UT 84112, USA.
Biochem Pharmacol. 2015 Aug 15;96(4):349-56. doi: 10.1016/j.bcp.2015.06.007. Epub 2015 Jun 11.
Although acetylcholine is widely utilized in vertebrate nervous systems, nicotinic acetylcholine receptors (nAChRs), including the α9α10 subtype, also are expressed in a wide variety of non-neuronal cells. These cell types include cochlear hair cells, adrenal chromaffin cells and immune cells. α9α10 nAChRs present in these cells may respectively play roles in protection from noise-induced hearing loss, response to stress and neuroprotection. Despite these critical functions, there are few available selective ligands to confirm mechanistic hypothesis regarding the role of α9α10 nAChRs. Conus, has been a rich source of ligands for receptors and ion channels. Here, we identified Conus geographus venom as a lead source for a novel α9α10 antagonist. The active component was isolated and the encoding gene cloned. The peptide signal sequence and cysteine arrangement had the signature of the σ-conotoxin superfamily. Previously isolated σ-conotoxin GVIIIA, also from Conus geographus, targets the 5-HT3 receptor. In contrast, αS-GVIIIB blocked the α9α10 nAChR with an IC50 of 9.8 nM, yet was inactive at the 5-HT3 receptor. Pharmacological characterization of αS-GVIIIB shows that it is over 100-fold selective for the α9α10 nAChR compared to other nAChR subtypes. Thus, the S-superfamily represents a novel conotoxin scaffold for flexibly targeting a variety of receptor subtypes. Functional competition studies utilized distinct off-rate kinetics of conotoxins to identify the α10/α9 nAChR interface as the site of αS-GVIIIB binding; this adds to the importance of the (+) face of the α10 rather than the (+) face of the α9 nAChR subunit as critical to binding of α9α10-targeted conotoxins.
尽管乙酰胆碱在脊椎动物神经系统中被广泛利用，但烟碱型乙酰胆碱受体（nAChRs），包括α9α10亚型，也在多种非神经元细胞中表达。这些细胞类型包括耳蜗毛细胞、肾上腺嗜铬细胞和免疫细胞。存在于这些细胞中的α9α10 nAChRs可能分别在预防噪声性听力损失、应激反应和神经保护中发挥作用。尽管有这些关键功能，但几乎没有可用的选择性配体来证实关于α9α10 nAChRs作用的机制假说。芋螺一直是受体和离子通道配体的丰富来源。在这里，我们鉴定出地纹芋螺毒液是一种新型α9α10拮抗剂的先导来源。分离出活性成分并克隆了编码基因。该肽的信号序列和半胱氨酸排列具有σ-芋螺毒素超家族的特征。之前同样从地纹芋螺中分离出的σ-芋螺毒素GVIIIA靶向5-HT3受体。相比之下，αS-GVIIIB以9.8 nM的IC50阻断α9α10 nAChR，但对5-HT3受体无活性。αS-GVIIIB的药理学特征表明，与其他nAChR亚型相比，它对α9α10 nAChR的选择性超过100倍。因此，S-超家族代表了一种新型芋螺毒素支架，可灵活靶向多种受体亚型。功能竞争研究利用芋螺毒素不同的解离速率动力学来确定α10/α9 nAChR界面是αS-GVIIIB的结合位点；这增加了α10亚基的（+）面而非α9 nAChR亚基的（+）面对于靶向α9α10的芋螺毒素结合至关重要的重要性。
