Azam Layla, Papakyriakou Athanasios, Zouridakis Marios, Giastas Petros, Tzartos Socrates J, McIntosh J Michael
Departments of Biology (L.A., J.M.M.) and Psychiatry (J.M.M.), University of Utah, Salt Lake City, Utah; George E. Wahlen Veterans Affair Medical Center, Salt Lake City, Utah (J.M.M.); National Center for Scientific Research "Demokritos," Athens, Greece (A.P.); and Department of Neurobiology, Hellenic Pasteur Institute, Athens, Greece (M.Z., P.G., S.J.T.)
Departments of Biology (L.A., J.M.M.) and Psychiatry (J.M.M.), University of Utah, Salt Lake City, Utah; George E. Wahlen Veterans Affair Medical Center, Salt Lake City, Utah (J.M.M.); National Center for Scientific Research "Demokritos," Athens, Greece (A.P.); and Department of Neurobiology, Hellenic Pasteur Institute, Athens, Greece (M.Z., P.G., S.J.T.).
Mol Pharmacol. 2015 May;87(5):855-64. doi: 10.1124/mol.114.096511. Epub 2015 Mar 4.
The α9α10 nicotinic acetylcholine receptor (nAChR) was first identified in the auditory system, where it mediates synaptic transmission between efferent olivocochlear cholinergic fibers and cochlea hair cells. This receptor gained further attention due to its potential role in chronic pain and breast and lung cancers. We previously showed that α-conotoxin (α-CTx) RgIA, one of the few α9α10 selective ligands identified to date, is 300-fold less potent on human versus rat α9α10 nAChR. This species difference was conferred by only one residue in the (-), rather than (+), binding region of the α9 subunit. In light of this unexpected discovery, we sought to determine other interacting residues with α-CTx RgIA. A previous molecular modeling study, based on the structure of the homologous molluscan acetylcholine-binding protein, predicted that RgIA interacts with three residues on the α9(+) face and two residues on the α10(-) face of the α9α10 nAChR. However, mutations of these residues had little or no effect on toxin block of the α9α10 nAChR. In contrast, mutations of homologous residues in the opposing nAChR subunits (α10 Ε197, P200 and α9 T61, D121) resulted in 19- to 1700-fold loss of toxin activity. Based on the crystal structure of the extracellular domain (ECD) of human α9 nAChR, we modeled the rat α9α10 ECD and its complexes with α-CTx RgIA and acetylcholine. Our data support the interaction of α-CTx RgIA at the α10/α9 rather than the α9/α10 nAChR subunit interface, and may facilitate the development of selective ligands with therapeutic potential.
α9α10烟碱型乙酰胆碱受体（nAChR）最初是在听觉系统中被发现的，它介导传出性橄榄耳蜗胆碱能纤维与耳蜗毛细胞之间的突触传递。由于其在慢性疼痛以及乳腺癌和肺癌中的潜在作用，该受体受到了更多关注。我们之前表明，α-芋螺毒素（α-CTx）RgIA是迄今为止鉴定出的少数几种α9α10选择性配体之一，对人α9α10 nAChR的效力比对大鼠α9α10 nAChR低300倍。这种物种差异仅由α9亚基（-）而非（+）结合区域中的一个残基导致。鉴于这一意外发现，我们试图确定与α-CTx RgIA相互作用的其他残基。先前基于同源软体动物乙酰胆碱结合蛋白结构的分子模拟研究预测，RgIA与α9α10 nAChR的α9（+）面的三个残基以及α10（-）面的两个残基相互作用。然而，这些残基的突变对α9α10 nAChR的毒素阻断作用几乎没有影响。相反，相对的nAChR亚基（α10的E197、P200和α9的T61、D121）中同源残基的突变导致毒素活性丧失19至1700倍。基于人α9 nAChR胞外域（ECD）的晶体结构，我们对大鼠α9α10 ECD及其与α-CTx RgIA和乙酰胆碱的复合物进行了建模。我们的数据支持α-CTx RgIA在α10/α9而非α9/α10 nAChR亚基界面处的相互作用，这可能有助于开发具有治疗潜力的选择性配体。
