Department of Chemistry, Faculty of Sciences, University of Chile, Las Palmeras 3425, Santiago, Chile.
Mol Pharmacol. 2010 Sep;78(3):366-75. doi: 10.1124/mol.110.065490. Epub 2010 Jun 14.
The Erythrina alkaloids erysodine and dihydro-beta-erythroidine (DHbetaE) are potent and selective competitive inhibitors of alpha4beta2 nicotinic acetylcholine receptors (nAChRs), but little is known about the molecular determinants of the sensitivity of this receptor subtype to inhibition by this class of antagonists. We addressed this issue by examining the effects of DHbetaE and a range of aromatic Erythrina alkaloids on [(3)H]cytisine binding and receptor function in conjunction with homology models of the alpha4beta2 nAChR, mutagenesis, and functional assays. The lactone group of DHbetaE and a hydroxyl group at position C-16 in aromatic Erythrina alkaloids were identified as major determinants of potency, which was decreased when the conserved residue Tyr126 in loop A of the alpha4 subunit was substituted by alanine. Sensitivity to inhibition was also decreased by substituting the conserved aromatic residues alpha4Trp182 (loop B), alpha4Tyr230 (loop C), and beta2Trp82 (loop D) and the nonconserved beta2Thr84; however, only alpha4Trp182 was predicted to contact bound antagonist, suggesting alpha4Tyr230, beta2Trp82, and beta2Thr84 contribute allosterically to the closed state elicited by bound antagonist. In addition, homology modeling predicted strong ionic interactions between the ammonium center of the Erythrina alkaloids and beta2Asp196, leading to the uncapping of loop C. Consistent with this, beta2D196A abolished sensitivity to inhibition by DHbetaE or erysodine but not by epierythratidine, which is not predicted to form ionic bonds with beta2Asp196. This residue is not conserved in subunits that comprise nAChRs with low sensitivity to inhibition by DHbetaE or erysodine, which highlights beta2Asp196 as a major determinant of the receptor selectivity of Erythrina alkaloids.
安石榴生物碱白屈菜红碱和二氢-β-石蒜碱（DHbetaE）是强效且选择性的α4β2 烟碱型乙酰胆碱受体（nAChR）竞争性抑制剂，但人们对该受体亚型对这类拮抗剂抑制的敏感性的分子决定因素知之甚少。我们通过检查 DHbetaE 和一系列芳香安石榴生物碱对[3H]烟碱结合和受体功能的影响，结合α4β2 nAChR 的同源模型、突变和功能测定来解决这个问题。DHbetaE 的内酯基团和芳香安石榴生物碱中 C-16 位的羟基被确定为效力的主要决定因素，当α4 亚基环 A 中的保守残基 Tyr126 被丙氨酸取代时，效力会降低。对抑制的敏感性也会因取代保守的芳香残基α4Trp182（环 B）、α4Tyr230（环 C）和β2Trp82（环 D）和非保守的β2Thr84 而降低；然而，只有α4Trp182 被预测与结合的拮抗剂接触，表明α4Tyr230、β2Trp82 和β2Thr84 共同作用于结合的拮抗剂引起的闭合状态。此外，同源建模预测安石榴生物碱的铵中心与β2Asp196 之间存在强烈的离子相互作用，导致环 C 去帽。与此一致的是，β2D196A 消除了对 DHbetaE 或白屈菜红碱的敏感性，但对不预测与β2Asp196 形成离子键的表 Erythratidine 没有影响。该残基在由 DHbetaE 或白屈菜红碱抑制敏感性低的 nAChR 组成的亚基中不保守，这突出了β2Asp196 作为安石榴生物碱受体选择性的主要决定因素。
