Seo Seungmae, Henry Jonathan T, Lewis Amanda H, Wang Nan, Levandoski Mark M
Department of Chemistry, Grinnell College, Grinnell, Iowa 50112, USA.
J Neurosci. 2009 Jul 8;29(27):8734-42. doi: 10.1523/JNEUROSCI.1859-09.2009.
We are interested in the positive allosteric modulation of neuronal nicotinic acetylcholine (ACh) receptors and have recently shown that the anthelmintic compound morantel potentiates by enhancing channel gating of the alpha3beta2 subtype. Based on the demonstration that morantel-elicited currents were inhibited by the classic ACh competitor dihydro-beta-erythroidine in a noncompetitive manner and that morantel still potentiates at saturating concentrations of agonist (Wu et al., 2008), we hypothesized that morantel binds at the noncanonical beta2(+)/alpha3(-) subunit interface. In the present study, we created seven cysteine-substituted subunits by site-directed mutagenesis, choosing residues in the putative morantel binding site with the aid of structural homology models. We coexpressed the mutant subunits and their respective wild-type partners in Xenopus oocytes and characterized the morantel potentiation of ACh-evoked currents, as well as morantel-evoked currents, before and after treatment with a variety of methanethiosulfonate (MTS)-based compounds, using voltage-clamp recordings. The properties of four of the seven mutants, two residues on each side of the interface, were changed by MTS treatments. Coapplication with ACh enhanced the extent of MTS modification for alpha3A106Cbeta2 and alpha3beta2S192C receptors. The activities of two mutants, alpha3T115Cbeta2 and alpha3beta2T150C, were dramatically altered by MTS modification. For alpha3beta2T150C, while peak current amplitudes were reduced, potentiation was enhanced. For alpha3T115Cbeta2, both current amplitudes and potentiation were reduced. MTS modification and morantel were mutually inhibitory: MTS treatment decreased morantel-evoked currents and morantel decreased the rate of MTS modification. We conclude that the four residues showing MTS effects contribute to the morantel binding site.
我们对神经元烟碱型乙酰胆碱（ACh）受体的正变构调节感兴趣，最近发现驱虫化合物莫仑太尔通过增强α3β2亚型的通道门控作用来发挥增强效应。基于以下证据：经典的ACh竞争性拮抗剂二氢β-刺桐啶以非竞争性方式抑制莫仑太尔诱发的电流，且在激动剂饱和浓度下莫仑太尔仍能发挥增强作用（Wu等人，2008年），我们推测莫仑太尔结合于非典型的β2(+)/α3(-)亚基界面。在本研究中，我们通过定点诱变创建了七个半胱氨酸取代的亚基，借助结构同源模型在推测的莫仑太尔结合位点选择残基。我们在非洲爪蟾卵母细胞中共表达突变亚基及其各自的野生型伴侣，并使用电压钳记录，在使用各种基于甲硫基磺酸盐（MTS）的化合物处理前后，表征莫仑太尔对ACh诱发电流以及莫仑太尔诱发电流的增强作用。七个突变体中的四个，即界面两侧各两个残基，其性质因MTS处理而改变。与ACh共同应用增强了α3A106Cβ2和α3β2S192C受体的MTS修饰程度。两个突变体α3T115Cβ2和α3β2T150C的活性因MTS修饰而发生显著改变。对于α3β2T150C，虽然峰值电流幅度降低，但增强作用增强。对于α3T115Cβ2，电流幅度和增强作用均降低。MTS修饰与莫仑太尔相互抑制：MTS处理降低莫仑太尔诱发的电流，而莫仑太尔降低MTS修饰的速率。我们得出结论，显示出MTS效应的四个残基构成了莫仑太尔的结合位点。
