From the Biochemistry and Molecular Biophysics Option and; the Division of Biology, California Institute of Technology, Pasadena, California 91125.
the Division of Biology, California Institute of Technology, Pasadena, California 91125.
J Biol Chem. 2013 Jul 19;288(29):21029-21042. doi: 10.1074/jbc.M112.423921. Epub 2013 May 29.
A modified invertebrate glutamate-gated Cl(-) channel (GluCl αβ) was previously employed to allow pharmacologically induced silencing of electrical activity in CNS neurons upon exposure to the anthelmintic drug ivermectin (IVM). Usefulness of the previous receptor was limited by 1) the high concentration of IVM necessary to elicit a consistent silencing phenotype, raising concern about potential side effects, and 2) the variable extent of neuronal spike suppression, due to variations in the co-expression levels of the fluorescent protein-tagged α and β subunits. To address these issues, mutant receptors generated via rational protein engineering strategies were examined for improvement. Introduction of a gain-of-function mutation (L9'F) in the second transmembrane domain of the α subunit appears to facilitate β subunit incorporation and substantially increase heteromeric GluCl αβ sensitivity to IVM. Removal of an arginine-based endoplasmic reticulum retention motif (RSR mutated to AAA) from the intracellular loop of the β subunit further promotes heteromeric expression at the plasma membrane possibly by preventing endoplasmic reticulum-associated degradation of the β subunit rather than simply reducing endoplasmic reticulum retention. A monomeric XFP (mXFP) mutation that prevents fluorescent protein dimerization complements the mutant channel effects. Expression of the newly engineered GluCl opt α-mXFP L9'F + opt β-mXFP Y182F RSR_AAA receptor in dissociated neuronal cultures markedly increases conductance and reduces variability in spike suppression at 1 nm IVM. This receptor, named "GluClv2.0," is an improved tool for IVM-induced silencing.
先前使用改良的无脊椎动物谷氨酸门控 Cl(-) 通道 (GluCl αβ)，使中枢神经系统神经元在接触驱虫药伊维菌素 (IVM) 时可通过药理诱导沉默其电活动。该先前受体的有用性受到以下因素的限制：1）为引起一致的沉默表型，需要高浓度的 IVM，这引起了对潜在副作用的担忧；2）由于荧光蛋白标记的 α 和 β 亚基的共表达水平的变化，神经元尖峰抑制的程度不同。为了解决这些问题，通过合理的蛋白质工程策略生成了突变体受体以进行检查和改进。研究了通过理性蛋白工程策略产生的突变受体，以改善其性能。在 α 亚基的第二个跨膜结构域中引入功能获得性突变 (L9'F) 似乎有助于 β 亚基的掺入，并显著增加异源 GluCl αβ 对 IVM 的敏感性。从 β 亚基的细胞内环中去除基于精氨酸的内质网保留基序 (RSR 突变为 AAA) 进一步促进了质膜上的异源表达，这可能是通过防止 β 亚基的内质网相关降解，而不是简单地减少内质网保留，从而促进了异源表达。防止荧光蛋白二聚化的单体 XFP (mXFP) 突变互补了突变通道的作用。在分离的神经元培养物中表达新设计的 GluCl opt α-mXFP L9'F + opt β-mXFP Y182F RSR_AAA 受体，可显著增加电导并减少在 1nm IVM 时尖峰抑制的可变性。该受体被命名为“GluClv2.0”，是用于 IVM 诱导沉默的改进工具。
