Department of Biosciences, Division of Biochemistry and Biotechnology, University of Helsinki, Helsinki, Finland.
PLoS One. 2012;7(11):e49282. doi: 10.1371/journal.pone.0049282. Epub 2012 Nov 14.
Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This "autoinactivation" has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1-A4 AMPA receptors (all flip isoform) expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.
天然的α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）受体的激动剂反应和通道动力学受跨膜辅助蛋白调节。Stargazin 是典型的辅助蛋白，可降低 AMPA 受体的脱敏作用并增加激动剂效力。此外，在 Stargazin 存在的情况下，AMPA 受体的稳态反应在较高谷氨酸浓度下呈逐渐下降趋势。这种“自失活”已被分配给 Stargazin-AMPA 受体复合物的物理解离，并被认为是一种防止过度激活的保护机制。在这里，我们分析了在 Stargazin 存在下表达的 GluA1-A4 AMPA 受体（所有翻转异构体）的自失活。同型 GluA1、GluA3 和 GluA4 通道表现出明显的自失活，表现为谷氨酸的稳态剂量反应曲线呈钟形。相比之下，同型 GluA2i 通道没有明显的自失活。GluA2 对自失活的抵抗力表现出对剪接形式的惊人依赖性，因为 GluA2-flop 受体显示出明显的自失活。有趣的是，GluA2-flip 受体对自失活的抵抗力以显性方式转移到异源受体上。为了研究自失活与 Stargazin 与 AMPA 受体物理分离的关系，我们分析了 GluA4-Stargazin 融合蛋白。值得注意的是，共价连接的复合物和单独表达的蛋白质表达出相似水平的自失活。我们得出结论，自失活是 AMPA 受体-Stargazin 复合物的亚基和剪接形式依赖性特性，涉及复合物内的结构重排，而不是任何物理解离。
