Quirk Jennifer C, Nisenbaum Eric S
Neuroscience Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
J Neurosci. 2003 Nov 26;23(34):10953-62. doi: 10.1523/JNEUROSCI.23-34-10953.2003.
Positive allosteric regulation of glutamate AMPA receptors involves conformational changes that can attenuate receptor desensitization and enhance ion flux through the channel pore. Many allosteric modulators (e.g., cyclothiazide and aniracetam) preferentially affect the flip (i) or flop (o) alternatively spliced isoform of AMPA receptors, implicating residues in the flip-flop domain as critical determinants of splice variant sensitivity. Indeed, previous mutational analyses have demonstrated that the differential sensitivity to cyclothiazide and aniracetam depends on a single amino acid, Ser (flip) and Asn (flop), suggesting that this residue may be solely responsible for differences in modulation of AMPA receptor isoforms. The present studies tested this hypothesis by investigating the molecular determinants of modulation of AMPA receptor splice variants by a structurally distinct compound, LY404187, which displays strikingly different and opposing kinetics of allosteric regulation characterized by a time-dependent enhancement in potentiation of homomeric GluR1-GluR4i and a time-dependent reduction in potentiation of GluR1-GluR4o. Site-directed mutagenesis of residues in the flip-flop domain of GluR2 revealed that, although exchange of Asn775 for Ser in GluR2o was sufficient to confer the GluR2i phenotype of potentiation, the corresponding mutation, Ser775Asn, in GluR2i did not impart the GluR2o response. In fact, the GluR2o kinetics of modulation depended on a novel set of substitutions in GluR2i, including Thr765Asn, Pro766Ala, and Val779Leu in combination with Ser775Asn. Collectively, these results show that, unlike cyclothiazide and aniracetam, the residues that confer splice variant differences in modulation by LY404187 are not identical and indicate that allosteric regulation of AMPA receptors can arise from multiple molecular determinants.
谷氨酸AMPA受体的正向变构调节涉及构象变化，这种变化可减弱受体脱敏并增强通过通道孔的离子通量。许多变构调节剂（如环噻嗪和阿尼西坦）优先影响AMPA受体的翻转（i）或摆动（o）选择性剪接异构体，这表明翻转-摆动结构域中的残基是剪接变体敏感性的关键决定因素。事实上，先前的突变分析表明，对环噻嗪和阿尼西坦的不同敏感性取决于单个氨基酸，即翻转异构体中的丝氨酸（Ser）和摆动异构体中的天冬酰胺（Asn），这表明该残基可能是AMPA受体异构体调节差异的唯一原因。本研究通过研究一种结构不同的化合物LY404187对AMPA受体剪接变体调节的分子决定因素来检验这一假设，该化合物表现出明显不同且相反的变构调节动力学，其特征是同源GluR1-GluR4i的增强呈时间依赖性，而GluR1-GluR4o的增强呈时间依赖性降低。对GluR2翻转-摆动结构域中的残基进行定点诱变表明，虽然在GluR2o中将Asn775替换为Ser足以赋予增强的GluR2i表型，但在GluR2i中相应的突变Ser775Asn并未赋予GluR2o反应。事实上，GluR2o的调节动力学取决于GluR2i中的一组新的替代，包括Thr765Asn、Pro766Ala和Val779Leu与Ser775Asn的组合。总的来说，这些结果表明，与环噻嗪和阿尼西坦不同，赋予LY404187调节剪接变体差异的残基并不相同，这表明AMPA受体的变构调节可能源于多个分子决定因素。
