Lee J C, Greig A, Ravindranathan A, Parks T N, Rao M S
Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.
Brain Res Brain Res Protoc. 1998 Nov;3(2):142-54. doi: 10.1016/s1385-299x(98)00035-x.
Glutamate activates three distinct classes of ionotropic receptors: AMPA, kainate and NMDA. AMPA receptors (AMPARs) are of particular importance as they mediate the majority of fast excitatory synaptic transmission and are implicated in a variety of neurological disorders [B. Bettler, C. Mulle, AMPA and kainate receptors, Neuropharmacology 34 (1995) 123-139]. Functional AMPARs are believed to be a heteromer comprising a combination of four closely related subunits, GluRs1-4 [B. Bettler, C. Mulle, AMPA and kainate receptors, Neuropharmacology 34 (1995) 123-139]. Diversity of AMPARs is obtained through multiple combinations of AMPAR subunits, by alternative splicing of subunits at the flip/flop and/or C-terminal sites, and by mRNA editing of a single amino acid at multiple sites [M. Hollmann, M. Hartley, S. Heinemann, Ca2+ permeability of KA-AMPA-gated glutamate receptor channel depends on subunit composition, Science 252 (1991) 851-853; B. Sommer, K. Keinanen, T.A. Verdoorn, W. Wisden, N. Burhashev, A. Herb, M. Kohler, T. Takagi, B. Sakmann, P.H. Seeburg, Flip and flop: a cell-specific functional switch in glutamate-operated channels in the CNS, Science 249 (1990) 1580-1585; B. Sommer, M. Kohler, R. Sprengel, P.H. Seeburg, RNA editing in brain controls a determinant of ion flow in glutamate-gated channels, Cell 67 (1991)]. The subunit combination, editing status, and splice variant expression have profound effects on channel kinetics and can serve as predictors of the channel's properties [M. Hollmann, M. Hartley, S. Heinemann, Ca2+ permeability of KA-AMPA-gated glutamate receptor channel depends on subunit composition, Science 252 (1991) 851-853; B. Sommer, K. Keinanen, T.A. Verdoorn, W. Wisden, N. Burhashev, A. Herb, M. Kohler, T. Takagi, B. Sakmann, P.H. Seeburg, Flip and flop: a cell-specific functional switch in glutamate-operated channels in the CNS, Science 249 (1990) 1580-1585; B. Sommer, M. Kohler, R. Sprengel, P.H. Seeburg, RNA editing in brain controls a determinant of ion flow in glutamate-gated channels, Cell 67 (1991)]. In this manuscript, we detail procedures for profiling AMPAR composition, namely: relative subunit ratios, expression of flip/flop isoforms, Q/R and R/G editing status, and Ca2+ permeability using small amounts of cDNA from identified cell populations.
AMPA、海人藻酸和NMDA。AMPA受体（AMPARs）尤为重要，因为它们介导了大部分快速兴奋性突触传递，并与多种神经系统疾病有关[B. 贝特勒，C. 米勒，AMPA和海人藻酸受体，《神经药理学》34 (1995) 123 - 139]。功能性AMPARs被认为是一种异聚体，由四个密切相关的亚基GluRs1 - 4组合而成[B. 贝特勒，C. 米勒，AMPA和海人藻酸受体，《神经药理学》34 (1995) 123 - 139]。AMPARs的多样性通过AMPAR亚基的多种组合、亚基在翻转/摆动和/或C末端位点的可变剪接以及多个位点单个氨基酸的mRNA编辑来实现[M. 霍尔曼，M. 哈特利，S. 海涅曼，KA - AMPA门控谷氨酸受体通道的Ca2 +通透性取决于亚基组成，《科学》252 (1991) 851 - 853；B. 索默，K. 凯南宁，T.A. 韦尔东，W. 维斯登，N. 布尔哈舍夫，A. 赫布，M. 科勒，T. 高木，B. 萨克曼，P.H. 西伯尔格，翻转和摆动：中枢神经系统中谷氨酸操纵通道的细胞特异性功能开关，《科学》249 (1990) 1580 - 1585；B. 索默，M. 科勒，R. 施普伦格尔，P.H. 西伯尔格，大脑中的RNA编辑控制谷氨酸门控通道中离子流的一个决定因素，《细胞》67 (1991)]。亚基组合、编辑状态和剪接变体表达对通道动力学有深远影响，并且可以作为通道特性的预测指标[M. 霍尔曼，M. 哈特利，S. 海涅曼，KA - AMPA门控谷氨酸受体通道的Ca2 +通透性取决于亚基组成，《科学》252 (1991) 851 - 853；B. 索默，K. 凯南宁，T.A. 韦尔东，W. 维斯登，N. 布尔哈舍夫，A.赫布，M. 科勒，T. 高木，B. 萨克曼，P.H. 西伯尔格，翻转和摆动：中枢神经系统中谷氨酸操纵通道的细胞特异性功能开关，《科学》249 (1990) 1580 - 1585；B. 索默，M. 科勒，R. 施普伦格尔，P.H. 西伯尔格，大脑中的RNA编辑控制谷氨酸门控通道中离子流的一个决定因素，《细胞》67 (1991)]。在本论文中，我们详细介绍了分析AMPAR组成的方法，即：相对亚基比例、翻转/摆动异构体的表达、Q/R和R/G编辑状态以及使用来自已鉴定细胞群体的少量cDNA测定Ca2 +通透性。
