Electrophysiological and molecular identification of glutamate receptor subunits expressed in hippocampal neurons

哺乳动物中枢神经系统中的谷氨酸受体通道分为三种亚型：NMDA、AMPA和海人藻酸受体。其中，AMPA受体是快速兴奋性神经传递的主要介体。AMPA受体是由GluR1、GluR2、GluR3和GluR4四种亚基蛋白的不同组合形成的。为了追踪大鼠海马神经元AMPA受体功能特性差异的分子基础，我们将全细胞膜片钳记录与逆转录(RT)和聚合酶链式反应(RT-PCR)相结合。我们先前在培养的大鼠海马神经元中发现了两种不同类型的AMPA受体。在I型神经元中，AMPA受体呈外向整流性，钙离子通透性极低。相反，II型神经元的AMPA受体表现出强烈的内向整流性和高的钙离子通透性。膜片钳反转录-聚合酶链式反应显示只有…在II型神经元中有较多的GluR1和GluR4亚基表达，未检测到GluR2亚基。而在I型神经元中，GluR2与GluR1一起高水平表达，偶尔与GluR3或GluR4共同表达。我们进一步将这项技术应用于分析大鼠海马片不同类型神经元中AMPA受体的亚单位组成。在海马区的非锥体中间神经元中，发现携带AMPA受体的II型神经元具有较强的内向整流和高的钙离子通透性。在所有被测试的脑片中的II型神经元(16个神经元)中，GluR1、GluR3或GluR4都有表达，但没有检测到GluR2的表达。因此，GluR2的存在或不存在可能决定了培养和切片中的海马神经元的整流特性和天然AMPA受体的钙通透性。膜片钳RT-PCR方法可用于广泛的生理学研究，在单细胞水平上阐明功能特性的分子基础。较少

The glutamate receptor channels in the mammalian central nervous system are classified into three subtypes : NMDA,AMPA and kainate receptors. Among them, AMPA receptors are the principal mediators of fast excitatory neurotransmission. AMPA receptors are formed by various combinations of four subunit proteins, GluRl, GluR2, GluR3 and GluR4. To trace the molecular basis underlying differences in the functional properties of AMPA receptors in rat hippocampal neurons, we coupled whole-cell patch-clamp recordings with reverse transcription (RT) followed by polymerase chain reaction amplification (patch-clamp RT-PCR method).We have previously found two distinct types of AMPA receptors in cultured rat hippocampal neurons. In type I neurons, AMPA receptors displayd an outward rectification and little Ca^<2+> permeability. In contrast, type II neurons had AMPA receptors that displayd strong inward rectification and high Ca^<2+> permeability. The patch-clamp RT-PCR method revealed that only the … More GluRl and GluR4 subunits were expressed and that no GluR2 subunit was detected in type II neurons. In contrast, GluR2 was expressed at a high level together with GluR1, and occasionally with GluR3 or GluR4, in type I neurons. We further applied this technique for analyzing subunit compositions of AMPA receptors in various types of neurons in rat hippocampal slices. Type II neurons carrying AMPA receptors with strong inward rectification and high Ca^<2+> permeability were found in non-pyramidal interneurons in the hippocampus. In all type II neurons tested (16 neurons) in slices, either GluR1, GluR3 or GluR4 was expressed, but no GluR2 expression was detected.Thus, It is likely that the presence or absence of GluR2 determines rectification properties and Ca^<2+> permeability in native AMPA receptors in hippocampal neurons both in cultures and slices. The patch-clamp RT-PCR method will be useful for a wide range of physiological studies to elucidate the molecular basis of functional properties at the single cell level. Less