GATING KINETICS OF GLYCINERGIC NMDA RECEPTORS

说明(申请人提供)：甘氨酸能N-甲基-D-天冬氨酸受体(NRs)是一种独特的兴奋性通道。它们不同于传统的谷氨酸能神经递质，它们只被甘氨酸激活，对谷氨酸不敏感，具有较低的单位电导、钙通透性和对电压依赖的镁离子阻断的敏感性。它们是GluN1(N1)和GluN3(N3A或N3B)亚基的四聚体。N1亚基有八个剪接变体(N1-1a到N1-4b)，每个剪接变体在大脑中都有特定的时空表达模式。N3A亚单位在新生儿中特异表达，对脊柱发育和突触可塑性至关重要。N3A的表达变化与精神分裂症的阴性症状有关。与在谷氨酸能NRS(N1/N2)中观察到的相反，根据N3A组装的N1剪接变体产生明显不同的宏观电流。我认为，这些差异可能部分源于N1剪接变体的C末端盒对N1/N3A受体的不同门控动力学。为了验证这一假说，我将追求以下三个目标：1)使用动力学分析和状态建模来充分表征高活性的N1-4a/N3亚型。2)通过系统比较选定的N1/N3A亚型的反应机理，确定每个C-端盒的动力学贡献。3)结合基于序列的突变和动力学分析，确定每个盒上负责门控调制的元件。这一提议的结果将为N1亚基的差异切片控制N1/N3A受体功能输出的机制提供洞察力。尽管近20年前已经克隆了N1/N3A受体，但人们对N1/N3A受体的作用机制及其在生理和病理状态中的作用知之甚少。这一提议的见解将刺激理性假说来解决这些知识差距，并将提供更全面的了解精神分裂症病理生理学的潜在分子机制。

DESCRIPTION (provided by applicant): Glycinergic N-methyl-D-aspartate receptors (NRs) are a unique type of excitatory channels. They differ from the traditional glutamatergic NRs in that that they are activated by glycine alone, are insensitive to glutamate, and have a lower unitary conductance, Ca2+ permeability, and sensitivity to voltage-dependent Mg2+ block. They are tetramers of GluN1 (N1) and GluN3 (N3A or N3B) subunits. The N1 subunit has eight splice variants (N1-1a through N1-4b), each with specific spatiotemporal expression patterns in the brain. The N3A subunit is specifically expressed neonatally and is critical for spine development and synaptic plasticity. Altered expression of N3A has been linked to the negative symptoms of schizophrenia. Contrary to what has been observed in glutamatergic NRs (N1/N2), strikingly distinct macroscopic current is produced dependent on the N1 splice variant with which N3A assembles. I propose that these differences may arise in part from distinct gating kinetics conferred onto N1/N3A receptors by the C-terminal cassettes of N1 splice variants. To test this hypothesis, I will pursue the following three aims: 1) Fully characterize the high-activity N1-4a/N3 isoform using kinetic analyses and state modeling. 2) Identify the kinetic contributions of each C-terminal cassette by systematically comparing reaction mechanisms of selected N1/N3A isoforms. 3) Identify the elements on each cassette responsible for gating modulation by combining sequence-based mutagenesis and kinetic analyses. The results from this proposal will provide insights into the mechanism by which differential slicing of N1 subunits controls the functional output of N1/N3A receptors. Despite having been cloned almost two decades ago, very little is known about the functional mechanism of N1/N3A receptors and how they contribute to both physiological and pathological states. Insights from this proposal will spur rational hypotheses to address these gaps in knowledge and will afford a more comprehensive understanding of the underlying molecular mechanism of the pathophysiology of schizophrenia.