GATING KINETICS OF GLYCINERGIC NMDA RECEPTORS

描述（由申请人提供）：甘氨酸能N-甲基-D-天冬氨酸受体（NR）是一种独特类型的兴奋性通道。它们与传统的谷氨酸能NR的不同之处在于，它们仅由甘氨酸激活，对谷氨酸不敏感，并且具有较低的单位电导、Ca 2+渗透性和对电压依赖性Mg 2+阻断的敏感性。它们是GluN 1（N1）和GluN 3（N3 A或N3 B）亚基的四聚体。N1亚基有8个剪接变体（N1-1a至N1- 4 b），每个变体在大脑中具有特定的时空表达模式。N3 A亚基特异性表达于脊髓，对脊髓发育和突触可塑性至关重要。N3 A的表达改变与精神分裂症的阴性症状有关。相反，已经观察到的nRs（N1/N2），显着不同的宏观电流产生依赖于N1剪接变异体与N3 A组装。我建议，这些差异可能部分来自不同的门控动力学赋予N1/N3 A受体的C-末端盒的N1剪接变异体。为了验证这一假设，我将追求以下三个目标：1）充分表征高活性N1-4a/N3异构体使用动力学分析和状态建模。2)通过系统地比较所选N1/N3 A亚型的反应机制，确定每个C-末端盒的动力学贡献。3)通过结合基于序列的诱变和动力学分析，鉴定每个盒上负责门控调节的元件。从这个建议的结果将提供洞察N1亚基的差异切片控制N1/N3 A受体的功能输出的机制。尽管在近二十年前已经被克隆，但对N1/N3 A受体的功能机制以及它们如何促进生理和病理状态知之甚少。从这个建议的见解将刺激理性的假设，以解决这些知识的差距，并将提供一个更全面的了解精神分裂症的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.