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（NRs）是一种独特的兴奋通道。它们与传统的谷氨酸能NRs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，Ca2+通透性和对电压依赖性Mg2+阻滞的敏感性。它们是GluN1 （N1）和GluN3 （N3A或N3B）亚基的四聚体。N1亚基有8个剪接变体（N1-1a至N1-4b），每一个都在大脑中具有特定的时空表达模式。N3A亚基在新生儿特异性表达，对脊柱发育和突触可塑性至关重要。N3A表达的改变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，显著不同的宏观电流的产生依赖于与N3A组装的N1剪接变体。我认为这些差异可能部分源于N1剪接变异体的c端盒传递给N1/N3A受体的不同门控动力学。为了验证这一假设，我将追求以下三个目标：1)利用动力学分析和状态建模充分表征高活性N1-4a/N3异构体。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)结合基于序列的诱变和动力学分析，确定每个磁带上负责门控调制的元件。本研究的结果将有助于深入了解N1亚基的差异切片控制N1/N3A受体功能输出的机制。尽管已经克隆了近20年，但人们对N1/N3A受体的功能机制以及它们如何影响生理和病理状态知之甚少。从这一建议中获得的见解将激发理性假设，以解决这些知识空白，并将对精神分裂症病理生理学的潜在分子机制提供更全面的理解。

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.