Genetic analysis to determine the functional role of GRID1

摘要 谷氨酸受体基因家族编码AMPA、海人藻酸和NMDA受体，它们介导 中枢神经系统中的兴奋性突触传递。另外两个基因家族成员GRID1 和GRID2，编码神秘的Delta受体GluD1和GluD2亚基，它们可以结合D-丝氨酸，但不能 似乎不会激活传统的信号系统。已知的Delta有四种功能效应 受体：(1)两种Delta受体(GluD1和GluD2)在细胞内发生某些突变时被激活 跨膜螺旋，它将非门控受体转化为结构性开放的通道， 产生一种强音内向电流。此外，谷氨酸结合域中的嵌合受体 从红藻氨酸或AMPA受体取代类似的D-丝氨酸结合结构域的GluD1和GluD2可以 被谷氨酸激活，这表明高度专门化的机制需要将激动剂结合 进入孔道开放是保守的，在德尔塔GluD1受体。(2)D-丝氨酸与GluD1受体的结合 携带TM3突变使其具有结构性活性可能会关闭该通道，从而增加这种可能性 D-丝氨酸结合在WT中产生有意义的构象变化，其生理作用未知 感受器。(3)钙离子与相邻两个D-丝氨酸结合区之间二聚体界面的结合 增强突变受体的构成电流，提示钙离子可以调节GluD1的构象和 功能。(4)远端的胞外区作为突触前Cbln2和neuresin的配体，可以 改变突触的形成。尽管Cbln2和neuresin与Glud1的结合似乎是至关重要的，但它仍然 目前尚不清楚GluD1的通道门控、D-丝氨酸结合或钙离子调节是否在脑内发挥重要作用。 群体遗传学的一种独特的力量是它可以在没有偏见的情况下识别蛋白质的关键功能 举止。GRID1是人体内耐力最差的基因之一，因缺乏而落在前2%的位置 变异，表明它起着至关重要的作用。与这一想法一致的是，患有神经疾病的患者 已被确定为GRID1中的错义变体，而普通人群中没有这种变体。我们会 除了通常观察到的耐受性良好的变异外，还评估与疾病相关的变异的影响 与GRID1功能相关的三种模式的健康人群--结构性激活、D-丝氨酸 结合、钙离子结合。如果这些功能属性中的任何一个是重要的，那么我们预计会发现疾病- 干扰它们的相关变异，而存在于现有种群中的变异应该没有 效果。三个电生理实验将回答以下问题： 目的1：错义变异能产生参与神经病理的活性离子通道吗？ 目的2：错义突变是否会改变D-丝氨酸和钙离子对结构性活性通道的作用？ 目的3：错义突变是否会改变谷氨酸对GluD1-GluK2嵌合受体的作用？

Summary The glutamate receptor gene family encodes AMPA, kainate, and NMDA receptors, which mediate excitatory synaptic transmission in the central nervous system. Two additional gene family members, GRID1 and GRID2, encode the enigmatic delta receptor GluD1 and GluD2 subunits, which can bind D-serine but do not appear to activate conventional signaling systems. There are four functional effects known for delta receptors: (1) Both delta receptors (GluD1 and GluD2) are activated when certain mutations occur in the transmembrane helices, which convert a non-gating receptor into a channel that is constitutively open, producing a tonic inward current. In addition, chimeric receptors in which the glutamate binding domain from a kainate or AMPA receptor replaces the analogous D-serine binding domain for GluD1 and GluD2 can be activated by glutamate, suggesting that the highly specialized machinery needed to convert agonist binding into pore opening is conserved in delta GluD1 receptors. (2) The binding of D-serine to GluD1 receptors harboring a TM3 mutation that renders them constitutively active can close the channel, raising that possibility that D-serine binding produces meaningful conformational changes with unknown physiological roles in WT receptors. (3) Ca2+ binding to a site at the dimer interface between two adjacent D-serine binding domains potentiates constitutive current in mutant receptors, suggesting Ca2+ could regulate GluD1 conformation and function. (4) The distal extracellular domain serves as a ligand for presynaptic Cbln2 and neurexin, which can alter synapse formation. Whereas Cbln2 and neurexin binding to GluD1 appears to be critical, it remains unclear whether channel gating, D-serine binding, or Ca2+ regulation of GluD1 play important roles in brain. A unique power of population genetics is that it can identify key functions of a protein in an unbiased manner. GRID1 is one of the least tolerant genes in the body, falling in the top 2 percentile for lacking variation, suggesting it plays essential roles. Consistent with this idea, patients with neurological conditions have been identified with missense variants in GRID1 that are absent in the general population. We will evaluate the effects of disease-associated variants in addition to well-tolerated variants commonly observed in the healthy population on three modalities associated with GRID1 function—constitutive activation, D-serine binding, Ca2+ binding. If any of these functional attributes are important, then we expect to find disease- associated variants that perturb them, while variants present in the standing population should be without effect. Three electrophysiological experiments will answer the following questions: Aim 1: Can missense variants produce active ion channels that are involved in neuropathology? Aim 2: Do missense variants alter the actions of D-serine and Ca2+ on constitutively active channels? Aim 3: Do missense variants alter the actions of glutamate on GluD1-GluK2 chimeric receptors?