红藻氨酸受体GluK1-3(谷氨酸受体离子型,红藻氨酸受体1-3)属于离子型谷氨酸受体家族,对于大脑中的快速兴奋性神经传递至关重要,并与神经和精神疾病有关。这些受体如何被小分子药物调节还没有很好的理解,尤其是GluK3.我们表明,正变构调节剂BPAM344可用于建立强大的钙敏感荧光为基础的检测激动剂,GluK1-3的拮抗剂和正变构调节剂。用于增强100μM红藻氨酸反应的BPAM344的半最大有效浓度(EC50)对于GluK1为26.3μM,对于GluK2为75.4μM,对于GluK3为639μM。发现Domoate是GluK1和GluK2的有效激动剂,EC50为0.77μM和1.33μM,分别,共同应用150μMBPAM344。在GluK3时,多莫酸作为一种非常弱的激动剂或拮抗剂,其半数最大抑制浓度(IC50)为14.5μM,在500μMBPAM344和100μM海藻酸盐存在下进行竞争结合。使用H523A突变的GluK3,我们通过X射线晶体学确定了配体结合域的第一个二聚体结构,允许定位BPAM344以及锌-,二聚体界面处的钠离子和氯离子结合位点。分子动力学模拟支持离子位点的稳定性以及Asp761,Asp790和Glu797在锌离子结合中的参与。使用电子显微镜,我们证明,在存在谷氨酸和BPAM344的情况下,全长GluK3采用二聚体的二聚体排列。
The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists, and positive allosteric modulators of GluK1-3. The half-maximal effective concentration (EC50) of BPAM344 for potentiating the response of 100 μm kainate was determined to be 26.3 μm for GluK1, 75.4 μm for GluK2, and 639 μm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC50 of 0.77 and 1.33 μm, respectively, upon co-application of 150 μm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC50) of 14.5 μm, in presence of 500 μm BPAM344 and 100 μm kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, as well as zinc-, sodium-, and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.