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Abstract：
Numerous rare variants that cause neurodevelopmental disorders (NDDs) occur within genes encoding synaptic proteins, including ionotropic glutamate receptors. However, in many cases, it remains unclear how damaging missense variants affect brain function. We determined the physiological consequences of an NDD causing missense mutation in the GRIK2 kainate receptor (KAR) gene, that results in a single amino acid change p.Ala657Thr in the GluK2 receptor subunit. We engineered this mutation in the mouse Grik2 gene, yielding a GluK2(A657T) mouse, and studied mice of both sexes to determine how hippocampal neuronal function is disrupted. Synaptic KAR currents in hippocampal CA3 pyramidal neurons from heterozygous A657T mice exhibited slow decay kinetics, consistent with incorporation of the mutant subunit into functional receptors. Unexpectedly, CA3 neurons demonstrated elevated action potential spiking because of downregulation of the small-conductance Ca2+ activated K+ channel (SK), which mediates the post-spike afterhyperpolarization. The reduction in SK activity resulted in increased CA3 dendritic excitability, increased EPSP-spike coupling, and lowered the threshold for the induction of LTP of the associational-commissural synapses in CA3 neurons. Pharmacological inhibition of SK channels in WT mice increased dendritic excitability and EPSP-spike coupling, mimicking the phenotype in A657T mice and suggesting a causative role for attenuated SK activity in aberrant excitability observed in the mutant mice. These findings demonstrate that a disease-associated missense mutation in GRIK2 leads to altered signaling through neuronal KARs, pleiotropic effects on neuronal and dendritic excitability, and implicate these processes in neuropathology in patients with genetic NDDs.SIGNIFICANCE STATEMENT Damaging mutations in genes encoding synaptic proteins have been identified in various neurodevelopmental disorders, but the functional consequences at the cellular and circuit level remain elusive. By generating a novel knock-in mutant mouse, this study examined the role of a pathogenic mutation in the GluK2 kainate receptor (KAR) subunit, a subclass of ionotropic glutamate receptors. Analyses of hippocampal CA3 pyramidal neurons determined elevated action potential firing because of an increase in dendritic excitability. Increased dendritic excitability was attributable to reduced activity of a Ca2+ activated K+ channel. These results indicate that a pathogenic KAR mutation results in dysregulation of dendritic K+ channels, which leads to an increase in synaptic integration and backpropagation of action potentials into distal dendrites.
摘要：
许多罕见的变异导致神经发育障碍（NDD）发生在基因编码突触蛋白，包括离子型谷氨酸受体。然而,在许多情况下,目前尚不清楚破坏性错觉变异如何影响大脑功能。我们确定了NDD导致GRIK2红藻氨酸受体（KAR）基因错义突变的生理后果，这导致GluK2受体亚基中的单个氨基酸变化p.Ala657Thr。我们在小鼠Grik2基因中设计了这种突变，产生GluK2（A657T）小鼠，并研究了两种性别的小鼠，以确定海马神经元功能是如何被破坏的。来自杂合A657T小鼠的海马CA3锥体神经元中的突触KAR电流表现出缓慢的衰减动力学，与突变亚基掺入功能受体一致。出乎意料的是,由于小电导Ca2激活的K通道（SK）的下调，CA3神经元表现出升高的动作电位尖峰，介导后尖峰超极化。SK活性的降低导致CA3树枝状兴奋性增加,增加的EPSP-尖峰耦合，并降低了CA3神经元中缔合连合突触的LTP诱导阈值。WT小鼠中SK通道的药理学抑制增加了树突兴奋性和EPSP-尖峰偶联，模拟A657T小鼠的表型，并提示在突变小鼠中观察到的异常兴奋性中SK活性减弱的致病作用。这些发现表明，GRIK2中的疾病相关错义突变导致通过神经元KAR的信号改变，对神经元和树突兴奋性的多效性作用，并将这些过程与遗传性NDD患者的神经病理学联系起来。已经在各种神经发育障碍中鉴定了编码突触蛋白的基因中的破坏性突变,但是细胞和电路层面的功能后果仍然难以捉摸。通过产生一种新型敲入突变小鼠，这项研究检查了致病性突变在GluK2红藻氨酸受体（KAR）亚基中的作用，离子型谷氨酸受体的一个亚类。海马CA3锥体神经元的分析表明，由于树突兴奋性的增加，动作电位放电升高。树突兴奋性的增加归因于Ca2激活的K通道的活性降低。这些结果表明，致病性KAR突变导致树突状K+通道的失调，这导致突触整合的增加和动作电位向远端树突的反向传播。
