Calcium channel and glutamate receptor signaling at synapses

 描述(申请人提供)：T型CaV3.2钙通道广泛表达于各种类型的神经元中，CaV3.2通道功能障碍与人类儿童失神癫痫(CAE)密切相关。然而，这些钙通道在神经元中的作用仍不清楚。更令人惊讶的是，约50%的CAE患者对用于治疗CAE的T型钙通道拮抗剂和一线药物乙琥胺没有反应，大多数无反应的患者携带功能获得性Cav3.2突变。在这个项目中，我们计划研究CaV3.2通道在神经细胞中的功能作用以及~20个CAE连锁的人类CaV3.2通道突变的发病机制。在初步研究中，我们从遗传学和药理学的角度对CaV3.2通道的活性进行了调控，并通过电生理、双光子成像、电子显微镜和行为学分析对其影响进行了监测。我们的初步结果一致表明，与其他钙通道不同，CaV3.2通道主要调节NMDA-R介导的突触传递。因此，我们假设CaV3.2通道调节突触NMDA传递，CAE连锁的CaV3.2通道突变通过增强谷氨酸能传递增强失神发作的易感性。具体地说，我们将研究CaV3.2通道的活性是否在体外和体内增强多种不同类型的大鼠神经元的NMDA和AMPA反应(目标1a)。此外，我们计划研究CaV3.2通道活动耦合的突触钙内流是否增强NMDA反应，从而导致AMPA反应的继发性增强(目标1b)。这些结果表明，神经元CaV3.2通道的主要生理功能是调节谷氨酸能突触传递。此外，我们将研究~20个与CAE相关的人类CaV3.2突变如何影响突触谷氨酸能传递(目标2a)。最后，我们计划研究大约20个与CAE相关的人类CaV3.2突变是否可以增加2-4赫兹棘波放电和失神样癫痫的易感性，以及谷氨酸受体拮抗剂是否可以抑制癫痫发作(目标2b)。这些结果将为这一机制提供新的线索，并为人类CaV3.2突变相关的CAE提供新的干预措施。

 DESCRIPTION (provided by applicant): T-type CaV3.2 calcium channels are widely expressed in various types of neurons and dysfunction of CaV3.2 channels has been strongly implicated in human childhood absence epilepsy (CAE). However, the role of these calcium channels in neurons remains unknown. More surprisingly, ~50% CAE patients did not respond to ethosuximide, a T-type Ca2+ channel antagonist and first-line drug used to treat CAE, and most of the nonresponsive patients carry gain-of-function Cav3.2 mutations. In this project, we plan to investigate the functional role of CaV3.2 channels in neuronal cells and the pathogenesis of ~20 CAE-linked human CaV3.2 channel mutations. In the preliminary investigation, we manipulated the activity of CaV3.2 channels genetically and pharmacologically, and monitored the effects with electrophysiological, two-photon imaging, electron microscopic and behavioral analyses. Our preliminary results consistently show that unlike other calcium channels, CaV3.2 channels function primarily to regulate NMDA-R-mediated transmission at synapses. Therefore, we hypothesize that CaV3.2 channels regulate synaptic NMDA transmission and that CAE- linked CaV3.2 channel mutations enhance susceptibility to absence seizures by potentiating glutamatergic transmission. Specifically, we will examine whether the activity of CaV3.2 channels enhances NMDA and AMPA responses in multiple different types of rat neurons in vitro and in vivo (Aim 1a). Moreover, we plan to study whether CaV3.2 channel activity-coupled synaptic calcium influx enhances NMDA responses that lead to the secondary potentiation of AMPA responses (Aim 1b). These results will define that the primary physiological function of neuronal CaV3.2 channels is to regulate glutamatergic synaptic transmission. In addition, we will examine how each of ~20 CAE- linked human CaV3.2 mutations may affect synaptic glutamatergic transmission (Aim 2a). Finally, we plan to investigate whether each of ~20 CAE-linked human CaV3.2 mutations may enhance the susceptibility to 2-4 Hz spike-and-wave discharges and absence-like seizures and if the seizures can be suppressed by glutamate receptor antagonists (Aim 2b). These results will shed new light on the mechanism and suggest new intervention for human CaV3.2 mutation-associated CAE.