ION CHANNELS IN EPILEPSY AND AS TARGETS FOR ANTIEPILEPTIC DRUGS

癫痫中的离子通道及其作为抗癫痫药物的靶标

• 批准号: 6290637
• 负责人: MICHAEL A. ROGAWSKI
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6290637
• 关键词: 6,7 benzomorphan; NMDA receptors; anticonvulsants; chemical structure function; chloride channels; drug interactions; drug metabolism; evoked potentials; glutamate receptor; hippocampus; inhibitor /antagonist; neurons; neuropharmacology; tissue /cell culture; voltage /patch clamp; voltage gated channel

The objective of this project is to explore new strategies for the rational development of antiepileptic drugs based upon their interaction with neuronal ion channel systems. Cellular electrophysiological recording techniques are used to study drug modulation of neurotransmitter-gated and voltage-activated ion channels in brain slices, cultured neurons and heterologous cells transfected with cloned ion channel subunit genes. In the present reporting period, we continued our focus on glutamate receptors and glutamate receptor-mediated synaptic transmission. Functional activity of NMDA-type glutamate receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine. However, several other amino acids including D-serine can activate NMDA receptors when coapplied with glutamate. Although D-amino acids are prominent in bacteria, they generally are thought not to occur in mammals. However, high levelsof D-serine have recently been found in mammalian brain, suggesting that the amino acid could serve as an endogenous coagonist for NMDA receptors. We observed that D-amino acid oxidase, an enzyme that selectively degrades D-serine, greatly attenuates NMDA receptor-mediated neurotransmission as assessed using whole-cell patch-clamp recordings in cultured hippocampal neurons. The inhibitory effects of the enzyme were fully reversed by exogenously applied D-serine which did not by itself potentiate NMDA receptor-mediated synaptic responses. These observations indicate that D-serine is an endogenous modulator at the glycine site of NMDA receptors and that it may fully occupy this site at some functional synapses. Agents that modify the activity of D-serine metabolic enzymes, including the recently cloned mammalian serine racemase, could be useful to diminish excessive activation of NMDA receptors as may occur in epilepsy. Several widely used antiepileptic drugs are believed to act via use-dependent block of voltage-activated sodium channels. However, the way in which sodium channel modulation protects against seizures is not well understood. We examined the hypothesis that sodium channel blocking anticonvulsants, including phenytoin and zonisamide, act through inhibition of synaptic glutamate release. AMPA receptor-mediated spontaneous and evoked synaptic currents were recorded in CA1 pyramidal neurons of the rat hippocampal slice using whole-cell voltage clamp techniques. Clinically-relevant concentrations of zonisimide induced a large reduction in the frequency and amplitude of spontaneous excitatory synaptic currents without altering their kinetic properties. In contrast, the amplitude and frequency of miniature excitatory postsynaptic currents was unaffected by the drug, indicating that zonisimide acts presynaptically. Zonisimide had minimal effects on excitatory synaptic currents evoked by low frequency stimulation of the stratum radiatum. However, with high frequency stimulation there was a dramatic use-dependent inhibitory action on the release of excitatory transmitter. The widely used sodium channel blocking anticonvulsant phenytoin had similar actions. We conclude that sodium channel blocking anticonvulsants have a common action to reduce glutamate-mediated excitatory synaptic transmission through effects on glutamate release mechanisms.Studies were also continued examining glutamate receptor mediated neurotransmission and synaptic plasticity mechanisms in the amygdala, a key brain site for epileptogenesis in animal models and a common primary focus for seizures in human epilepsy. We previously demonstrated that a component of the excitatory synaptic response evoked in basolateral amygdala neurons by stimulation of the external capsule is mediated by kainate receptors containing the GluR5 subunit. Moreover, we observed that kainate receptor activation induces a novel form of NMDA receptor-independent enduring synaptic facilitation. We now demonstrate using in situ hybridization histochemistry that GluR5 kainate receptor mRNA is expressed at high levels in the basolateral amygdala; other kainate receptor subunit mRNAs were expressed at lower levels. GluR5 kainate receptors may be calcium permeable. To examine the hypothesis that calcium entry via GluR5 kainate receptors mediates GluR5 kainate receptor-dependent enduring synaptic facilitation, amygdala slices were treated with the membrane permeant calcium chelator BAPTA-AM. Slices exposed to BAPTA-AM failed to show GluR5 kainate receptor-mediated synaptic plasticity although synaptic responses were largely unaffected. Fura-2 imaging confirmed that BAPTA-AM produced a substantial reduction in intracellular calcium. We conclude that GluR5 containing kainate receptors mediate a novel form of calcium-dependent enduring synaptic facilitation. We propose that GluR5 kainate receptors could serve as mediators of epileptic hyperexcitability and epileptogenesis, such as occurs in response to kainate receptor agonists including the neurotoxins kainate and domoate, and we further suggest that drugs that block GluR5 kainate receptors could be useful in the prevention and treatment of some forms of epilepsy.

本项目的目的是探索基于与神经元离子通道系统相互作用的抗癫痫药物的合理开发新策略。细胞电生理记录技术被用来研究药物调制的神经递质门控和电压激活的离子通道在脑切片，培养的神经元和异源细胞转染克隆的离子通道亚基基因。在本报告期内，我们继续关注谷氨酸受体和谷氨酸受体介导的突触传递。NMDA型谷氨酸受体的功能活性需要谷氨酸结合和被推测为甘氨酸的内源性促凝剂的结合。然而，包括D-丝氨酸在内的其他几种氨基酸在与谷氨酸共同应用时可以激活NMDA受体。虽然D-氨基酸在细菌中很突出，但它们通常被认为不存在于哺乳动物中。然而，最近在哺乳动物脑中发现了高水平的D-丝氨酸，这表明该氨基酸可以作为NMDA受体的内源性促凝剂。我们观察到，D-氨基酸氧化酶，一种酶，选择性降解D-丝氨酸，大大减弱NMDA受体介导的神经传递使用全细胞膜片钳记录在培养的海马神经元进行评估。酶的抑制作用被完全逆转外源性应用的D-丝氨酸本身并不增强NMDA受体介导的突触反应。这些观察结果表明，D-丝氨酸是一个内源性的NMDA受体的甘氨酸位点的调节剂，它可能完全占据这个网站在一些功能性突触。改变D-丝氨酸代谢酶活性的药物，包括最近克隆的哺乳动物丝氨酸消旋酶，可用于减少可能发生在癫痫中的NMDA受体的过度活化。几种广泛使用的抗癫痫药物被认为通过使用依赖性阻断电压激活的钠通道起作用。然而，钠通道调节防止癫痫发作的方式还不清楚。我们研究了钠通道阻断抗惊厥药，包括苯妥英钠和唑尼沙胺，通过抑制突触谷氨酸释放发挥作用的假设。用全细胞电压钳技术记录大鼠海马脑片CA 1区锥体神经元AMPA受体介导的自发性和诱发性突触电流。临床相关浓度的唑尼酰亚胺诱导自发兴奋性突触电流的频率和振幅大幅降低，而不改变其动力学特性。相比之下，微型兴奋性突触后电流的振幅和频率不受药物的影响，这表明唑尼西胺在突触前发挥作用。唑尼酰胺对低频刺激辐射层诱发的兴奋性突触电流的影响极小。然而，高频刺激对兴奋性递质的释放有显著的使用依赖性抑制作用。广泛应用的钠通道阻断剂苯妥英钠也有类似作用。我们的结论是，钠通道阻断抗惊厥药物有一个共同的行动，以减少谷氨酸介导的兴奋性突触传递通过影响谷氨酸释放mechanism.Studies还继续检查谷氨酸受体介导的神经传递和突触可塑性机制的杏仁核，一个关键的大脑部位癫痫动物模型和癫痫发作的共同主要焦点在人类癫痫。我们以前证明，兴奋性突触反应的一个组成部分引起的基底外侧杏仁核神经元的刺激外囊介导的红藻氨酸受体含有GluR 5亚基。此外，我们观察到红藻氨酸受体激活诱导一种新形式的NMDA受体独立持久的突触易化。我们现在证明，使用原位杂交组织化学，GluR 5红藻氨酸受体mRNA的表达在基底外侧杏仁核高水平，其他红藻氨酸受体亚基mRNA的表达水平较低。GluR 5红藻氨酸受体可以是钙可渗透的。为了检验钙通过GluR 5红藻氨酸受体进入介导GluR 5红藻氨酸受体依赖性持久突触促进的假设，用膜渗透钙螯合剂BAPTA-AM处理杏仁核切片。暴露于BAPTA-AM的切片未能显示GluR 5红藻氨酸受体介导的突触可塑性，尽管突触反应在很大程度上不受影响。Fura-2成像证实，BAPTA-AM产生细胞内钙的大幅减少。我们得出结论，GluR 5含有红藻氨酸受体介导的钙依赖持久的突触促进的一种新形式。我们认为GluR 5红藻氨酸受体可以作为癫痫过度兴奋和癫痫发生的介质，例如发生在响应红藻氨酸受体激动剂，包括神经毒素红藻氨酸和domoate，我们进一步建议，药物，阻止GluR 5红藻氨酸受体可能是有用的，在预防和治疗某些形式的癫痫。