Role of trace metal sensitive voltage-gated Ca2+ channels for neuroprotective and neurotoxic actions of endogenous Zn2+ and Cu2+ in the limbic system.

疏松结合(组织化学反应)的锌和铜离子可能是神经元传递的调节者，限制边缘通路的病理性高兴奋性，但它们也可能导致癫痫发作相关的兴奋性细胞死亡。更新建议继续关注两个电压门控钙通道(Cav2.3和Cav3.2)，这两个通道在突触传递中起关键作用，也是少数几个对大脑中静息Cu2+和Zn2+水平敏感的分子靶点之一。我们以前已经证明，并在我们的报告中证实，它们的遗传或药物消融对海人酸(KA)诱导的边缘癫痫具有显著的保护作用。到目前为止，我们的实验主要集中在体外锌离子对Cav2.3通道的影响以及在Cav2.3缺陷和Cav2.3功能正常的小鼠中的作用。微摩尔水平的锌离子以浓度依赖的方式改变克隆的人Cav2.3通道的门控和渗透。在癫痫发作期间，pH的病理生理变化会以不同的方式改变这些锌离子对Cav2.3的影响。在Cav2.3功能正常的小鼠中，微摩尔浓度的锌离子会引起行为和电生理的改变，这在Cav2.3缺陷小鼠中是没有的。在伸展阶段，我们将主要评估这些通道在静息时Cu2+的抗惊厥和神经保护作用以及在癫痫发作期间细胞内Cu2+(和Zn2+)进入的毒性效应。体内遥测皮质脑电图术将由免疫组织化学和死后自动移位分析补充，以评估Cu2+(和Zn2+)对野生型小鼠和缺乏相应钙通道亚单位的野生型小鼠和KA诱导的癫痫发作的影响。我们将继续在稳定转基因的HEK-293细胞中进行体外膜片钳记录，并将从天然神经元和海马片开始描述在其他参数干扰最小的条件下的细胞和分子作用机制。我们的项目仍然提供了在生理条件下和边缘系统过度激活期间锌与神经元兴奋性的相关性的功能洞察，这将扩展到铜与这些过程的相关性。这一应用推广不仅可以更好地理解大脑中保护性和毒性的锌离子(和铜)作用的明显二元性，而且还可以帮助指导基于对大脑中铜或锌离子水平的操纵而开发的预防性或干预性方法。

Losely bound (histochemically reactive) Zn2+ and Cu2+ ions are putative regulators of neuronal transmission and limit pathologic hyper-excitability in limbic pathways, but they can also contribute to seizure-related excitotoxic cell death. The renewal proposal continues to focus on the two voltage-gated Ca2+ channels (Cav2.3 and Cav3.2), which are critically involved in synaptic transmission and among the few molecular targets sensitive to resting Cu2+ and Zn2+ levels in the brain. We have previously shown and are confirming in our report that their genetic or pharmacologic ablation provides significant protection from kainic acid (KA)-induced limbic seizures. Up to now our experiments have focused on the effects of Zn2+ on Cav2.3 channels in-vitro and in Cav2.3-deficient and Cav2.3-competent mice. Micromolar levels of Zn2+ alter gating and permeation of the cloned human Cav2.3-channel in a concentration-dependent manner. Pathophysiological changes in pH, as they occur during epileptic seizures, differently alter the potency of these Zn2+ effects on Cav2.3. In Cav2.3-competent mice, micromolar Zn2+ concentrations provoke behavioral and electrophysiological changes, which are not found in Cav2.3-deficient mice.During the extension phase, we will mainly assess the role of these channels for the anti-convulsive and neuroprotective effects of Cu2+ at rest, and for the toxic effects of cellular Cu2+ (and Zn2+) entry during seizures. In vivo telemetric electrocorticography will be complemented by immunhistochemical and autometallographic post-mortem analysis to assess effects of Cu2+ (and Zn2+) on normal neurotransmission and KA-induced seizures in wild-type mice and mice lacking the respective Ca2+ channel subunits. We will continue with in vitro patch-clamp recordings in stably transfected HEK-293 cells, and will begin with native neurons and hippocampal slices to characterize cellular and molecular mechanisms of action under conditions of minimal interference from other parameters. Our project still provides functional insights into the relevance of Zn2+ for neuronal excitability under physiological conditions and during excessive activation of the limbic system, which will be extended to the relevance of Cu2+ for these processes. The applied extension may not only provide a better understanding of the apparent dualism of protective vs. toxic Zn2+ (and Cu2+) actions in the brain but could also help in guiding the development of preventive or interventional approaches based on a manipulation of brain Cu2+ or Zn2+ levels.