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

松散结合（组织化学反应）的Zn 2+和Cu 2+离子是神经元传递的假定调节剂，并限制边缘系统通路中的病理性超兴奋性，但它们也可能导致脑缺血相关的兴奋性毒性细胞死亡。更新建议继续关注两个电压门控Ca 2+通道（Cav2.3和Cav3.2），这两个通道在突触传递中起着关键作用，并且是对大脑中的静息Cu 2+和Zn 2+水平敏感的少数分子靶标之一。我们以前已经表明，并在我们的报告中证实，他们的遗传或药理学消融提供了显着的保护，从红藻氨酸（KA）诱导的边缘癫痫发作。到目前为止，我们的实验主要集中在体外和Cav2.3缺陷和Cav2.3感受态小鼠中Zn 2+对Cav2.3通道的影响。微摩尔水平的Zn 2+以浓度依赖性方式改变克隆的人Cav2.3通道的门控和渗透。pH的病理生理变化，因为它们发生在癫痫发作期间，不同地改变这些Zn 2+对Cav2.3的影响的效力。在Cav2.3感受态小鼠中，微摩尔Zn 2+浓度引起行为和电生理变化，这在Cav2.3缺陷小鼠中没有发现。在延伸阶段，我们将主要评估这些通道在休息时Cu 2+的抗惊厥和神经保护作用中的作用，以及在癫痫发作期间细胞Cu 2+（和Zn 2+）进入的毒性作用。体内遥测皮层电描记术将通过免疫组织化学和自显影尸检分析进行补充，以评估Cu 2+（和Zn 2+）对野生型小鼠和缺乏相应Ca 2+通道亚单位的小鼠的正常神经传递和KA诱导癫痫发作的影响。我们将继续在稳定转染的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切片开始，以在其他参数干扰最小的条件下表征细胞和分子作用机制。我们的项目仍然提供了功能性的见解锌+神经元兴奋性的相关性在生理条件下，并在过度激活的边缘系统，这将扩展到铜+这些过程的相关性。应用的扩展不仅可以更好地理解大脑中保护性与毒性Zn 2+（和Cu 2+）作用的明显二元论，而且还可以帮助指导基于操纵大脑Cu 2+或Zn 2+水平的预防或干预方法的发展。

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.