N-type Calcium Channels in Nocipeptive Neurons

 描述（由申请人提供）：N型CaV2.2钙通道是调节脊髓浅背角伤害感受器释放谷氨酸和P物质的至关重要的蛋白质。伤害感受器对有害信号（如高温）做出反应，并且在正常工作时具有高度保护性。在某些慢性疼痛状况下，例如在外周神经损伤后，正常的热和触摸被认为是疼痛的，并且疼痛回路的持续自发活动可以引起持续的疼痛。了解从正常到慢性疼痛状态的转变过程中发生的分子和细胞变化是改善目前治疗慢性疼痛的治疗方法的关键。脊髓背角中的突触前N型钙通道是包括吗啡在内的用于治疗神经性和慢性疼痛综合征的药物的主要靶点。在这个项目的前两个阶段，我们发现，在哺乳动物神经系统不同的神经元群体表达不同亚型的N型钙通道。最重要的是，我们在伤害感受器中发现了一种新的N型通道亚型，我们预测当神经元高速放电时，这种通道对吗啡的抑制特别敏感。这一令人兴奋的发现提出了一种可能性，即可能开发出选择性作用于伤害感受器中的N型通道的药物或疗法，这些通道负责慢性疼痛，而对神经系统中其他地方的N型通道影响较小。在我们项目的第三阶段，我们将i）鉴定控制伤害感受器中N型钙通道亚型表达模式的基因组DNA和前mRNA机制，以及这些机制如何实现细胞特异性，ii）鉴定在脊髓背角中突触前神经末梢起作用的精确的N型钙通道亚型，并建立它们对阿片类药物和其他镇痛剂的独特反应性，和iii）显示突触前N-型钙通道的活性和性质在慢性疼痛状态中如何改变。为了完成这个项目，我们已经产生了几种转基因小鼠品系，其中我们去除了单个剪接选择，减少了可用的N型通道亚型的数量。此外，我们使用光遗传学结合retrolabeling来检查背角投射神经元中的突触事件，这些事件是由光诱导的热敏传入激活引起的。我们整合基因调控，离子通道功能，突触传递，行为和药理学的分析，以促进我们对这一高度重要的钙离子通道组的理解。我们的目标是确定精确的钙通道亚型，控制正常和异常的疼痛信号从外周伤害感受器到大脑中的中央处理部位的传输。我们的研究结果应该导致新的策略，以帮助数以百万计的神经性和慢性疼痛患者。

 DESCRIPTION (provided by applicant): N-type, CaV2.2 calcium channels are critically important proteins that regulate release of glutamate and substance P from nociceptors in the superficial dorsal horn of the spinal cord. Nociceptors respond to harmful signals such as high heat and, when functioning normally, are highly protective. In certain chronic pain conditions, such as after peripheral nerve injury, normal heat and touch are perceived as painful, and ongoing spontaneous activity of pain circuits can cause unrelenting pain. Understanding the molecular and cellular changes that occur during the transition from normal to chronic pain states is the key to improving current - and inadequate - therapies to treat chronic pain. Presynaptic N-type calcium channels in the spinal dorsal horn are major targets of drugs, including morphine, that are used to treat neuropathic and chronic pain syndromes. In the first two phases of this project we discovered that distinct neuronal populations in the mammalian nervous system express different isoforms of N-type calcium channels. Most importantly, we discovered a novel N-type channel isoform in nociceptors, which we predicted is particularly sensitive to inhibition by morphine when neurons fire at high rates. This exciting discovery has raised the possibility that drugs or therapies might be developed that act selectively on the N-type channels in the nociceptors that are responsible for chronic pain with less influence on N-type channels elsewhere in the nervous system. In the third phase of our project we will i) identify genomic DNA and pre-mRNA mechanisms that control expression patterns of N-type calcium channel isoforms in nociceptors and how these mechanisms achieve cell-specificity, ii) identify the precise N-type calcium channel isoforms that function at presynaptic nerve terminals in the spinal dorsal horn and establish their unique responsiveness to opiates and other analgesics, and iii) show how the activity and properties of presynaptic N-type calcium channels are altered in chronic pain states. To complete this project we have generated several genetically modified strains of mice in which we remove individual splice options, reducing the number of N-type channel isoforms available. In addition we use optogenetics combined with retrolabeling to examine synaptic events in dorsal horn projection neurons that arise from light-induced activation of thermosensing afferents. We integrate analyses of gene regulation, ion channel function, synaptic transmission, behavior and pharmacology to advance our understanding of this highly important group of calcium ion channels. We aim to identify the precise calcium channel isoforms that control transmission of normal and abnormal pain signals from peripheral nociceptors to central processing sites in the brain. Our results should lead to new strategies to help millions of neuropathic and chronic pain sufferers.