N-type Calcium Channels in Nociceptive Neurons

描述(申请人提供)：N型钙通道调节脊髓背角浅层初级感觉神经传入的谷氨酸和P物质的释放。脊髓背角突触前N型钙通道是治疗神经病理性和慢性疼痛综合征药物的主要靶点。然而，我们还不知道为什么N型钙通道阻滞剂对神经病理性疼痛如此有效，我们也不知道它们在介导阿片类药物的脊髓镇痛作用中有多重要。在我们项目的第二阶段，我们将检验我们的假设，即一种不同的N型钙通道对于包括阿片类药物在内的一系列药物在体内的脊髓水平止痛作用是必不可少的。在这笔赠款的第一个资助期内，我们发现这种新型剪接异构体N型钙通道Cav2.2-e37a对通过G蛋白偶联受体作用的药物明显更敏感，包括吗啡。这一点很重要，因为我们先前已经证明，Cav2.2-e37a通道富含伤害性感受器，而Cav2.2-e37b通道遍布整个神经系统，以及ii)吗啡和其他药物的脊髓水平镇痛作用至少部分是通过抑制背角突触前N型通道介导的。我们现在准备在体内测试Cav2.2-e37a在正常和疾病状态下的功能意义。我们利用单外显子基因打靶技术开发了四个新的小鼠品系。这些小鼠经过基因改造，只表达含有Cav2.2-e37a或Cav2.2-e37b的N型通道，但不能同时表达这两种通道。我们已经开发出所有必要的技术专长，在电生理、生化、免疫组织化学和行为水平上对这些小鼠进行评估。我们将询问Cav2.2-e37a通道是否介导吗啡的基础伤害性感受和基础镇痛效应。我们还将询问Cav2.2-e37a通道是否需要痛觉过敏与神经病理性疼痛的发展，以及它们是否有必要在神经病理性疼痛中介导吗啡的止痛效果。我们的研究与寻找有效治疗神经性疼痛和更普遍的慢性疼痛的挑战高度相关。周围神经疾病可在急性神经损伤后发生，并与糖尿病、自身免疫性疾病、营养不良和感染密切相关。数以百万计的神经性和慢性疼痛患者没有有效的治疗方法。 公共卫生相关性：我们的研究与寻找治疗神经性疼痛和更普遍的慢性疼痛的有效方法高度相关。通过研究疼痛通路中N型钙通道的一种新形式，我们对新型敲击小鼠的研究应该会导致抑制这种N型钙通道活性和减轻疼痛患者的新策略。)

DESCRIPTION (provided by applicant): N-type calcium channels regulate release of glutamate and substance P from primary sensory afferents in the superficial dorsal horn of the spinal cord. Presynaptic N-type calcium channels in the spinal dorsal horn are major targets of drugs to treat neuropathic and chronic pain syndromes. However, we don't yet understand why N-type calcium channel blockade is so effective against neuropathic pain and we don't understand how important they are in mediating the spinal analgesic actions of opiates. In this second phase of our project we will test our hypothesis that a distinct isoform of N-type calcium channels is essential for spinal level analgesic actions of a sub-set of drugs including opiates in vivo. During the 1st funding period of this grant we discovered that this novel splice isoform the N-type calcium channel, CaV2.2-e37a, is significantly more sensitive to drugs that act through G protein coupled receptors, including morphine. This is important because i) we previously showed that CaV2.2-e37a channels are enriched in nociceptors whereas CaV2.2-e37b channels are found throughout the nervous system, and ii) spinal level analgesic actions of morphine and other drugs are, at least in part, mediated by inhibition of presynaptic N-type channels in the dorsal horn. We are now ready to test the functional significance of CaV2.2-e37a in vivo in normal and disease states. We have developed four novel mouse lines using single exon gene targeting. These mice are genetically modified to express exclusively N-type channels containing either CaV2.2-e37a or CaV2.2-e37b but not both. We have developed all the necessary technical expertise to assess these mice at electrophysiology, biochemical, immunohistochemical, and behavioral levels. We will ask if CaV2.2-e37a channels mediate basal nociception and basal analgesic effects of morphine. We will also ask if CaV2.2-e37a channels are required for hyperalgesia to develop with neuropathic pain, and if they are necessary to mediate the analgesic effects of morphine during neuropathic pain. Our studies are highly relevant to the challenge of finding effective treatment for neuropathic and more generally chronic pain. Peripheral neuropathies can develop following acute nerve injury, and are strongly associated with diabetes, autoimmune disorders, malnutrition, and infections. There are no effective treatments for the millions of neuropathic and chronic pain sufferers. PUBLIC HEALTH RELEVANCE: Our studies are highly relevant to finding effective treatment for neuropathic and more generally chronic pain. By studying a novel form of the N-type calcium channel in the pain pathway, our studies of novel knock-in mice should lead to new strategies for inhibiting the activity of this N-type calcium channel and alleviating pain sufferers. )