FUNCTIONAL ANALYSIS OF CALCIUM CHANNEL MUTATIONS IN TRIGEMINAL NOCICEPTION

描述（由申请人提供）：偏头痛是最常见的神经血管疾病之一，具有明显的遗传易感性。了解偏头痛的机制将导致更具体的治疗。家族性偏瘫偏头痛（Familial hemplegic Migraine， FHM）是一种罕见的遗传性偏头痛，伴有先兆和偏瘫，是探索偏头痛病理生理的良好模型。FHM 1型基因（FHM-1）编码P/Q型电压门控Ca2+通道的成孔1A亚基，这是支持电压依赖性Ca2+进入的关键角色，这对于神经递质在突触前末端的释放和突触后体细胞和树突中的Ca2+信号传导至关重要。FHM-1患者中多个1A突变的发现为阐明FHM-1以及偏头痛的机制提供了一个平台。我们已经启动了实验来探索T666M（最常见的FHM-1突变）对三叉神经痛觉通路中负责头痛产生的神经元中电压依赖性Ca2+内流的影响。我们的初步研究表明，T666M导致培养的三叉神经节和颈背角神经元中通过P/Q型通道的Ca2+内流减少。有趣的是，随之增加的低压门控t型电流仅在三叉神经节小肽能神经元中观察到。本课题的研究目的是进一步研究T666M突变对三叉神经节和背角神经元的功能影响。我们假设，以T666M为代表的FHM-1突变的功能丧失可能通过两种可能的情况增加三叉神经伤害感觉回路的增益：1)增加三叉神经初级传入神经元的兴奋性；2)优先削弱颈/髓背角的抑制性突触传递。在第一个目标中，我们将使用电流钳记录来检查T666M突变通道对神经元兴奋性的影响。该提案的第二个目的是解决T666M突变通道如何影响来自伤害性神经元的突触传递。此外，我们将以1A功能丧失突变的小鼠为实验模型，测试P/ q型通道缺陷对三叉神经损伤性神经元兴奋性和神经传递的影响。总之，这些实验不仅将增加我们对电压门控Ca2+通道对FHM-1和一般偏头痛病理生理的贡献的理解；但也揭示了其他形式的头痛的潜在机制。重要的是，这些研究将为未来在三叉神经伤害回路背景下FHM-1突变的功能后果的研究以及从系统神经生物学的角度奠定基础。公共卫生相关性：偏头痛是最常见的神经血管疾病之一，是医疗保健系统的巨大负担。了解疾病机制将极大地促进偏头痛预防和姑息治疗药物的开发。人类P/ q型电压门控Ca2+通道的多重突变与家族性偏瘫偏头痛1型(FHM-1) -一种偏头痛的遗传形式有关。我们建议研究T666M的功能后果，这是最常见的突变，作为了解偏头痛机制的途径。

Description (provided by applicant): Migraine is one of the most common neurovascular disorders with noticeable genetic predisposition. Understanding the mechanisms of migraine will lead to more specific treatments. Familial Hemiplegic Migraine (FHM), a rare hereditary form of migraine with aura and hemiparesis, serves as a good model for exploring migraine pathophysiology. The gene for FHM type 1 (FHM-1) encodes the pore-forming 1A subunit of P/Q- type voltage-gated Ca2+ channels - a key player in supporting voltage-dependent Ca2+ entry that is critical for neurotransmitter release at presynaptic terminals and postsynaptic Ca2+ signaling in soma and dendrites. The discovery of multiple 1A mutations in FHM-1 patients provides a platform for elucidating the mechanism underlying FHM-1 as well as migraine in general. We have initiated experiments to explore the effects of T666M, the most frequently occurring FHM-1 mutation, on voltage-dependent Ca2+ influx in neurons from the trigeminal nociceptive pathway responsible for headache pain generation. Our preliminary studies indicate that T666M results in a decrease of Ca2+ influx through P/Q- type channels in cultured neurons from trigeminal ganglion as well as cervical dorsal horn. Interestingly, a consequent increase of low-voltage gated T-type current is only observed in the small peptidergic trigeminal ganglion neurons. The research objective of this proposal is to further investigate the functional consequences of the T666M mutation in trigeminal ganglion and dorsal horn neurons. We hypothesize that loss-of-function FHM-1 mutations as represented by T666M may increase the gain of trigeminal nociceptive circuitry via two possible scenarios: 1) increasing the excitability of trigeminal primary afferent neurons and 2) preferentially weakening the inhibitory synaptic transmission at cervical/medullary dorsal horn. In the first aim, we will examine the effect of T666M mutant channels on neuronal excitability using current clamp recordings. The second aim of the proposal addresses how T666M mutant channels affect synaptic transmission from nociceptive neurons. In addition, we will use tottering, mice with a loss-of-function 1A mutation, as experimental model to test the effect of defect P/Q-type channels on the excitability and neurotransmission of trigeminal nociceptive neurons. Together, these experiments will not only increase our understanding of the contribution of voltage-gated Ca2+ channels to FHM-1 and general migraine pathophysiology; but also shed light on the mechanisms underlying other forms of headache. Importantly, these studies will lay ground work for future investigations of the functional consequences of FHM-1 mutations in the context of trigeminal nociceptive circuit as well as from a systems neurobiology perspective. PUBLIC HEALTH RELEVANCE: Migraine is one of the most common neurovascular disorders and an enormous burden to the healthcare system. Understanding the disease mechanisms will greatly facilitate drug development for both preventive and palliative therapies of migraine. Multiple mutations in human P/Q-type voltage-gated Ca2+ channels have been associated with familial hemiplegic migraine type 1 (FHM-1) - a hereditary form of migraine. We propose to study the functional consequences of T666M, the most frequently occurred mutation, as a gateway towards understanding the mechanisms underlying migraine headache.