Role of Shaker Channel Function in the Regulation of Sleep in Drosophila

描述(申请人提供)：睡眠无处不在[1]，对生活至关重要[2]，而睡眠不足会导致几种有害的影响[3]。然而，由于缺乏针对睡眠的药物，睡眠调节的机制尚不清楚。对果蝇(即果蝇)的研究表明，编码Shaker K通道[4]和两个Shaker通道调节器(高动力[5]和无眠[6])的基因突变极大地减少了日常睡眠时间，这一点意义重大，因为K通道似乎在调节哺乳动物睡眠方面也具有类似的作用[7-8]。这一建议包括三个系列的研究，以调查Shaker、其调节器和睡眠之间的关系。第一个目标是确定果蝇中负责Shaker依赖的睡眠调节的神经元群体。这将通过在选定的大脑区域异位表达显性负Shaker亚单位(通过两部分GAL4系统)和分析异常睡眠表型来实现。预期的结果将有助于我们理解果蝇睡眠的神经解剖学，并为未来对这些候选细胞中的睡眠调节机制(S)进行分子解剖奠定基础。第二个目的是探索上述睡眠减少等位基因导致睡眠表型异常的可能细胞机制。这将通过将每个等位基因对果蝇神经肌肉接头处记录的突触活动和细胞离子电流的影响与它们导致的睡眠损失的严重程度相关联来实现。第三个目的是确定无眠调节Shaker通道活动的机制是否通过质膜上的直接相互作用。识别这种机制不仅可以深入了解睡眠调节，而且因为这将是一种全新的方法，通过它可以特别增强Shaker通道的活动，可能会对非睡眠产生重大影响(例如，开发新的抗高血压或抗心律失常药物)。 与公共健康相关：大多数美国人没有睡到推荐的“每晚8小时”[9]，导致睡眠不足，每年给美国经济带来大约150-750亿美元的压力[10-11]，并与几个主要的健康问题(如糖尿病、心脏病、癌症)[12-14]有关。然而，我们对睡眠调节的生物学机制知之甚少，这可能有助于开发干预措施来控制我们的睡眠需求。这项拟议的研究将调查在调节果蝇睡眠方面发挥作用的大脑解剖和细胞活动，为未来在哺乳动物中的研究奠定基础。

DESCRIPTION (provided by applicant): Sleep is ubiquitous[1] and vital for life[2], while sleep loss leads to several detrimental effects[3]. However, as demonstrated by the absence of sleep-specific drugs, the mechanism of sleep regulation is unknown. Studies in Drosophila (i.e. fruit flies) show that mutations in genes encoding the Shaker K+ channel[4] and two Shaker channel modulators (Hyperkinetic[5] and Sleepless[6]) dramatically reduce daily sleep time, which is significant because K+ channels seem to have a similar role in the regulation of mammalian sleep as well[7-8]. This proposal includes three lines of studies to investigate the relationships between Shaker, its modulators, and sleep. The first aim is to identify the neuronal populations responsible for the Shaker-dependent regulation of sleep in Drosophila. This will be accomplished by ectopically expressing dominant negative Shaker subunits in select brain areas (via the bipartite GAL4 system) and assaying for abnormal sleep phenotypes. The anticipated results will contribute to our understanding of the neuroanatomy of sleep in Drosophila and allow for future molecular dissection of the sleep regulatory mechanism(s) in these candidate cells. The second aim is to explore the possible cellular mechanism by which the aforementioned sleep- reducing alleles are responsible for their abnormal sleep phenotypes. This will be accomplished by correlating the effects of each of the alleles on synaptic activity and cellular ionic currents recorded at the Drosophila neuromuscular junction with the severities of sleep loss that they induce. The third aim is to determine whether the mechanism by which Sleepless modulates Shaker channel activity is through a direct interaction at the plasma membrane. Identification of such a mechanism would not only provide insight into sleep regulation, but, because this would be a completely novel means by which to specifically enhance Shaker channel activity, could have significant non-sleep implications (e.g. the development of new anti-hypertensive or antiarrythmic agents). PUBLIC HEALTH RELEVANCE: The majority of Americans do not sleep for the recommended "8 hours a night"[9], resulting in sleep deprivation that incurs an estimated ~$15-75 billion per year strain[10-11] on the US economy and is linked to several major health problems (e.g. diabetes, heart disease, cancer)[12-14]. However, we know very little about the biological mechanism of sleep regulation, which could be useful to develop interventions to manipulate our need for sleep. The proposed studies will investigate the brain anatomy and cellular activity that play a role in regulating sleep in the fruit fly, laying the foundation for future studies in mammals.