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

睡眠是一种普遍存在的现象[1]，对生命至关重要[2]，而睡眠不足会导致几种不利影响[3]。然而，由于缺乏睡眠特异性药物，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]和两种Shaker通道调节剂（Hyperkinetic[5]和Sleepless[6]）的基因突变显著减少了每日睡眠时间，这是重要的，因为K+通道似乎在哺乳动物睡眠调节中也具有类似的作用[7-8]。该提案包括三个研究路线，以调查Shaker及其调节剂与睡眠之间的关系。 第一个目标是确定负责果蝇睡眠的Shaker依赖性调节的神经元群体。这将通过在选择的脑区域异位表达显性负性Shaker亚基（通过二分GAL 4系统）并测定异常睡眠表型来实现。预期的结果将有助于我们对果蝇睡眠神经解剖学的理解，并允许未来对这些候选细胞中的睡眠调节机制进行分子解剖。 第二个目的是探索上述睡眠减少等位基因负责其异常睡眠表型的可能细胞机制。这将通过将每个等位基因对突触活动和在果蝇神经肌肉接头处记录的细胞离子电流的影响与它们诱导的睡眠丧失的严重程度相关联来实现。 第三个目的是确定不眠调节振动通道活性的机制是否是通过质膜上的直接相互作用。这种机制的鉴定不仅可以提供对睡眠调节的深入了解，而且因为这将是一种完全新颖的方法，通过这种方法可以特异性地增强Shaker通道活性，因此可能具有重要的非睡眠意义（例如开发新的抗高血压或抗心律失常药物）。 公共卫生相关性：大多数美国人没有达到推荐的“每晚8小时”[9]，导致睡眠不足，估计每年对美国经济造成约150 - 750亿美元的压力[10-11]，并与几个主要健康问题（例如糖尿病，心脏病，癌症）有关[12-14]。然而，我们对睡眠调节的生物机制知之甚少，这可能有助于开发干预措施来控制我们的睡眠需求。这项研究将调查在调节果蝇睡眠中发挥作用的大脑解剖学和细胞活动，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.