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

描述（由申请人提供）：睡眠是无所不在的[1]，对生命至关重要，而睡眠不足会导致一些不利影响[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。对果蝇的研究表明，编码Shaker K+通道[4]和两种Shaker通道调节剂（Hyperkinetic[5]和失眠[6]）的基因突变显著减少了每天的睡眠时间，这一点很重要，因为K+通道似乎在哺乳动物睡眠调节中也起着类似的作用[7-8]。该提案包括三条研究线，以调查震动，它的调节性，和睡眠之间的关系。第一个目标是确定果蝇中负责依赖于shaker的睡眠调节的神经元群。这将通过在选定的脑区（通过双侧GAL4系统）异位表达显性阴性Shaker亚基并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，并允许未来对这些候选细胞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导致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果蝇神经肌肉连接处记录的细胞离子电流的影响与它们引起的睡眠缺失的严重程度相关联来完成。第三个目的是确定失眠调节激振器通道活性的机制是否通过质膜上的直接相互作用。这种机制的识别不仅将提供对睡眠调节的深入了解，而且，因为这将是一种全新的手段，可以专门增强震颤通道的活性，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）。

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.