Kv2.1 membrane corrals:Regulators of K+ channel function and trafficking

描述（由申请人提供）：离子通道在结构和功能方面的研究取得了巨大的进展。然而，缺乏关于活细胞如何实时处理离子通道蛋白的信息。这些信息是重要的，因为离子通道表面表达的快速调制可能代表了细胞电兴奋性调节的中心机制。人类疾病已经与离子通道定位和运输缺陷联系在一起。Kv2.1延迟整流K+通道靶向海马神经元独特的细胞表面簇。由于这些结构在体内受到与神经元损伤相关的刺激（如缺氧、缺血和过量的神经递质释放）的调节，因此这些微结构域可能在这种损伤中参与神经保护反应。然而，Kv2.1也在细胞凋亡中发挥作用，这种Kv亚型提供了完成皮质神经元凋亡程序所需的K+通透性增加。这些不同的功能要求严格管制Kv2.1的活动和贩运。我们的总体假设是，Kv2.1表面簇是调节Kv2.1运输和功能的核心。因此，更好地理解集群功能将提高我们管理中风相关问题的能力。通过将这些通道聚集在一个定义的表面隔间内，作为插入和检索的平台，运输的调节比通道均匀分布在细胞表面更有效。此外，我们的初步数据表明，表面簇内的通道在从这些细胞表面区域释放之前不会传导K+，这表明K+电流密度不仅仅是由细胞表面的运输控制的。拟议的研究将采用多学科方法，利用活细胞成像、单通道检测/跟踪、定点诱变和电生理学。这三个特定目标将验证以下假设：1)Kv2.1簇代表通道插入和恢复的细胞表面平台；2)刺激诱导的皮质细胞骨架修饰导致细胞表面簇的立即释放；3)Kv2.1表面簇是包含非导电通道的细胞表面存储位点。这项研究的意义将远远超出神经生物学，因为Kv2.1是最广泛表达的Kv通道，对心血管和内分泌系统也具有重要的生理意义。公共卫生相关性：拟议的研究考察了脑神经元中Kv2.1钾通道的定位。这种定位在脑卒中损伤和大脑多动的神经保护反应中是必不可少的。缺乏Kv2.1通道的小鼠是癫痫和过度兴奋的。了解Kv2.1定位的分子机制将有助于加强中风和癫痫的治疗。

DESCRIPTION (provided by applicant): Tremendous advances in ion channel research have been made with respect to channel structure and function. However, there is a lack of information regarding how living cells process ion channel proteins in real time. Such information is important since rapid modulation of ion channel surface expression is likely to represent a central mechanism in the regulation of cellular electrical excitability. Human diseases have already been linked to ion channel localization and trafficking defects. The Kv2.1 delayed-rectifier K+ channel targets to unique cell surface clusters in hippocampal neurons. Since these structures are regulated in vivo by stimuli associated with neuronal injury, i.e. hypoxia, ischemia, and excess neurotransmitter release, these microdomains are likely to participate in the neuro-protective response during such insults. However, Kv2.1 also plays a role in apoptosis, with this Kv isoform providing the increased K+ permeability required for the completion of the apoptotic program in cortical neurons. These diverse functions mandate that Kv2.1 activity and trafficking be tightly regulated. Our overall hypothesis is that the Kv2.1 surface clusters are central in the regulation of both Kv2.1 trafficking and function. Thus, a better understanding of cluster function will improve our ability to manage stroke-related issues. By clustering these channels within a defined surface compartment that serves as a platform for both insertion and retrieval, the regulation of trafficking is more efficient than if the channel is homogenously distributed over the cell surface. In addition, our preliminary data suggest that channels within the surface clusters do not conduct K+ until released from these cell surface domains, suggesting that K+ current density is controlled by more than transport to the cell surface. The proposed research will employ a multidisciplinary approach utilizing live cell imaging, single channel detection/tracking, site-directed mutagenesis and electrophysiology. The three Specific Aims will test the hypotheses that 1) Kv2.1 clusters represent cell surface platforms for channel insertion and retrieval, 2) stimulus-induced modification of the cortical cytoskeleton causes immediate release from the cell surface cluster and 3) Kv2.1 surface clusters are cell surface storage sites containing non-conducting channel. This research will have implications far beyond neurobiology, for Kv2.1 is the most widely expressed Kv channel and is also physiologically important to the cardiovascular and endocrine systems. PUBLIC HEALTH RELEVANCE: The proposed research examines the localization of the Kv2.1 potassium channel in brain neurons. This localization is essential in the neuro-protective response to stroke injury and hyperactivity in the brain. Mice lacking the Kv2.1 channel are epileptic and hyper-excitable. Understanding the molecular mechanisms underlying Kv2.1 localization will enhance the treatment of stroke and epilepsy.