Dynamic modulation of ionic and lipid signaling by neuronal Kv2 channels

本提案中概述的训练计划侧重于定义由电压门控K+通道Kv2.1控制的基本神经生理功能。Kv2.1通道在靠近内质网（ER）的神经元体细胞上形成突出的质膜（PM）簇。这些Kv2.1相关的ER- pm连接或epj通常含有Ca2+处理机制，包括l型Ca2+通道（ltcc）和ryanodine受体（RyR） ER Ca2+释放通道。除了作为Ca2+摄取和释放的重要位点外，epj还在调节细胞脂质处理中发挥重要作用。内质网和PM之间的脂质转移可以受到Ca2+的急性调节，epj上的脂质调节酶对细胞Ca2+动力学起相互作用。由于Kv2.1簇增强epj的形成并可能调节这些位点的Ca2+信号，Kv2.1完全准备好整合和控制神经元Ca2+和脂质信号。重要的是，临床研究结果表明，Kv2.1相关的epj对正常脑功能至关重要：Kv2.1中三种不同的突变破坏了其与epj聚集组织所需的通道结构域，导致严重的神经发育迟缓。然而，Kv2.1相关epj的分子结构、调控和功能作用仍然知之甚少。这是确定Kv2.1通道如何促进正常神经元功能的主要障碍，并限制了我们对其在衰弱性神经元疾病发病机制中的作用的理解。虽然其在神经元中的作用尚不清楚，但发现Kv2.1在神经内分泌细胞中的聚集可促进致密核囊泡（DCV）的胞吐，DCV是神经元中含有多种神经活性货物的分泌细胞器。由于神经元DCV释放缺陷与自闭症、焦虑症和癫痫有关，因此确定Kv2.1通道与DCV释放之间的分子交叉点非常重要。我假设kv2.1相关的epj控制神经元Ca2+和脂质信号来调节DCV的释放。我将通过确定Kv2.1通道调节局部Ca2+和脂质稳态和神经元信号传导的机制来验证中心假设（目的1）。这些发现将扩展到Kv2.1通道如何促进体树突DCV释放的详细研究（目的2）。本研究的成功完成将促进我们对调节神经元功能的基本机制的理解。此外，阐明Kv2.1通道对神经元DCV释放的影响将极大地扩展对DCV胞吐机制的理解，也可能提高对Kv2.1对神经系统疾病贡献的机制的理解。通过这个奖学金，我将发展1)对Kv2.1通道的生理功能的新认识，以及2)我作为一个专注于离子通道生物学的独立研究者的潜力。这些培训目标将通过详细的研究计划，在研究设计方面具有专业知识的特别合格的导师，以及加州大学戴维斯分校优秀的设施和培训资源来促进。

The training plan outlined in this proposal focuses on defining the fundamental neurophysiological functions controlled by the voltage-gated K+ channel Kv2.1. Kv2.1 channels form prominent plasma membrane (PM) clusters on the neuronal soma that are in close proximity to the endoplasmic reticulum (ER). These Kv2.1- associated ER-PM junctions, or EPJs, often contain Ca2+ handling machinery, including L-type Ca2+ channels (LTCCs) and ryanodine receptor (RyR) ER Ca2+ release channels. In addition to being significant sites of Ca2+ uptake and release, EPJs also serve important roles in modulating cellular lipid handling. Lipid transfer between the ER and PM can be acutely regulated by Ca2+, and lipid-modulating enzymes at EPJs exert a reciprocal effect on cellular Ca2+ dynamics. As Kv2.1 clusters enhance the formation EPJs and may modulate Ca2+ signaling at these sites, Kv2.1 is perfectly poised to integrate and control neuronal Ca2+- and lipid signals. Importantly, clinical findings suggest that Kv2.1-associated EPJs are critical for normal brain function: three distinct mutations in Kv2.1 that disrupt the channel domain required for its clustered organization with EPJs cause severe neurodevelopmental delay. However, the molecular architecture, regulation, and functional roles of Kv2.1- associated EPJs remain poorly understood. This presents a major obstacle to determining how Kv2.1 channels contribute to normal neuronal function and limits our understanding of its contributions to the pathogenesis of debilitating neuronal disorders. Although its role in neurons is not yet clear, Kv2.1 clustering in neuroendocrine cells was found to facilitate the exocytosis of dense-core vesicles (DCV), secretory organelles that in neurons contain diverse neuroactive cargo. As defects in neuronal DCV release are associated with autism, anxiety disorders, and epilepsy, it is important to define the molecular points of intersection between Kv2.1 channels and DCV release. I hypothesize that Kv2.1-associated EPJs control neuronal Ca2+ and lipid signals to regulate DCV release. I will test the central hypothesis by determining the mechanisms by which Kv2.1 channels modulate local Ca2+ and lipid homeostasis and signaling in neurons (Aim 1). These findings will be extended to detailed studies of how Kv2.1 channels contribute to the regulation of somatodendritic DCV release (Aim 2). Successful completion of the proposed research will advance our understanding of the fundamental mechanisms regulating neuron function. Moreover, elucidating the influence of Kv2.1 channels on neuronal DCV release will greatly expand comprehension of the mechanisms underlying DCV exocytosis and may also improve understanding of the mechanisms underlying Kv2.1’s contributions to neurological disorders. Through this fellowship, I will develop 1) a novel understanding of the physiological functions of Kv2.1 channels, and 2) my potential as an independent investigator focused on ion channel biology. These training goals will be facilitated by the detailed research plan, the exceptionally qualified mentors with expertise in the proposed study design, and the outstanding facilities and training resources available at UC Davis.