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)的神经元胞体上形成显著的质膜(PM)簇。这些Kv2.1相关的ER-PM连接或EPJ通常含有钙处理机制，包括L型钙通道(LTCC)和兰诺定受体(RyR)ER钙释放通道。除了是钙摄取和释放的重要部位外，EPJ还在调节细胞脂质处理方面发挥重要作用。内质网和质膜之间的脂质转移可以受到钙离子的强烈调控，而外周血中的脂质调节酶对细胞内钙动力学起着相互作用的作用。由于Kv2.1簇促进了EPJ的形成，并可能调节这些部位的钙信号，Kv2.1完全准备好整合和控制神经元的钙信号和脂质信号。重要的是，临床发现表明Kv2.1相关的EPJ对正常的大脑功能至关重要：Kv2.1的三个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延迟。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神经元疾病发病机制中的作用的理解。尽管Kv2.1在神经元中的作用尚不清楚，但已发现神经内分泌细胞中的Kv2.1聚集促进致密核小泡(DCV)的胞吐，致密核小泡是神经元中含有各种神经活性物质的分泌细胞器。由于神经元DCV释放的缺陷与自闭症、焦虑症和癫痫有关，因此确定Kv2.1通道和DCV释放的分子交叉点是很重要的。我假设Kv2.1相关的EPJ控制神经元的钙和脂信号来调节DCV的释放。我将通过确定Kv2.1通道调节局部钙离子和脂质动态平衡的机制以及神经元中的信号来检验中心假设(目标1)。这些发现将被扩展到详细研究Kv2.1通道如何有助于调节躯体树突状细胞DCV的释放(目标2)。这项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大对DCV胞吐机制的理解，也可能促进对Kv2.1‘S在神经疾病中作用的机制的理解。通过这一奖学金，我将1)对Kv2.1通道的生理功能有一个新的理解，以及2)我作为一名专注于离子通道生物学的独立研究员的潜力。这些培训目标将由详细的研究计划、在拟议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提供的出色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.