Modulation of Neuronal Ion Channels by 2nd Messengers

描述（由申请人提供）：离子通道电流是大多数生物体电活动的基本单位。在我们的神经系统中，K+和Ca2+通道是至关重要的，它们的调节提供了一种直接控制神经元兴奋性和突触神经递质释放的方法。这些调节对基本神经功能至关重要，对它们的理解应该有助于为一系列涉及大脑、神经和肌肉的疾病提供新的医疗干预模式。我们主要关注几个神经元K+电流基础的Kv7 （KCNQ/ m型）K+通道家族和Cav2.2 （n型）Ca2+通道。特别是，我们试图阐明作用于这些类型离子通道的几种调节途径的分子机制。我们使用了一种异源系统，其中通道、受体和信号分子的cDNA克隆在哺乳动物组织培养细胞中表达，并制备了初级交感神经元。我们所研究的两种途径在G蛋白的Gq/11家族中有共同的受体偶联，磷脂酶C的激活和磷脂酰肌醇4,5-二磷酸（PIP2）的水解。Kv7和Cav2.2通道都受到PIP2的调控，在这个项目中，我们将研究PIP2如何与这些通道相互作用，相互作用的位点，以及介导门控调节的通道蛋白部分。某些刺激提高细胞内Ca2+，并与钙调素（CaM）协同作用于Kv7通道。我们将进一步研究Ca2+/CaM作用的分子机制。其他Gq/11偶联受体不会升高细胞内Ca2+，而是通过耗尽PIP2膜起作用。使用多种方法，我们将探讨受体作用的这种特异性。使用的工具包括膜片钳电生理学、分子生物学、生物化学和先进的成像技术。我们研究的分子和信号通路与人类健康和疾病有着广泛的相关性。Kv7通道在调节神经元兴奋性中起主导作用，其调节可能是情绪状态、记忆和身体器官调节变化的基础。功能失调的Kv7通道引起特定的癫痫综合征。Cav2.2通道的调节调节神经递质的释放，神经递质是神经元间突触的基本信号。因此，我们的研究应该为开发针对各种神经疾病的新型治疗干预模式提供基础。概要：本项目研究神经细胞中介导电流的离子通道是如何被调节的。我们使用生物物理和分子工具在分子和个体活细胞水平上研究这些信号。我们试图理解神经系统的这种调节，它是人类思想、情感和行为的复杂现象的基础。我们的发现可能有助于减轻许多情绪、运动和意识疾病，这些疾病是神经功能障碍。

DESCRIPTION (provided by applicant): Ion channel currents are the fundamental units of electrical activity in most organisms. In our nervous system, K+ and Ca2+ channels are critical, and their regulation provides a way to directly control neuronal excitability and release of neurotransmitter at synapses. The modulations are crucial to basic nervous function and their understanding should contribute to novel modes of medical interventions for a range of disorders involving the brain, nerves and muscles. We focus primarily on the family of Kv7 (KCNQ/M-type) K+ channels that underlies several neuronal K+ currents and on Cav2.2 (N-type) Ca2+ channels. In particular, we seek to elucidate the molecular mechanisms of several modulatory pathways that act on these types of ion channels. We use both a heterologous system in which cDNA clones of the channels, receptors and signaling molecules are expressed in mammalian tissue-culture cells, and a preparation of primary sympathetic neurons. Two pathways that we study have in common receptors coupled to the Gq/11 family of G proteins, the activation of phospholipase C, and hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Both Kv7 and Cav2.2 channels have emerged as being regulated by PIP2, and in this project we will investigate how PIP2 interacts with the channels, the site(s) of the interaction, and the parts of the channel proteins that mediate the regulation of gating. Stimulation of certain raises intracellular Ca2+ and acts on Kv7 channels in concert with calmodulin (CaM). We will further investigate the molecular mechanism by which Ca2+/CaM acts. Other Gq/11-coupled receptors do not raise intracellular Ca2+ and act by depleting the membrane of PIP2. Using a variety of approaches, we will probe this specificity in receptor action. The tools to be used include patch-clamp electrophysiology, molecular biology, biochemistry, and advanced imaging techniques. The molecules and signaling pathways that we study have broad relevance to human health and disease. Kv7 channels play a dominant role in regulating excitability of neurons, and their regulation likely underlies changes in emotional state, memory and regulation of body organs. Dysfunctional Kv7 channels cause specific epileptic syndromes. The modulation of Cav2.2 channels regulates release of neurotransmitter which is the basic signal at synapses between neurons. Thus, our research should provide the basis for the development of novel modes of therapeutic intervention for a variety of nervous diseases. Lay summary: This project studies how ion channels, which mediate the electricity in nerve cells, are regulated. We investigate these signals at the level of the molecule and the individual living cell using biophysical and molecular tools. We seek to understand this regulation of the nervous system that underlies the complex phenomena of human thought, emotion and behavior. Our findings may help to alleviate the many diseases of mood, motion and consciousness that are disorders of nervous function.