Structure and Function of TRPV Ion Channels

描述(由申请人提供)：拟议研究的长期目标是在分子和结构水平上了解TRPV离子通道响应如何整合外部刺激和细胞状态。TRPV通道在感觉和痛觉以及钙稳态中都很重要。在分子水平上，TRPV通道的大的N-末端和C-末端的胞液结构域感知细胞状态的信息--例如，钙和磷脂酰肌醇水平--以调节通道的敏感性。我们最近在TRPV1-ARD中发现了一个重要的生理上重要的多配体结合位点，它调节着TRPV1通道的敏感性。这一建议扩展了这些最近的结果，以解决小分子和蛋白质配体与TRPV N-和C-末端胞浆区域的相互作用如何协同调节通道敏感性。TRPV离子通道是重要的药物靶点，TRPV离子通道的结构和功能研究结果将为药物开发提供新的基于结构的设计策略。目的1.对TRPV1胞液区域的调节配体结合位点进行结构表征：我们的工作模型是，当TRPV1通道开放时，随着钙离子进入细胞，PIP2从TRPV1-CT释放出来，ATP从TRPV1-ARD释放出来，这样钙结合的CaM就可以使TRPV1的N端和C端交联，从而使通道失活。我们将使用生化、结构和电生理实验来测试这一工作模式，并研究TRPV1中N-端和C-端配体结合位点之间的功能关系。目的2.研究TRPV通道中共有的和独特的调节相互作用：TRPV1是6个哺乳动物TRPV亚家族成员中研究得最好的。我们利用我们从所有家族成员中成功制备的TRPV-ARD来确定是否有其他TRPV-ARD共享在TRPV1中发现的ATP或CaM结合位点，如果是，是否共享ATP或CaM介导的通道调节机制。我们还将确定几个独特的TRPV-ARD相互作用的结构。这些结果将为TRPV通道的功能和调节的保守化和专门化的程度提供洞察力。目的3.确定TRPV1通道辣椒素敏感性的结构决定因素：最终，要了解通道敏感性是如何调节的，我们还需要了解该通道是如何激活的。这一目标的目的是通过突变、化学修饰和电生理学的结合，精确定位TRPV1内结合辣椒素或在通道激活时改变构象的区域。辣椒素结合位点是通道内潜在止痛药化合物靶点的主要位置。因此，我们获得的关于辣椒素结合位点以及辣椒素如何影响TRPV1通道构象的知识可以应用于药物开发，将我们的结果直接与保健研究联系起来。

DESCRIPTION (provided by applicant): The long term goal of the proposed research is to understand at a molecular and structural level how TRPV ion channel responses integrate external stimuli and cellular state. The TRPV channels are important in sensory and pain perception and in calcium homeostasis. At a molecular level, the large N- and C-terminal cytosolic domains of TRPV channels sense information about the cellular state - calcium and phosphoinositide levels, for example - to regulate channel sensitivity. We have recently identified a physiologically important multi-ligand binding site within the TRPV1-ARD that modulates TRPV1 channel sensitivity. This proposal expands on these recent results to address how interactions of small molecule and protein ligands with the TRPV N- and C-terminal cytosolic regions synergize to regulate channel sensitivity. TRPV ion channels are important drug targets and the results of the proposed structure and function studies of TRPV channels will enable new structure-based design strategies for drug development. The work can be divided in the following specific aims: Aim 1. To structurally characterize the regulatory ligand binding sites in the TRPV1 cytosolic regions: Our working model is that upon TRPV1 channel opening, as Ca2+ enters the cell, PIP2 is released from the TRPV1-CT and ATP from the TRPV1-ARD, such that Ca2+-bound CaM can crosslink the Nand C-termini of TRPV1, thereby inactivating the channel. We will use biochemical, structural and electrophysiological experiments to test this working model and address the functional relationship between the N- and C-terminal ligand binding sites in TRPV1. Aim 2. To characterize shared and unique regulatory interactions of the ARDs of TRPV channels: TRPV1 is the best studied of the six mammalian TRPV subfamily members. We take advantage of our success in preparing TRPV-ARDs from all family members to determine whether any other TRPV-ARD shares the ATP or CaM binding site found in TRPV1 and, if so, whether the ATP- or CaM-mediated channel regulation mechanisms are shared. We will also determine the structure of several unique TRPV-ARD interactions. The results will provide insights into the extent of conservation vs. specialization of the function and regulation of TRPV channels. Aim 3. To identify the structural determinants of capsaicin sensitivity in the TRPV1 channel: Ultimately, to understand how channel sensitivity is regulated, we also need to understand how the channel is activated. The goal of this aim is to pinpoint the regions within TRPV1 that bind capsaicin or change conformation upon channel activation, through a combination of mutagenesis, chemical modifications and electrophysiology. The capsaicin binding site is a prime location within the channel to target with potential pain relief drug compounds. Knowledge we gain about the capsaicin binding site and how capsaicin affects the conformation of the TRPV1 channel can therefore be applied to drug development, directly linking our results to healthcare research.