Surveying the landscape of ion channel regulatory partners

综述：离子通道是离子传导蛋白，是所有细胞电行为的重要调节因子。它们对于我们体内的电信号是必不可少的，并且是开发治疗方法的重要目标。虽然参与细胞信号传导的其他蛋白质家族通常在其组装成复杂的多蛋白质结构的背景下进行研究，但离子通道复合物的组装研究或理解较少。该提案描述了一种系统的/程序化的方法来识别和理解与电压门控离子通道相互作用的蛋白质的生物学作用。背景：生物电信号是由离子通道响应化学、机械或电刺激而打开和关闭而产生的。这些信号构成了我们与环境的瞬间互动，通过快速的电信号事件，这些事件对于兴奋细胞的生理控制至关重要。虽然已经发现了许多离子通道类型的身份，但它们与信号复合物中的调节蛋白的关联却少得多。因此，离子通道的研究往往在缺乏可能影响其功能或生物发生的调控伙伴。我们已经采取了各种策略来解决这一广泛的知识差距，总的目标是确定未被识别的蛋白质，调节不同的电压门控钾通道。我们已经实施了蛋白质组学的方法来确定接近电压门控钾离子通道的蛋白质，然后通过生物化学验证这些发现和各种实验来确定对电压依赖性门控或电压门控离子通道成熟的影响。使用这些方法，我们已经确定了以前未知的调节蛋白，影响生物发生，成熟和门控特性的电压门控离子通道。在这个建议中，我们描述了我们已经实施的基本方法，用于识别和筛选Kv1.2通道的候选相互作用物，并计划将这些方法扩展到其他生物相关通道。我们还提出了与TMEM 33相关的初步研究结果，TMEM 33是Kv1.2通道的候选调节剂，作为表征离子通道调节剂的案例研究和短期目标。所描述的方法和实验突出了基于蛋白质组学方法的工作流程，以深入了解蛋白质相互作用的结构细节及其生理意义。 影响：该提案描述了一个基于发现的程序，旨在识别未识别的信号复合物，调节电压门控离子通道。这种方法可能会有广泛的影响，因为它是不常见的离子通道在目前的调查。结合筛选和功能的方法将揭示新的离子通道的调节机制沿着潜在的新的生物学作用的电压门控离子通道及其监管伙伴。

OVERVIEW: Ion channels are ion-conducting proteins that are essential regulators of electrical behavior in all cells. They are essential for electrical signaling in our bodies, and are important targets for the development of therapeutics. While other protein families involved in cellular signaling are commonly studied in the context of their assembly in complex multi-protein structures, assembly of ion channel complexes is less studied or understood. This proposal describes a systematic/programmatic approach to identify and understand the biological roles of proteins that interact with voltage-gated ion channels. BACKGROUND: Bio-electrical signaling is generated by opening and closing of ion channels in response to chemical, mechanical, or electrical stimuli. These signals underlie our moment-to-moment interactions with the environment, via rapid electrical signaling events that are essential for physiological control of excitable cells. While the identity of many ion channel types has been discovered, their association with regulatory proteins in signaling complexes is much less understood. Therefore, ion channels are often studied in the absence of regulatory partners that may influence their function or biogenesis. We have adopted a variety of strategies to address this broad knowledge gap, with the general objective of identifying unrecognized proteins that regulate diverse voltage-gated potassium channels. We have implemented proteomic approaches to identify proteins in close proximity to voltage-gated potassium channels, followed by biochemical validation of these findings and a variety of experiments to identify effects on voltage-dependent gating or maturation of voltage-gated ion channels. Using these approaches, we have identified previously unknown regulatory proteins that influence the biogenesis, maturation, and gating properties of voltage-gated ion channels. In this proposal we describe the basic methods we have implemented for identification and screening of candidate interactors of Kv1.2 channels, and planned extension of these approaches to other biological relevant channels. We also present preliminary findings related to TMEM33, a candidate regulator of Kv1.2 channels, as a case study and short term objective to characterize ion channel regulators. The approaches and experiments described highlight workflow that builds on proteomic approaches to develop insights into structural details of protein interactions and their physiological significance. IMPACT: This proposal describes a discovery-based program designed to identify unrecognized signaling complexes that regulate voltage-gated ion channels. This approach may have a broad impact as it is not commonly addressed in current investigations of ion channels. The combination of screening and functional approaches will uncover novel ion channel regulatory mechanisms along with potential new biological roles of voltage-gated ion channels and their regulatory partners.