Surveying the landscape of ion channel regulatory partners

概述：离子通道是一种离子传导蛋白，是所有细胞中电行为的基本调节器。它们对我们体内的电信号是必不可少的，也是治疗学发展的重要靶点。虽然参与细胞信号传递的其他蛋白质家族通常是在复杂的多蛋白质结构中组装的，但对离子通道复合体的组装研究较少。这项建议描述了一种系统的/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：生物电信号是通过离子通道的开启和关闭来响应化学、机械或电刺激而产生的。这些信号构成了我们与环境时刻相互作用的基础，通过对可兴奋细胞的生理控制至关重要的快速电信号事件。虽然许多离子通道类型的特性已经被发现，但它们与信号复合体中的调节蛋白之间的联系却知之甚少。因此，离子通道通常是在没有调节伙伴的情况下进行研究的，这些伙伴可能会影响离子通道的功能或生物发生。我们已经采取了多种策略来解决这一广泛的知识鸿沟，总的目标是识别调节不同电压门控钾通道的未识别蛋白质。我们应用蛋白质组学方法来鉴定电压门控钾通道附近的蛋白质，然后对这些发现进行生化验证，并进行各种实验以确定对电压门控离子通道的电压依赖门控或成熟的影响。使用这些方法，我们已经确定了以前未知的调节蛋白，影响电压门控离子通道的生物发生、成熟和门控特性。在这项建议中，我们描述了我们已经实施的识别和筛选Kv1.2通道候选交互作用的基本方法，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TMEM33相关的初步发现，TMEM33是Kv1.2通道的候选调节器，作为一个案例研究和短期目标，以表征离子通道调节器。所描述的方法和实验突出了建立在蛋白质组学方法基础上的工作流程，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：这项建议描述了一个基于发现的计划，旨在识别调节电压门控离子通道的未被识别的信号复合体。这种方法可能会产生广泛的影响，因为它在目前对离子通道的研究中并不常见。筛选和功能方法的结合将揭示新的离子通道调节机制，以及电压门控离子通道及其调节伙伴潜在的新的生物学作用。

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.