Surveying the landscape of ion channel regulatory partners

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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.