Endogenous Ion Channel Activity Tracers to Monitor the Involvement of Kv2 Channels During Ischemic Attack Neuronal electrical signals are governed by the combined action of many ion channel subtypes. Different sets of ion channels sum to create a remarkable diversity in neuronal electrical excitability. However, due to technological limitations, dissecting the individual role of an ion channel subtype during a complex physiological or pathophysiological event remains difficult. Consequently we have a limited understanding of how the electrical dynamics of individual endogenous ion channel subtypes contribute to global signals, especially in intact tissue or in live animals. Technology developed in Dr. Jon Sack?s lab offers an opportunity to image the activity of ion channel subtypes throughout a complex tissue. Specifically, these Kv2 Activity Tracers (KATs) report activation of endogenous neuronal potassium voltage-gated ion channels of subtype 2. We have engineered these KATs for 2-photon imaging, and demonstrated that KATs can report activation endogenous neuronal Kv2 ion channels in brain slices. I propose to measure activation of a specific ion channel subtype in tissue slices and live animals under pathophysiological ischemic stress that mimics stroke. In the brain, Kv2 ion channels are highly expressed in most, if not all neurons. Kv2 channels are proposed to be crucial to suppress excitotoxic signaling events during many stresses, including ischemic attack. Previous studies have suggested that Kv2 ion channels become very active during ischemia, suppress electrical excitability, and provide neural protection from excitotoxic signaling. However, there has also been evidence that excessive efflux of potassium through Kv2 channels during ischemia can trigger apoptotic cascades leading to neuronal death. Both of these proposed roles assume a dramatic increase in the number of active Kv2 channels, yet this has never been observed in real time in complex tissues. For my doctoral studies I will probe a mechanism for mass activation, and image changes in Kv2 ion channel activity in models of ischemic stroke. My results will improve our understanding of the molecular mechanisms leading to neuronal cells death following stroke, and whether Kv2 channels are a potential drug target to increase neuronal survival following stroke. Further, my research will be the first attempt at using fluorescent probes to measure conformational change of specific ion channels in intact tissue and potentially transform the way protein activation is studied in a physiological context. Throughout this project the sponsor/co-sponsor team, will implement a comprehensive training plan focused on improving critical thinking, experimental and analytical skills, presentation and public speaking skills, and aid in creating a network of colleagues and collaborators.
Currently a large gap in knowledge exists in understanding the roles specific neuronal ion channel subtypes play during neural signaling. Delineating these molecular mechanisms is crucial to understand why ischemia produced by stroke has damaging consequences on neuronal health. The experiments proposed will allow us to track the activity of a specific ion channel during an ischemic attack, improve our understanding of the fundamental electrical responsibilities of this ion channel, to potentially aid in the development of targeted therapeutics for ischemia.
