The Structural and Mechanistic Basis of THIK1 K+ Channel Regulation

Ion channels facilitate the diffusion of charged ions such as Na+, K+ and Cl- across the cell membranes of almost every living cell thereby generating electrical potentials. In humans this electrical activity controls many important physiological processes and ion channel dysfunction contributes to a variety of diseases. Many therapeutic strategies target ion channels, so a greater understanding of how these channels function, how their activity is regulated, and how drugs can be used to modulate their properties is critical for a fundamental understanding of their role in health and disease and our ability to expand and exploit their potential as drug targets. The family of Two Pore-Domain (K2P/KCNK) Potassium Channels control the movement of K+ critical for regulation of cellular transmembrane potentials. In previous studies we have used structural, computational and electrophysiological approaches to dissect the molecular mechanisms controlling the activity of several members of this family (e.g. TREK/TASK/TRESK channels) and also demonstrated how these properties can become defective in the disease state. THIK1 is also a member of the K2P family, but despite being identified almost 25 years ago, it has been little studied until recently when its activity was found to control the function of microglia, the brain's innate immune cells, and to activate their pro-inflammatory response that can lead to neurodegeneration. However, this process is still poorly understood and most other aspects of THIK1 structure/function and physiology also remain unknown. In exciting new (unpublished) studies we have determined a 3D-structure of THIK1 using single-particle Cryo Electron Microscopy (CryoEM). This structure reveals several important features including an unusual extracellular ion exit pathway, a lower 'bundle-crossing' gate and a lipid bound to a key regulatory site near the selectivity filter. This is supported by our initial electrophysiological studies which confirm that these features play an important role in defining the unique biophysical and physiological properties of this channel. We also have recent evidence that small synthetic antibody fragments (Sybodies) can be generated against K2P channels and that their selection can be biased to enrich for those which modulate channel activity, thus offering useful tools for a variety of structure/function approaches both in vitro and in vivo. Our industrial partner (Cerevance, Cambridge UK) has an interest in CNS disorders, and one of their drugs (CVN293) currently in development for treatment of neurodegenerative disorders such as ALS and Alzheimers is a highly-selective THIK1 inhibitor. However, its binding site on THIK1 and the molecular mechanism(s) of its inhibitory action on channel activity remain unknown. Based on the preliminary evidence and new approaches we present, we aim to address the following broad questions: How does the THIK1 channel open and close, and how is this regulated by natural ligands and signalling pathways within the cell? How do small molecule drugs interact with THIK1 to regulate channel gating and can new or better approaches be designed for the modulation of channel activity? How do the unique structural and biophysical properties of THIK1 relate to its role in microglial function. Overall, this project will generate a step change in our fundamental understanding of THIK channels, how they are regulated, their biophysical properties and physiological role. This basic knowledge may also help inform the design of current and future strategies to treat neuroinflammatory disorders and so could have significant impact well beyond its immediate scientific goals.