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Title: Structural and functional studies of K2P channels
Author: Mackenzie, Alexandra
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2015
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Two-pore domain potassium channels (K2Ps) are a family of dimeric potassium channels responsible for regulation of cellular excitability. In 2012, crystal structures of TREK-2 were solved revealing this channel in two different conformational states. In the down state, transmembrane helices M2, 3 and 4 descend further into the cytoplasm than in the up state. This reveals two gaps (fenestrations) that link the membrane directly to the ion conduction pathway. However, the functional relevance of these two states was not known. In this work, I use state-dependent modulators of TREK-2 to investigate the functional relevance of helical movement in K2P channels. By solving the co-crystal structures of these modulators with TREK-2, I aimed to uncover which states these modulators bind to and how this modifies potassium conductance through the channel. As small molecule binders have weak affinities for TREK-2, brominated ligand derivatives were used to increase sensitivity and specificity of ligand detection. The co-crystal structures of TREK-2 with brominated fluoxetine (Prozac) derivatives revealed functionally inhibited down states with reduced occupancy in the selectivity filter. These inhibitors bind within the fenestration of TREK-2. Since the fenestration is absent in the up state, inhibition must work by stabilising the down state of the protein. This finding supports a gating mechanism of TREK-2 that links movement of the M2-4 helices with gating at the channel filter. TREK-2 was also co-crystallised with derivatives of the small molecule activator BL-1249. Crystals of this complex were produced using both vapour diffusion and lipidic cubic phase. These crystals produced diffraction at both synchrotron and free electron laser sources. Initial low-resolution structures provide the first insight into where BL-1249 may bind to TREK-2.
Supervisor: Tucker, Stephen ; Carpenter, Liz Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available