Members of the two-pore potassium-selective (K2P) ion channel superfamily control cell excitability by contributing to the resting membrane potential. Through this, K2P channels are involved in a variety of physiological processes and dysfunction of these channels has been linked to diseases such as epilepsy, depression and migraine. The aim of this study was to develop a greater understanding of how K2P channels, in particular TREK-1, are gated. In the initial stage of this study we hoped to identify mutations which alter the function of the TREK-1 channel by screening a random mutant library using a K+-auxotrophic strain of S. cerevisiae, SGY1528. From the assay we identified a number of gain-of-function (GoF) mutations, primarily distributed through the pore-lining transmembrane (TM) helices TM2 and TM4. The clustering of mutations in these regions suggested a major role for these helices in channel gating. Subsequent electrophysiological characterisation of these mutations revealed an increase in basal channel activity and altered sensitivity to modulation by extracellular pH, as well as by activators DEPC and BL-1249. The publication of two K2P channel crystal structures, TWIK-1 (pdb code: 3UKM) and TRAAK (3UM7), enabled us to build an accurate homology model of TREK-1 and more accurately interpret these functional studies. This approach revealed a number of interesting points, the most important being an interaction between TM4 and pore-helix 1 (PH1). Further mutagenesis studies of this region confirmed that this interaction is essential for normal channel function. Another interaction was identified involving a number of residues within the interface between TM helices TM2, TM3 and TM4 emphasised the importance of helical movements for gating TREK-1 channels. Based on our findings we therefore present a model for gating of the TREK-1 channel, which suggests that the movement of TM4 in particular, is transduced to the selectivity filter gate via PH1.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:581091 |
| Date | January 2012 |
| Creators | Sharma, Chetan |
| Contributors | Tucker, Stephen John |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:c449026b-a8a4-4501-9b75-386658a2035e |
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