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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Grimes, Leanne M
Languages: English
Types: Doctoral thesis
Subjects: QR
P2X receptors and pannexins (Panx) are eukaryotic ion channels that are implicated in a range of diseases and conditions including cancer, inflammation and pain sensation and as a result, are important therapeutic targets. Deducing their 3D-structures would enable the use of structure-based drug design to identify novel agonists or antagonists. However, solving eukaryotic membrane protein structures is a significant challenge due to the requirement for high yields of purified folded, functional protein, which are not readily obtainable with conventional over-expression systems. By using P2X receptors and pannexins as model ion channel targets, this thesis aims to test Drosophila melanogaster as a system for the over-expression and functional analysis of eukaryotic ion channels. A number of epitope-tagged P2X and Panx protein constructs were generated and first expressed in HEK-293 cells (rat P2X2-GFP, human P2X4-GFP, rat Panx1-GFP and human P2X4-int-CBD (chitin binding domain)) to allow their expression, glycosylation and oligomeric states to be investigated as markers of protein folding and quality. Subsequently, rat P2X2-GFP and rat Panx1-GFP constructs were successfully expressed in the photoreceptor cells of Drosophila melanogaster, where the photoreceptive membrane in the visual system is organised into a densely packed brush of microvilli, the rhabdomere. This system provides a large surface area of membrane for protein expression. Although the yields of purified protein were lower than expected, rat Panx1-GFP was successfully purified and used for low resolution structural studies with transmission electron microscopy. Rat P2X2-GFP was also expressed in the nervous system of Drosophila under control of a pan-neural, C155-Gal4 driver and was shown to be functional by measuring ATP-evoked action potentials using electrophysiological recordings of the Drosophila taste sensilla. This system was also used to test the activity of an adenosine nucleotide library of 80 compounds. Three nucleotides were identified that elicited responses similar to ATP; these were 2F-ATP, ATPαS and ATPγS.
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