The characterisation of 5-BDBD antagonism of human P2X4 ion channels.

Hickey, Harry (2020) The characterisation of 5-BDBD antagonism of human P2X4 ion channels. Masters thesis, University of East Anglia.

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The purinergic receptor P2X4 is a ligand-gated, non-selective cation channel, activated by ATP. This receptor is involved in pathophysiological processes including inflammatory pain and cardiac function. Selective inhibition of P2X4 is seen as a possible therapy, however little is known about the limited number of selective antagonists available. This study characterises the activity of 5-BDBD (one such antagonist). There is conflicting evidence for the action of this molecule with one prominent paper suggesting that 5-BDBD is a competitive antagonist (Balasz et al, 2012) and another suggesting that it interacts allosterically (Abdelrahman, 2017). The aims of this study were, to determine the mode of action for 5-BDBD and to identify residues responsible for mediating the effects of 5-BDBD.

When challenging an ATP dose response curve with various concentrations of 5-BDBD, a reduction in the maximal response (at 20 and 30µM of 5-BDBD) and a decrease in potency (at 10, 20 and 30µM of 5-BDBD) were found. This suggested that 5-BDBD acts allosterically. A scan of the receptor was conducted using 10 amino acid chimeras (P2X4 substituted for aligned insensitive P2X2). This scan used 30µM of ATP and 20µM of 5-BDBD to test for inhibition of the chimeric receptors. There was one chimera (at residues 81-90) which produced a significantly lower inhibition of response than the wildtype (p=4.8x10-4). An inhibition of 29.43% was found, compared to the wildtype inhibition of 72.1% with a range for the remaining chimeras of 70.5% to 85.6%. Within this region residue 81 was unique to P2X4 and when tested, gave a significantly lower inhibition of 42.73% compared to the wild type at 79.2% (P=0.009). This implicates residue 81 with eliciting the inhibitory effect of 5-BDBD on P2X4.

The hope is that these findings can aid in understanding the mechanism of action for antagonism of the P2X4 receptor.

Item Type: Thesis (Masters)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 15 Apr 2021 08:46
Last Modified: 15 Apr 2021 08:46


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