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Title: Characterisation of expression and functional properties of P2Xz receptors
Author: Bradley, Helen Joanne
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2010
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Abstract P2X7 receptors form non-selective cation channels at the plasma membrane and are gated by the binding of extracellular ATP. Prolonged P2X7 receptor activation also leads to formation of a pore that allows permeation of large molecules. Many cell types express P2X7 receptors. Expression is particularly high in cells of hematopoietic origin, where they have important roles in many biological processes, including release of pro-inflammatory cytokines and apoptosis. Furthermore, theĀ· properties of P2X7 receptors, including their functional expression and pharmacology, exhibit striking species differences. However, the underlying molecular bases for these differences are not fully understood. The gene encoding human P2X7 receptors contains numerous single nucleotide polymorph isms (SNP), several of which are non-synonymous (ns- SNP). Two ns-SNP mutations, H155Y and A348T, were of particular interest due to their gain-of function effect on P2X7 receptors. Furthermore, a previous study found ATP-induced secretion of cytokines was enhanced in immune cells from individuals harbouring the A348T mutation. These two mutations were therefore examined in detail. Whole-cell patch-clamp current recordings clearly showed both mutations increased agonist-induced maximal currents, with no or very mild effect on agonist sensitivity. Cells expressing human P2X7 receptors had lower ATP-evoked maximal currents in comparison to the rat receptor. Mutations H155Y and A348T change the residues to those in the corresponding positions of the WT rat P2X7 receptor. Introduction of reciprocal mutations, Y155H and T348A into the rat P2X7 receptor reduced agonist-evoked current amplitudes. Substitution of residues surrounding His 155 and Ala348 in the human P2X7 receptor with the corresponding residues of the rat P2X7 receptor did not result in gain-of-function, with the exception F353L. However, the reciprocal mutation L353F in the rat P2X7 receptor had no effect. Taken together, these results strongly indicate that residues 155 and 348 are important in determining the functional expression of P2X7 receptors. In addition, they reveal that the residues at these two positions contribute to the differences in functional expression of human and rat P2X7 receptors. Further investigations into the functional roles of His 155 and Ala348 were performed by studying the effects of mutating to residues with side chains of distinct properties. Substitution of His 155 with leucine and aspartic acid reduced iv ATP-induced current amplitudes, whilst phenylatanine, arginine and alanine had no significant effect. Mutation of Ala348 revealed a discernible effect on the human P2Xy receptor in that substitution with residues with larger side chains reduced, whereas changes to residues with small side chains increased, the amplitude of ATP-evoked currents. Immunostaining and biotin labelling revealed the H155Y mutation of human P2Xy receptors increased, whilst Y155H of rat receptors decreased surface expression. No such effect on surface expression resulted from reciprocal mutations at position 348. A human P2Xy receptor model, based on the recently determined crystal structure of the zebrafish P2X4 receptor, indicates that His 155 is in the extracellular region, distant from the agonist-binding site and ion-permeating pore, whilst Ala348 is located immediately intracellular to the narrowest part of the ion-conducting pathway. Therefore, the simple and most consistent explanation for the effects of mutating residues at 155 and 348 is that the residue at 155 is important in determining receptor surface expression, and the residue at 348 is involved in single channel function. The monkey P2Xy receptor, which shares 96% sequence homology with the human P2Xy receptor, has been functionally characterised, and its pharmacological properties were similar to that of the human receptor. The monkey P2Xy receptor had a 14-fold higher sensitivity to BzATP over ATP. Furthermore, the sensitivity to ATP and BzATP was slightly lower than in comparison to human P2Xy receptors (2.5- and 2-fold, respectively). The sensitivity of the monkey P2Xy receptor to the P2Xy receptor antagonists KN-62, AZ11645373 and A-438079 was virtually indistinguishable from that of the human P2Xy receptor. Therefore the five amino acids in the extracellular domain that differ between human and monkey P2Xy receptors do not critically interact with these antagonists. The similar pharmacological profiles of human and monkey P2Xy receptors suggests the monkey provides a suitable model for to investigate P2Xy receptor involvement in human diseases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available