Characterization of ATP Analogue Cross-Linking Compounds and Biochemical Analysis of P2X Receptors

The aim of this thesis was to test whether cross-linking ATP analogues could be used to provide direct evidence of the amino acid residues which contribute to the ATP binding domain of P2X receptors. The UV irradiation of 2-azido ATP caused the compound to cross-link to the P2X1 receptor and caused a significant reduction in the response to ATP. The reduction of cross-linking following the pre-treatment of cells in excess ATP suggested competition between ATP and 2-azido ATP for occupancy of the P2X receptor binding site. This was proven with the radio-labelling of the P2X1 receptor with 2-azido [y32p] ATP. Similar techniques were used to identify other photo-reactive compounds with activity at the P2X1 receptor. These photo-reactive compounds had reactive sites at different coordinates around the ATP molecule and can potentially cross-link to different regions within the ATP binding domain. In an attempt to localize the ATP binding site, the P2X1 receptor was tagged with flag and his epitopes and purified. Protein mass fingerprinting showed that the digestion of the P2X1 protein did not provide enough coverage of the protein to guarantee successful analysis by mass spectrometry. Additionally, initial studies using digestion to identify peptide fragments which had bound to 2-azido [y32P] ATP showed that experimental conditions caused the cleavage of the label from the peptide. This thesis has demonstrated that photo-reactive ATP analogues can be used to label the P2X receptors. Further work should identify a suitable combination of enzymes to efficiently fragment the protein and allow spectral analysis to identify the amino acid residues which covalently bind to each photo-reactive compound. This would give the first direct evidence of the contributing amino acid residues in the ATP binding domain of the P2X receptors.