Location of Repository

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

By Kelvin Christopher Agboh

Abstract

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.\ud 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.\ud 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.\ud 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

Publisher: University of Leicester
Year: 2007
OAI identifier: oai:lra.le.ac.uk:2381/8243

Suggested articles

Preview

Citations

  1. (1998). 2', 3'-0-(2,4,6- trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) -a nanomolar affinity antagonist at rat mesenteric artery P2X receptor ion channels.
  2. (2002). A conformational analysis of Walker motif A [GXXXXGKT (S)] in nucleotide-binding and other proteins.
  3. (1998). A domain contributing to the ion channel of ATP-gated P2X2 receptors identified by the substituted accessibility method.
  4. (1994). A new class of ligand-gated ion channel defined by P2X receptor for extracellular ATP.
  5. (1995). A P2X purinoceptor cDNA conferring a novel pharmacological profile. Federation of European Biochemistry,
  6. (1995). A P2X receptor expressed by a subset of sensory neurons of special interest.
  7. (2003). A role of the fast ATPgated P2XI cation channel in thrombosis of small arteries in vivo.
  8. (2002). A study of P2XI receptor function in murine megakaryocytes and human platelets reveals synergy with P2Y receptors.
  9. (2003). Adenosine 5'-tetraphosphate (Ap(4)), a new agonist on rat midbrain synaptic terminal P2 receptors.
  10. (1997). Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology, and pharmacology.
  11. (1974). Adenosine triphosphate -a constituent of cholinergic, synaptic vesicles.
  12. (2002). Adenosine-receptor Subtypes: their relevance to adenosine-mediated responses in asthma and chronic obstructive pulmonary disease.
  13. (2000). ADP is not an agonist at P2XI receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets.
  14. (1983). Affinity labeling of purine nucleotide sites in proteins.
  15. (1977). Affinity labeling of rabbit muscle pyruvate kinase by 5'-p-fluorosulfonylbenzoyladenosine.
  16. (1998). Affinity labeling of two nucleoticle sites on Na, K-ATPase using 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-[ot- 32p] diphosphate (TNP-8N3 _[Ot_32p]ADP) as a photoactivatable probe.
  17. (2003). Affinity purification-mass spectrometry: Powerful tools for the characterization of protein complexes.
  18. (2001). Agonist-stimulated intemalization of the ligandgated ion channel P2X, in rat vas deferens Federation of European Biochemical Societies,
  19. (2001). Amino acid residues involved in gating identified in the first membrane-spanning domain of the rat P2X2 receptor.
  20. (1996). An antagonistinsensitive P2X receptor expressed in epithelia and brain.
  21. (2000). Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labeling: binding characteristics and amino acid sequences.
  22. (2005). Atomic force microscopy imaging demonstrates that P2X2 receptors are trimers but that P2X6 receptor subunits do not oligomerize.
  23. (2003). ATP analogues with modified phosphate chains and their selectivity for rat P2X2 and P2X2/3 receptors.
  24. (2003). ATP and sensory transduction in the enteric nervous system.
  25. (2000). ATP as a presynaptic modulator. Life Sciences,
  26. (2004). ATP binding at human P2XI receptors. Contribution of aromatic and basic amino acids revealed using mutagenesis and partial agonists.
  27. (1998). ATP binding site of P2X channel proteins: structural similarities with class 11 aminoacyl-tRNA synthetases.
  28. (1992). ATP mediates fast synaptic transmission in mammalian neurons.
  29. (1992). ATP receptor-mediated synaptic currents in the central nervous system.
  30. (2005). ATP receptors in pain sensation: Involvement of spinal microglia and P2X4 receptors. Purinergic Signalling,
  31. (1990). ATP-activated channels in rat and bullfrog sensory neurons: concentration dependence and kinetics.
  32. (1998). b ). Co-expression of P2XI and P2X5 receptor subunits reveals a novel ATP-gated ion channel.
  33. (1998). b). Identification of amino acid residues contributing to desensitization of the P2X2 receptor channel.
  34. (2004). b). Molecular properties of ATP-gated P2X receptor ion channels. Trends in Pharmacological Sciences,
  35. (2002). b). P2XI receptor- defi ci ent mice establish the native P2X receptor and a P2Y6-like receptor in arteries. Molecular Pharmacology,
  36. (1997). Baculovirus expression provides direct evidence for heteromeric assembly of P2X2 and P2X3 receptors.
  37. (1986). Benzophenone-ATP: A photoaffinity label for the active site of ATPases.
  38. (2003). Bladder and cutaneous sensory neurons of the rat express different functional P2X receptors.
  39. (1963). Catecholarnines and adenosine triphosphate in isolated adrenergic nerve granules.
  40. (1998). Central P2X4and P2X6channeI subunits Coassemble into a novel heteromeric ATP receptor.
  41. (1994). Characterization of P2-purinoceptors in the smooth muscle of the rat tail artery: a comparison between contractile and electrophysiological responses.
  42. (1995). Cited in:
  43. (1929). Cited in: Mubagwa, et al
  44. (1996). Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels.
  45. (1995). Co-expression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons.
  46. (1993). Combined application of site-directed mutagenesis, 2-azido-ATP labeling, and lin-benzo-ATP binding to study the noncatalytic sites of Escherichia coli FI-ATPase.
  47. (2002). Combined in-gel tryptic digestion and CNBr cleavage for the generation of peptide maps of an integral membrane protein with MALDI-TOF mass spectrometry.
  48. Complementary role of extracellular ATP and adenosine in ischemic preconditioning in the rat heart.
  49. (2001). Conserved Negatively Charged Residues Are Not Required for ATP Action at P2XI Receptors Biochemical and
  50. (1981). Contribution by purines to the neurogenic response of the vas deferens of the guinea pig.
  51. (2004). Contribution of Calcium Ions to P2X Channel Responses.
  52. (2006). Contribution of conserved polar glutarnine, asparagine and threonine residues and glycosylation to agonist action at human P2X, receptors for ATP.
  53. (1999). Contributions of the Cterminal domain to the control of P2X receptor desensitization.
  54. (2007). Cysteine substitution mutants give structural insight and identify ATP binding and activation sites at P2X receptors.
  55. (2004). Desensitization masks nanomolar potency of ATP for the P2XI receptor.
  56. (1997). Different sensitivities to pH of ATPinduced currents at four cloned P2X receptors.
  57. (1996). Differential distribution of two ATP-gated ion channels (P2X receptors) determined by immunocyto chemistry.
  58. (1987). Direct evidence for the interaction of the nucleotide affinity analog 5'-FSBA with a platelet ADP receptor.
  59. (1999). Direct photoaffinity labelling of the Kir6.2 subunit of the ATPsensitive K+ channel by 8-azido-ATP.
  60. (2005). Disruption of Lipid Rafts Inhibits P2Xj Receptormediated Currents and Arterial Vasoconstriction.
  61. (2005). Disruption of the P2X7 purinoceptor gene abolishes chronic inflammatory and neuropathic pain.
  62. (1982). Distantly related sequences in the -(-- and B-subunits of ATP synthase, myosin, kinases and other ATPrequiring enzymes and a common nucleotide binding fold.
  63. (2001). Distinct Localization of P2X Receptors at Excitatory Postsynaptic Specializations.
  64. (2005). Dual presynaptic control by ATP of glutarnate release via facilitatory P2Xj, P2X2/3, and P2X3 and inhibitory P2Yj, P2Y2, and/or P2Y4 receptors in the rat hippocampus.
  65. (2001). Ectonucleotidases: some recent developments and a note on nomenclature.
  66. (1991). Effect of presynaptic P2 receptor stimulation on transmitter release.
  67. (2003). Effects of A-317491, a novel and selective P2X3/P2X2/3 receptor antagonist, on neuropathic, inflammatory and chemogenic nociception following intrathecal and intraplantar administration.
  68. (2000). Effects of diadenosine polyphosphates (Ap(n)As) and adenosine polyphospho guanosines (Ap(n)Gs) on rat mesenteric artery P2X receptor ion channels.
  69. (1989). Effects of suramin and alpha, beta-methylene ATP indicate noradrenaline-ATP co-transmission in the response of the mouse vas deferens to single and low frequency pulses.
  70. (1970). Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by nonadrenergic inhibitory nerves in the gut.
  71. (2002). Expression and function of P2 receptors in bone.
  72. (1996). Families of ion channels with two hydrophobic segments.
  73. (2003). Fluorescent and colored trinitrophenylated analogs of ATP and GTP.
  74. (2004). Functional regulation of P2X6 receptors by N-linked glycosylation: identification of a novel f alphal I beta) -methylene ATP-sensitive phenotype.
  75. (1998). Functional role of alternative splicing in pituitary P2X2 receptor-channel activation and desensitization.
  76. (1995). Further development of the locus control region/murine erythroleukemia expression system: high level expression and characterization of recombinant human calcitonin receptor. Protein Expression and Purification,
  77. (2004). G-protein-coupled receptor regulation of P2XI receptors does not involve direct channel phosphorylation.
  78. (2004). Gain and loss of channel function by alanine substitutions in the transmembrane segments of the rat ATPgated P2X2 receptor.
  79. (2002). Heteromultimeric P2XI/2 receptors show a novel sensitivity to extracellular pH. Pharmacology and Experimental Therapeutics,
  80. (2002). Heteromultimerization modulates P2X receptor functions through participating extracellular and C-terminal subdomains.
  81. (1993). High-level expression of secreted proteins from cells adapted to serum-free suspension culture.
  82. (2006). Historical Review: ATP as a neurotransmitter.
  83. (1999). Identification of a domain involved in ATP-gated ionotropic receptor subunit assembly.
  84. (1981). Identification of a lysine residue at a nucleotide binding site in the firefly luciferase with p-fluorosulfonyl[
  85. (2004). Identification of a trafficking motif involved in the stabilization and polarization of P2X receptors.
  86. (1997). Identification of amino acid residues contributing to the pore of a P2X receptor.
  87. (2004). Identification of Negative Residues in the P2X3ATP Receptor Ectodomain as Structural Determinants for Desensitization and the Ca2+-sensing Modulatory Sites.
  88. (1992). Identification of peptides from the adenine binding domains of ATP and ANW in adenylate kinase: isolation of photoaffinity-label led peptides by metal chelate chromatography.
  89. (1996). Identification of the ATP binding domain of recombinant human 40-kDa 2', 5'-oligoadenylate synthetase by photoaffinity labeling with 8-azido-[alpha-32P]ATP.
  90. (1997). Immunohistochernical study of the P2X2 and P2X3 receptor subunits in rat and monkey sensory neurons and their central terminals.
  91. (2001). Increased release of ATP from endothelial cells during acute inflammation.
  92. (2001). Inflammatory mediators potentiate ATP-gated channels through the P2X3 subunit.
  93. (1985). Is there a basis for distinguishing two types of P2 Purinoceptor? General Pharmacology,
  94. (2004). KATP channel interaction with adenine nucleotides.
  95. (1998). Membrane topology of an ATP-gated ion channel (P2X receptor).
  96. (2005). Molecular determinants of the agonist binding domain of a P2X receptor channel. Molecular Pharmacology,
  97. (2004). Molecular physiology of P2 receptors in the central nervous system.
  98. (2006). Molecular properties of P2X receptors.
  99. (2006). Molecular properties of P2X receptors. Pflugers Arch -
  100. (2004). Molecular Structure of P2X Receptors. Current Topics in Medicinal Chemistry,
  101. (2005). Mutagenesis studies of conserved proline residues of human P2XI receptors for ATP indicate that proline 272 contributes to channel function.
  102. (1998). N-Linked glycosylation is essential for the functional expression of the recombinant P2X2 receptor.
  103. (1975). Neurogenic release of purine compounds in blood vessels.
  104. (1996). Neuronal synthesis, storage and release of ATP,
  105. (1994). New structural motif for ligandgated ion channels defined by an ionotropic ATP receptor,
  106. (1998). Nucleotide binding to the C-terminal nucleotide binding domain of ArsA.
  107. (1977). Observations on the algogenic actions of adenosine compounds on the human blister base preparation.
  108. (1956). Observations on the distribution of catechol amines and adenosinetriphosphate in the bovine adrenal medulla.
  109. (2001). On the contribution of the first transmembrane domain to whole-cell current through an ATPgated ionotropic P2X receptor.
  110. (1951). On the transmitter responsible for antidromic vasodilation in the rabbit's ear.
  111. (1999). Other peripheral mediators: 5-hydroxytryptamine and purines. In: Pharmacology fourth
  112. (2001). P2X Receptors in peripheral neurons.
  113. (2004). P2X3 receptor localizes into lipid rafts in neuronal cells.
  114. (1998). P2XI and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels.
  115. (2003). P2XI receptor currents after disruption of the PKC site and its surroundings by dominant negative mutations in HEK293 cells. Autonomic Neuroscience: Basic and Clinical,
  116. (2002). P2XI Receptor Subunit Contribution to Gating Revealed by a Dominant Negative PKC Mutant.
  117. (1979). Partial non-cleavage by cyanogen bromide of a methionine-cysteine bond from human serum albumin and bovine a-lactalbumin.
  118. (1998). Partial peripheral nerve injury leads to activation of astroglia and microglia which parallels the development of allodynic behaviour.
  119. (1995). Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2X purinoceptors). Molecular Pharmacology,
  120. (1992). Pharmacological evidence that adenosine triphosphate and noradrenaline are co-transmitters in the guinea-pig vas deferens.
  121. (2000). Pharmacology of cloned P2X receptors. Annual Review of Pharmacology and Toxicology,
  122. (2006). Pharmacology of P2X receptors.
  123. (2004). Photoactive benzophenone labelled pepticle.
  124. (1994). Photoaffinity labeling of creatine kinase with 2-azido- and 8-azidoadenosine triphosphate: identification of two peptides from the ATP-binding domain.
  125. (2001). Polar residues of the second transmembrane domain influence cation permeability of the ATP-gated P2X2.
  126. (1997). Presence of P2XI purinoceptors in human platelets and megakaryoblastic cell lines.
  127. (2006). Protein cleavage strategies for an improved analysis of the membrane proteome.
  128. (1972). Purinergic nerves.
  129. (2001). Purinergic signalling: ATP Release.
  130. (1994). Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacological Theories,
  131. (1996). Purinoceptors: ontogeny and phylogeny.
  132. (2006). Regulation of platelet functions by P2 Receptors. Annual Review of Pharmacology and Toxicology,
  133. (2000). Respiration and energy. In: Instant notes in biochemistry second
  134. (1996). Role of adenosine in the heart and circulation.
  135. (2000). Roles of individual N-glycans for ATP potency and expression of the rat P2X, receptor.
  136. (2000). Sensing arterial C02 levels: a role for medullary P2X receptors.
  137. (1996). Signalling by extracellular nucleotides.
  138. (1999). Single channel properties of P2X2purinoceptors.
  139. (2000). Smooth muscles do not have a common P2X receptor phenotype: expression, ontogeny and function of P2XI receptors in mouse ileum, bladder and reproductive systems.
  140. (2003). Storage and release of ATP from astrocytes in culture.
  141. (1996). Structure and function in rhodopsin: High level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines.
  142. (1990). Subtypes of P2-purinoceptors. Studies using analogues of ATP.
  143. (2003). Subunit arrangement in P2X receptors.
  144. (1944). The action of adenosine triphosphate and related compounds on mammalian skeletal muscle.
  145. (1986). The active site structure of Na+/K+-transporting ATPase: location of the 5-(p-fluorosulfonyl)benzoyladenosine binding site and soluble peptides released by trypsin.
  146. (1966). The Amino acid sequence of an extracellular Nuclease of Staphylococcus aureus.
  147. (1933). The biological significance of the linkages in adenosine triphosphate acid.
  148. (1954). The capillary dilator substance in dry powders of spinal roots: a possible role of adenosine triphosphate in chemical transmission from nerve endings.
  149. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7)-Science,
  150. (1998). The distribution of single P2XI-receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder.
  151. (1931). The effects of adenoisne and adenosine phosphates on the isolated guinea-pig uterus. Pflugers Archive,
  152. (1998). The expression of P2X3 purinoreceptors in sensory neurons: effects of axotomy and glial-derived neurotrophic factor. Molecular and cellular neurosciences,
  153. (2001). The FLAG pepticle, a versatile fusion tag for the purification of recombinant proteins.
  154. (1959). The liberation of adenosine triphosphate on anticiromic stimulation of sensory nerves.
  155. (2002). The nucleotide-binding site of human sphingosine kinase 1.
  156. (2003). The P2X3 subunit: a molecular target in pain therapeutics.
  157. (1978). The Post-junctional effects and neural release of purine compounds in the guinea-pig vas deferens.
  158. (2006). The purinergic component of human vas deferens contraction.
  159. (1958). The release of catechol amines from the amine containing granules of the adrenal medulla.
  160. (1995). The role of N-glycans in the secretory pathway.
  161. (2000). The Role of Positively Charged Amino Acids in ATP Recognition by Human P2XI Receptors.
  162. (1961). The transmission of excitation from autonomic nerve to smooth muscle.
  163. (1998). Topological analysis of the ATPgated ionotrophic P2X2 receptor subunit.
  164. (2000). Urinary bladder hypo-reflexia and reduced painrelated behaviour in P2X3 receptor-deficient mice.

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.