Characterization of affinity-tagged fluorescent P2X and P2Y receptors in BAC transgenic mice in health and disease
Final Report Abstract
The main objectives of FOR 748 project 3 were (i) to generate BAC transgenic mice that faithfully mirror the expression of P2X2, P2X3 and P2Y1 receptors and, by using these mice, (ii) to determine the spatial and temporal expression patterns of P2X2, P2X3 and P2Y1 receptors in healthy mice and (iii) expression changes in mice with chronic inflammatory or neuropathic pain. We have successfully generated three BAC transgenic mice strains, each expressing one of the following P2 receptor transgenes under the control of their genomic regulatory elements: (1) the mP2X2-Strep-His-TagRFP fusion protein (2) P2X3 promotor-driven soluble GFP (3) and P2Y1 promotor-driven soluble TagRFP. By using the strains exhibiting the strongest fluorescence, we have characterized in healthy adult mice the basal expression of P2X2, P2X3 and P2Y1 receptors in several CNS areas including DRG neurons, spinal cord, hippocampus, olfactory bulb and cerebellum. The main novel findings are as follows. Firstly, with the P2X2-TagRFP mice we found that the P2X2 receptor expression on cerebellar Purkinje cells is mainly restricted to the somata of these cells and is low in dendrites. This finding is important for the understanding of the role of P2X2 receptors in the cerebellar neuronal network. Secondly, in the testis of our adult P2X3 reporter mice we found strong P2X3 expression in spermatocytes, spermatides and mature sperm cells, but not in spermatogonia. This finding discloses a so far unknown role of P2X3 receptors in maturation and differentiation in male gametogenesis. Thirdly, with our P2Y1 reporter mice we could show for the first time that a subset of mitral cells and external tufted cells of the adult olfactory bulb express P2Y1 receptors. We have established mouse models of chronic inflammatory pain and neuropathic pain using injection of complete Freunds adjuvant (CFA) and chronic constriction injury (CCI), respectively. The preliminary data from the ongoing work show that the number of P2X3 expressing neurons in dorsal root ganglia of segment L4-S3 is unchanged in mice with unilateral constriction injury of the sciatic nerve. This suggests that the well-known increase in P2X3 receptor-mediated neurotransmission in neuropathic pain must result from an increased number of P2X3 receptors per neuron or enhanced signal propagation rather than the de novo expression of P2X3 receptors in sensory neurons that normally lack P2X3 receptors. The presentation of data of our P2X3 and P2Y1 receptor reporter mice during the international ‘Purines 2014’ conference in Bonn resulted in five requests from national and international scientists to use our mice for addressing pertinent questions. We searched the inevitable breaks imposed by breeding the BAC mice to conduct a series of related studies that are briefly outlined here. (i) We have elucidated the role of astrocyte P2Y1 and GPR17 receptors in traumatic brain injury in human tissue and P2X7 receptors in astrocyte–neuron communication in cooperation with project 1. (ii) By combining X-ray structure-based homology modeling and site-directed mutagensis, we have identified in collaboration with TP 1 amino acid residues responsible for the potency of P2X agonists and antagonists at the P2X2, P2X2+6 and P2X3 receptor and involved in spontaneous and ATP-induced conformational changes during activation of the P2X2 or P2X3 receptor.
Publications
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Cross-inhibition between native and recombinant TRPV1 and P2X(3) receptors. Pain. 2009; 143: 26–36
Stanchev D, Blosa M, Milius D, Gerevich Z, Rubini P, Schmalzing G, Eschrich K, Schaefer M, Wirkner K, Illes P
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Amino acid residues constituting the agonist binding site of the human P2X3 receptor. J Biol Chem. 2011; 286: 2739–49
Bodnar M, Wang H, Riedel T, Hintze S, Kato E, Fallah G, Groger-Arndt H, Giniatullin R, Grohmann M, Hausmann R, Schmalzing G, Illes P, Rubini P
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Molecular determinants of potent P2X2 antagonism identified by functional analysis, mutagenesis, and homology docking. Mol Pharmacol. 2011; 79: 649–61
Wolf C, Rosefort C, Fallah G, Kassack MU, Hamacher A, Bodnar M, Wang H, Illes P, Kless A, Bahrenberg G, Schmalzing G, Hausmann R
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P2X Receptors and Their Roles in Astroglia in the Central and Peripheral Nervous System. The Neuroscientist. 2011; 18: 422–38
Illes P, Verkhratsky A, Burnstock G, Franke H
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Rodent cortical astroglia express in situ functional P2X7 receptors sensing pathologically high ATP concentrations. Cereb Cortex. 2011; 21: 806–20
Oliveira JF, Riedel T, Leichsenring A, Heine C, Franke H, Krügel U, Nörenberg W, Illes P
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ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors. J Biol Chem. 2012; 287: 13930–43
Hausmann R, Bodnar M, Woltersdorf R, Wang H, Fuchs M, Messemer N, Qin Y, Günther J, Riedel T, Grohmann M, Nieber K, Schmalzing G, Rubini P, Illes P
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Molecular and functional properties of P2X receptors--recent progress and persisting challenges. Purinergic Signal. 2012; 8: 375–417
Kaczmarek-Hajek K, Lorinczi E, Hausmann R, Nicke A
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P2X1 and P2X2 receptors in the central nervous system as possible drug targets. CNS Neurol Disord Drug Targets. 2012; 11: 675–86
Hausmann R, Schmalzing G
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Pathophysiology of astroglial purinergic signalling. Purinergic Signal. 2012; 8: 629–57
Franke H, Verkhratsky A, Burnstock G, Illes P
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Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors. Br J Pharmacol. 2012; 167: 48–66
Nörenberg W, Sobottka H, Hempel C, Plotz T, Fischer W, Schmalzing G, Schaefer M
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Changes of the GPR17 receptor, a new target for neurorepair, in neurons and glial cells in patients with traumatic brain injury. Purinergic Signal. 2013; 9: 451–62
Franke H, Parravicini C, Lecca D, Zanier ER, Heine C, Bremicker K, Fumagalli M, Rosa P, Longhi L, Stocchetti N, De Simoni MG, Weber M, Abbracchio MP
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Heteromeric assembly of P2X subunits. Front Cell Neurosci. 2013; 7: 250
Saul A, Hausmann R, Kless A, Nicke A
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Salt bridge switching from Arg290/Glu167 to Arg290/ATP promotes the closedto-open transition of the P2X2 receptor. Mol Pharmacol. 2013; 83: 73–84
Hausmann R, Günther J, Kless A, Kuhlmann D, Kassack MU, Bahrenberg G, Markwardt F, Schmalzing G
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A hydrophobic residue in position 15 of the rP2X3 receptor slows desensitization and reveals properties beneficial for pharmacological analysis and high-throughput screening. Neuropharmacology. 2014; 79: 603–15
Hausmann R, Bahrenberg G, Kuhlmann D, Schumacher M, Braam U, Bieler D, Schlusche I, Schmalzing G
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Astrocyte-neuron interaction in the substantia gelatinosa of the spinal cord dorsal horn via P2X7 receptor-mediated release of glutamate and reactive oxygen species. Glia. 2014; 62: 1671–786
Ficker C, Rozmer K, Kató E, Andó RD, Schumann L, Krügel U, Franke H, Sperlágh B. Riedel T, Illes P
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Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening. Br J Pharmacol. 2014; 171: 5093–112
Kowalski M, Hausmann R, Dopychai A, Grohmann M, Franke H, Nieber K, Schmalzing G, Illes P, Riedel T
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Natural compounds with P2X7 receptormodulating properties. Purinergic Signal. 2014; 10:313–326
Fischer W, Urban N, Immig K, Franke H, Schaefer M
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Pathological potential of astroglial purinergic receptors. In: Glutamate and ATP at interface of metabolism and signaling in the brain, Advances in Neurobiology, Vol. 9. Eds.: Parpura V, Schousboe A, Verkhratsky A. 2014; 11: 213–56
Franke H, Illes P
