Characterization of affinity-tagged fluorescent P2X and P2Y receptors in BAC transgenic mice in health and disease

• Antragstellerinnen / Antragsteller: Privatdozentin Dr. Heike Franke; Professor Dr. Günther Schmalzing
• Fachliche Zuordnung: Klinische Neurologie; Neurochirurgie und Neuroradiologie
• Förderung: Förderung von 2007 bis 2015
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 22935240

Information about the precise distribution of specific receptors on neurons is crucial for the understanding of pain processing. Converging evidence from electrophysiological, genetic and pharmacological studies supports the notion of an important role for P2X2 and, particularly, P2X3 receptors in nociception. To facilitate the morphological and functional identification of neurons carrying P2X2 or P2X3 receptors, we plan to generate transgenic mice, which express these subunits as fluorescent fusion proteins (P2X2-CFP, P2X3-YFP) under the control of their own promotors. Double transgenic mice will be obtained by crossbreeding the single transgenic mice. The expressed fluorescence will (i) allow for the comprehensive microscopic mapping of the distribution of P2X2, P2X3, and P2X2+3 receptors in mice, and (ii) guide the identification of neurons for functional analysis by patch clamping. In addition, using incorporated tandem affinity tags, the fluorescent receptors can be isolated under non-denaturing conditions from specific tissues for biochemical analysis. By exploiting the advantages of recombinant protein technology, this additional approach has the potential to resolve the subunit stoichiometry of heteromeric P2X2+3 receptors and the P2X2/P2X3 receptor asset of individual tissues. Furthermore, it enables co-purification of receptorinteracting proteins from native tissues for mass spectrometric identification. Overall, these transgenic mice should provide a rich resource for new insights in P2X2/P2X3 receptor localization and function and later developmental and pathophysiological studies.

• DFG-Verfahren: Forschungsgruppen
• Teilprojekt zu: FOR 748: Neuronal and glial P2 receptors; molecular basis and functional significance
• Beteiligte Person: Dr. Ralf Hausmann