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The contribution of the intracellular domains to P2X1 receptor regulation

By Hairuo Wen

Abstract

P2X1 receptors are expressed throughout the body and contribute to a range of physiological process, e.g. thrombosis and smooth muscle tone. The intracellular terminals of the P2X1 receptor have been shown to be involved in channel regulation.\ud The aim of this thesis was to explore the contribution of the intracellular amino and carboxy termini to channel regulation, using concatenation, site-directed mutagenesis and biochemical methods. Trimeric concatenated P2X1 receptors were non-functional.The interactions between the intracellular termini were revealed to be essential for subunit aggregation and channel expression. Subsequently, over 30 cysteine point mutations at both the amino and carboxy domains of the P2X1 receptors were generated and examined by electrophysiological studies with methanethiosulfonate modifications. The conserved TXTXK/R at the amino terminal and the residues around the YXXXK motif at the carboxy terminal were highlighted to be important for channel gating and protein trafficking. Residues close to the P2X1 channel pore were shown to be essential in ion conduction and implied interactions between the conserved regulatory motifs and the channel pore of adjacent residues.\ud Roles of the intracellular domains of P2X1 receptor in regulation by protein kinase were revealed by co-expression of minigenes encoding the sequences of amino/carboxy terminus with WT receptors. The results indicated both amino and carboxy termini contribute to the phorbol ester PMA and GPCR mediated response regulations, and demonstrated the important roles of the conserved TXTXK/R motif and the residues around the YXXXK motif. This research sheds light on the possible interactions between the amino and carboxy termini to P2X1 receptor function. The results will be useful for understanding the intracellular topology and signaling modulation mechanism of P2X1 receptor

Publisher: University of Leicester
Year: 2009
OAI identifier: oai:lra.le.ac.uk:2381/9054

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