Research Paper
Subject Category: Molecular and cellular mechanisms
British Journal of Pharmacology (2008) 153, 544–556; doi:10.1038/sj.bjp.0707596; published online 26 November 2007
Cloning and pharmacological characterization of the guinea pig P2X7 receptor orthologue
E Fonfria1, W C Clay2, D S Levy3,4, J A Goodwin1, S Roman1, G D Smith1, J P Condreay2 and A D Michel1
- 1Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research & Development Limited, New Frontiers Science Park, Harlow, Essex, UK
- 2Department of Biochemical & Cellular Targets, Molecular Discovery Research, GlaxoSmithKline, Research Triangle Park, NC, USA
- 3Department of Genomic & Proteomic Sciences, Genetics Research, GlaxoSmithKline Research and Development, Collegeville, PA, USA
Correspondence: Dr E Fonfria, Neurology & GI Centre of Excellence for Drug Discovery, GlaxoSmithKline Research & Development Limited, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK. E-mail: Elena.2.Fonfria@gsk.com
4Current address: Oncology Center of Excellence for Drug Discovery, GlaxoSmithKline Research and Development, South Collegeville Road, Collegeville, PA 19426, USA.
Received 24 July 2007; Revised 12 September 2007; Accepted 30 October 2007; Published online 26 November 2007.
Abstract
Background and purpose:
The human, rat, and mouse P2X7 receptors have been previously characterized, and in this study we report the cloning and pharmacological properties of the guinea pig orthologue.
Experimental approach:
A cDNA encoding for the guinea pig P2X7 receptor was isolated from a guinea pig brain library. The receptor was expressed in U-2 OS cells using the BacMam viral expression system. A monoclonal antibody was used to confirm high levels of cell surface expression and the functional properties were determined in ethidium bromide accumulation studies.
Key results:
The predicted guinea pig protein is one amino acid shorter than the human and rat orthologues and over 70% identical to the rat and human receptors. In contrast to human and rat P2X7 receptors, 2'-&3'-O-(4benzoylbenzoyl)ATP (BzATP) was a partial agonist of the guinea pig P2X7 receptor when compared to ATP and acted as an antagonist in some assays. However, as at other species orthologues, BzATP was more potent than ATP. The guinea pig P2X7 receptor possessed higher affinity for 1-[N,O-bis(5-isoquinoline sulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62), suramin and Coomassie Brilliant Blue than human or rat P2X7 receptors suggesting that it is pharmacologically different to other rodent or human P2X7 receptors.
Conclusions and implications:
The guinea pig recombinant P2X7 receptor displays a number of unique properties that differentiate it from the human, rat and mouse orthologues and this structural and functional information should aid in our understanding of the interaction of agonists and antagonist with the P2X7 receptor.
Keywords:
ion channel, BacMam, ATP, BzATP, U-2 OS
Abbreviations:
BacMam, recombinant baculovirus in which the polyhedrin promoter has been replaced with a mammalian promoter; BzATP, 2'-&3'-O-(4benzoylbenzoyl) ATP; CBB, Coomassie brilliant blue; KN62, 1-[N, O-bis(5-isoquinolinesulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; mAb, monoclonal antibody; PFU, plaque-forming units; PPADS, pyridoxal phosphate-6-azophenyl-2', 4'-disulphonic acid; U-2 OS, human osteosarcoma cell line
