Genetic and Functional Analysis of Human P2X5 Reveals a Distinct Pattern of Exon 10 Polymorphism with Predominant Expression of the Nonfunctional Receptor Isoform Smita Kotnis, Brendan Bingham, Dmitry V. Vasilyev, Scott W. Miller, Yuchen Bai, Sarita Yeola, Pranab K. Chanda, Mark R. Bowlby, 1 Edward J. Kaftan, Tarek A. Samad, and Garth T. Whiteside Pfizer Global Research and Development, Princeton, New Jersey Received January 19, 2010; accepted March 11, 2010 ABSTRACT P2X5 is a member of the P2X family of ATP-gated nonselec- tive cation channels, which exist as trimeric assemblies. P2X5 is believed to trimerize with another member of this family, P2X1. We investigated the single-nucleotide polymor- phism (SNP) at the 3' splice site of exon 10 of the human P2X5 gene. As reported previously, presence of a T at the SNP location results in inclusion of exon 10 in the mature transcript, whereas exon 10 is excluded when a G is present at this location. Our genotyping of human DNA samples reveals predominance of the G-bearing allele, which was exclusively present in DNA samples from white American, Middle Eastern, and Chinese donors. Samples from African American donors were polymorphic, with the G allele more frequent. Reverse transcription-polymerase chain reaction analysis of lymphocytes demonstrated a 100% positive cor- relation between genotype and P2X5 transcript. Immuno- staining of P2X1/P2X5 stably coexpressing cell lines showed full-length P2X5 to be expressed at the cell surface and the exon 10-deleted isoform to be cytoplasmic. Fluorometric imaging-based pharmacological characterization indicated a ligand-dependent increase in intracellular calcium in 1321N1 astrocytoma cells transiently expressing full-length P2X5 but not the exon 10-deleted isoform. Likewise, electrophysiolog- ical analysis showed robust ATP-evoked currents when full- length but not the exon 10-deleted isoform of P2X5 was expressed. Taken together, our findings indicate that most humans express only a nonfunctional isoform of P2X5, which is in stark contrast to what is seen in other vertebrate species in which P2X5 has been studied, from which only the full- length isoform is known. P2X receptors belong to the family of nonselective cation channels gated by extracellular ATP (Burnstock, 2006; Surprenant and North, 2009). To date, seven P2X recep- tors have been cloned, denoted P2X1–P2X7 (MacKenzie et al., 1999). P2X receptors form trimeric assemblies (Kawate et al., 2009) with both homomeric and heteromeric forms having been described previously (Virginio et al., 1998; Haines et al., 1999; Cockayne et al., 2005; Guo et al., 2007; Nicke, 2008). P2X5 is widely expressed in immune and nervous system tissue (Le ˆ et al., 1997) and heart and skeletal muscle (Collo et al., 1996; Garcia-Guzman et al., 1996; Cox et al., 2001; North, 2002; Ryten et al., 2002; Guo et al., 2008; Musa et al., 2009). Among the known human P2X5 splice variants is a poly- morphism for exon 10. The 22 amino acids encoded by exon 10, including the carboxyl terminus of the extracellular ATP- binding site (Hansen et al., 1997) and the amino terminus of the second transmembrane domain (Fig. 1A), are important to ion channel function. Absence of these residues results in a nonfunctional protein that is prone to aggregation (Le ˆ et al., 1997; Bo et al., 2003; Duckwitz et al., 2006). The human polymorphism for exon 10 is in fact a polymorphism for functional P2X5 receptor. The human P2X5 exon 10 polymorphism arises from a single-nucleotide polymorphism (SNP) at the 3' splice site of exon 10 (Fig. 1B). The original P2X5 cDNA cloned from human brain lacks an exon 10 sequence (Le ˆ et al., 1997), 1 Current affiliation: Pain and Migraine Research, Merck Research Labs, West Point, Pennsylvania. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.110.063636. ABBREVIATIONS: SNP, single-nucleotide polymorphism; FLIPR, fluorometric imaging plate reader; -me-ATP, --methyl-ATP; PPADS, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid; GFP, green fluorescent protein; BCC, basal cell carcinoma; SCC, squamous cell carci- noma; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; TNP, 2',3'-O- (2,4,6-trinitrophenyl); TM, transmembrane. 0026-895X/10/7706-953–960$20.00 MOLECULAR PHARMACOLOGY Vol. 77, No. 6 Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics 63636/3589044 Mol Pharmacol 77:953–960, 2010 Printed in U.S.A. 953 at ASPET Journals on July 19, 2018 molpharm.aspetjournals.org Downloaded from