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Identification of a loss-of-function polymorphism in the human P2X4 receptor

L. Stokes,1 K.K. Skarratt,1 B.J. Gu1 and J.S. Wiley,2 1Sydney Medical School - Nepean, University of Sydney, Penrith, NSW 2750, Australia and 2Howard Florey Institute, Alan Gilbert Building, Carlton South, VIC 3053, Australia.

The P2X4 receptor is a ligand-gated ion channel activated by extracellular ATP. The P2RX4 gene lies adjacent to the highly polymorphic P2RX7 gene on chromosome 12q24.3. To date four non-synonymous single nucleotide polymorphisms (SNPs) have been found in P2RX4 however the functional effects associated with these mutations in the receptor are unknown. Site directed mutagenesis was used to introduce mutations into a GFP-tagged human P2X4 plasmid and functional P2X4 responses were measured using whole cell patch clamp electrophysiology in transfected HEK-293 cells. The Tyr 315>Cys mutation showed a dramatic loss-of-function with a response of only 10.9% of wild-type P2X4 receptors (p= 0.0002, n=4-8 cells). This tyrosine residue is predicted to contribute to ATP binding in the extracellular domain and the Tyr 315>Cys mutant displayed a reduced sensitivity to ATP (EC50 of 192 μM compared to wild-type P2X4 EC50 of 5 μM). The Ala 6>Ser, Ile 119>Val and Ser 242>Gly mutations showed no significant difference in ATP sensitivity. We genotyped 200-500 Caucasian subjects at four SNPs in the P2RX4 gene to determine allele frequencies in the population. We found the Tyr 315>Cys polymorphism was rare with a frequency of 1.1% (n=416 subjects) and was only found in heterozygous dosage.