Genetic and functional analysis of human P2X5 reveals a distinct pattern of exon 10 polymorphism with predominant expression of the nonfunctional receptor isoform.
P2X5 is a member of the P2X family of ATP-gated nonselective cation channels, which exist as trimeric assemblies. P2X5 is believed to trimerize with another member of this family, P2X1. We investigated the single-nucleotide polymorphism (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 correlation between genotype and P2X5 transcript. Immunostaining 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, electrophysiological 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.
Kotnis
Smita
S
Pfizer Global Research and Development, CN8000, Princeton, NJ 08543-8000, USA.
Bingham
Brendan
B
Vasilyev
Dmitry V
DV
Miller
Scott W
SW
Bai
Yuchen
Y
Yeola
Sarita
S
Chanda
Pranab K
PK
Bowlby
Mark R
MR
Kaftan
Edward J
EJ
Samad
Tarek A
TA
Whiteside
Garth T
GT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D005091:Exons; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D058485:Receptors, Purinergic P2X5; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
C546465:P2RX5 protein, human; D020033:Protein Isoforms; D018048:Receptors, Purinergic P2; D058485:Receptors, Purinergic P2X5