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The human P2X4 receptor gene is alternatively spliced
P D Dhulipala1, Y X Wang, M I Kotlikoff
1Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6046, USA.
Gene
|March 25, 1998
Summary
Researchers cloned human P2X4 receptor variants, including a full-length and an alternatively spliced form. Only the full-length P2X4 gene expressed ATP-gated cation currents in oocytes, suggesting functional differences.
Area of Science:
- Molecular Biology
- Neuroscience
- Cell Biology
Background:
- Adenosine triphosphate (ATP) functions as an excitatory neurotransmitter.
- P2X receptors are ligand-gated, non-selective cation channels crucial for ATP signaling.
- Understanding P2X receptor subtypes, like P2X4, is vital for elucidating cellular responses to ATP.
Purpose of the Study:
- To clone and characterize full-length and alternatively spliced forms of the human P2X4 gene.
- To investigate the expression patterns of human P2X4 variants in different tissues.
- To determine the functional consequences of P2X4 splicing on ATP-gated ion channel activity.
Main Methods:
- Cloning of human P2X4 cDNA from a stomach library using a rat P2X4 probe.
- Nucleotide sequence analysis to identify full-length and alternatively spliced variants.
- In vitro translation to confirm open reading frames.
- Northern analysis and RT-PCR with RNAse protection assays to determine tissue expression.
- Xenopus oocyte expression system to assess functional ATP-gated currents.
Main Results:
- Identified and sequenced full-length human P2X4 cDNA (388 residues, 82% homology to rat P2X4) and an alternatively spliced variant.
- The splice variant replaces the N-terminus with a sequence homologous to hsp-90 chaperonin proteins.
- Northern analysis revealed full-length P2X4 expression in smooth muscle, heart, and skeletal muscle.
- Alternatively spliced P2X4 RNAs were detected in smooth muscle and brain.
- Only the full-length P2X4 cRNA, when injected into Xenopus oocytes, produced ATP-gated non-selective cation currents.
Conclusions:
- The human P2X4 gene exists in at least two forms: full-length and alternatively spliced.
- The alternatively spliced P2X4 variant likely results from alternative splicing involving a sequence related to hsp-90.
- Functional studies in Xenopus oocytes indicate that the full-length P2X4 receptor mediates ATP-gated cation currents, while the alternatively spliced form does not, suggesting differential roles in cellular signaling.