Abstract

We isolated a cDNA from human brain encoding a purinergic receptor that shows a high degree of homology to the rat P2X₄ receptor (87% identity). By fluorescence in situ hybridization, the human P2X₄ gene has been mapped to region q24.32 of chromosome 12. Tissue distribution analysis of human P2X₄transcripts demonstrates a broad expression pattern in that the mRNA was detected not only in brain but also in all tissues tested. Heterologous expression of the human P2X₄ receptor inXenopus laevis oocytes and human embryonic kidney 293 cells evoked an ATP-activated channel. Simultaneous whole-cell current and Fura-2 fluorescence measurements in human embryonic kidney 293 cells transfected with human P2X₄ cDNA allowed us to determine the fraction of the current carried by Ca²⁺; this was ∼8%, demonstrating a high Ca²⁺ permeability. Low extracellular Zn²⁺ concentrations (5–10 μm) increase the apparent gating efficiency of human P2X₄ by ATP without affecting the maximal response. However, raising the concentration of the divalent cation (>100 μm) inhibits the ATP-evoked current in a non-voltage-dependent manner. The human P2X₄ receptor displays a very similar agonist potency profile to that of rat P2X₄ (ATP ≫ 2-methylthio-ATP ≥ CTP > α,β-methylene-ATP > dATP) but has a notably higher sensitivity for the antagonists suramin, pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid, and bromphenol blue. Chimeric constructs between human and rat isoforms as well as single-point mutations were engineered to map the regions responsible for the different sensitivity to suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid.