CHARACTERIZATION OF RECOMBINANT HUMAN P2X(4) RECEPTOR REVEALS PHARMACOLOGICAL DIFFERENCES TO THE RAT HOMOLOG

We isolated a cDNA from human brain encoding a purinergic receptor tha t shows a high degree of homology to the rat P2X(4) receptor (87% iden tity). By fluorescence in situ hybridization, the human P2X(4) gene ha s been mapped to region q24.32 of chromosome 12. Tissue distribution a nalysis of human P2X(4) transcripts demonstrates a broad expression pa ttern in that the mRNA was detected not only in brain but also in all tissues tested. Heterologous expression of the human P2X(4) receptor i n Xenopus laevis oocytes and human embryonic kidney 293 cells evoked a n ATP-activated channel. Simultaneous whole-cell current and Fura-2 fl uorescence measurements in human embryonic kidney 293 cells transfecte d with human P2X(4) cDNA allowed us to determine the fraction of the c urrent carried by Ca2+; this was similar to 8%, demonstrating a high C a2+ permeability. Low extracellular Zn2+ concentrations (5-10 mu M) in crease the apparent gating efficiency of human P2X(4) by ATP without a ffecting the maximal response. However, raising the concentration of t he divalent cation (>100 mu M) inhibits the ATP-evoked current in a no n-voltage-dependent manner. The human P2X(4) receptor displays a very similar agonist potency profile to that of rat P2X(4) (ATP much greate r than 2-methylthio-ATP greater than or equal to CTP > alpha,beta-meth ylene-ATP > dATP) but has a notably higher sensitivity for the antagon ists suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, a nd 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-pho sphate-6-azophenyl-2',4'-disulfonic acid.