Abstract

P2X receptors are a family of ATP-gated ion channels. Four cDNAs with a high degree of homology to the rat P2X₂ receptor were isolated from human pituitary and pancreas RNA. Genomic sequence indicated that these cDNAs represent alternatively spliced messages. Northern analysis revealed high levels of human P2X₂(hP2X₂) message in the pancreas, and splice variants could be detected in a variety of tissues. Two cDNAs encoded functional ion channels when expressed in Xenopus oocytes, a receptor structurally homologous to the prototype rat P2X₂ receptor (called hP2X_2a) and a variant containing a deletion within its cytoplasmic C terminus (called hP2X_2b). Pharmacologically, these functional human P2X₂ receptors were virtually indistinguishable, with the P2X receptor agonists ATP, 2-methylthio-ATP, 2′ and 3′-O-(4-benzoylbenzoyl)-ATP, and ATP5′-O-(3-thiotriphosphate) being approximately equipotent (EC₅₀ = 1 μM) in eliciting extracellular Ca²⁺ influx. The P2 receptor agonists α,β-methylene ATP, adenosine, adenosine 5′-O-(2-thiodiphosphate), and UTP were inactive at concentrations up to 100 μM. Both hP2X_2a and hP2X_2b receptors were sensitive to the P2 receptor antagonist pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulfonic acid (IC₅₀ = 3 μM). In contrast to the analogous rat P2X₂ and P2X_2b receptors, the desensitization rates of the hP2X_2a and hP2X_2b receptors were equivalent. Both functional forms of the human P2X₂receptors formed heteromeric channels with the human P2X₃receptor. These data demonstrate that the gene structure and mRNA heterogeneity of the P2X₂ receptor subtype are evolutionarily conserved between rat and human, but also suggest that alternative splicing serves a function other than regulating the desensitization rate of the human receptor.