Molecular cloning and characterization of a novel ATP P2X receptor subtype from embryonic chick skeletal muscle

Abstract

We have cloned a new P2X ligand-gated ion channel receptor from embryonic chick skeletal muscle, which is tentatively named as chick P2X8 (cP2X8) receptor. The cloned cDNA encodes a protein with 402 amino acids. Electrophysiological study of the recombinant cP2X8 receptor expressed in Xenopus oocytes showed that 10 μM ATP induced a fast inward current followed by rapid and long lasting desensitization in medium containing 1.8 mM Ca2+. In medium with 0.3 mM Ca2+ ATP induced a bi-phasic response as follows: a slower inward current succeeded the initial fast one. 2-Methylthio-ATP, α,β-methylene-ATP, and adenosine 5'-O-(thio)triphosphate were potent agonists, whereas ADP was a very weak agonist. ATP-induced currents were blocked by 100 μM suramin and pyridoxal phosphate 6-azophenyl-2',4'- disulfonic acid. Northern blot analysis and reverse transcription-polymerase chain reaction showed that cP2X8 RNA transcripts were mainly expressed in skeletal muscle, brain, and heart of Day 10 chick embryos. A moderate level of expression was also detected in gizzard and retina. Whole mount in situ hybridization showed that cP2X8 RNA transcripts were expressed mainly in neurotube, notochord, and stomach in Day 3 embryos. In Day 4 and Day 6 embryos, the cP2X8 RNA transcripts were highly expressed in the myotome and premuscle mass. The physiological role of this receptor in the establishment of the skeletal muscle innervation will be studied.