Cloning and expression of a P2X receptor from the chick embryo.

P2X receptors are ATP-gated ion channels. A number of P2X receptor subunits (P2X$\sb{1-7}$) have been cloned from a variety of tissues, including rat vas deferens, PC12 cells, rat dorsal root ganglia, and rat brain. All the cloned P2X receptors contain two putative transmembrane domains and therefore are distinct from other ligand-gated ion channels such as the nicotinic acetylcholine receptors and the ionotropoic glutamate receptors. P2X receptors have been suggested to function in fast synaptic transmission, nociception, ATP-mediated cell lysis and programmed cell death. ATP has a potent depolarizing action on embryonic chick skeletal muscle, but has no effect on skeletal muscle in hatched chicks. In a search for the P2X receptor responsible for the depolarizing action of ATP on embryonic chick skeletal muscle, we screened a 10 day chick embryo cDNA library with P2X$\sb1$ as the probe. We identified a full length P2X cDNA clone which we have tentatively named as P2XCE. The full length P2XCE cDNA contains an open-reading frame coding for 385 amino acids. P2XCE has the highest sequence homology to P2X$\sb4$ which was cloned from rat brain, sharing 75% amino acid sequence identity with P2X$\sb4.$ We conducted three lines of investigation to study the expression of P2XCE in vivo and in vitro. RNase protection assays indicated that P2XCE mRNA is expressed in several tissues at different developmental stages, including skeletal muscle of E10 embryo and newly hatched chicks. When voltage clamped at $-$100 mV, ATP elicits an inward current on HEK293 cells transfected with P2XCE cDNA. P2XCE is different from the endogenous P2X receptor of embryonic chick skeletal muscle and the rat P2X$\sb4$ receptor in properties such as desensitization, affinity for ATP, agonist and antagonist specificity, and expression level in specific tissues. Based on these differences, we conclude that P2XCE by itself does not form the endogenous P2X receptor. P2XCE appears to be a novel member of the P2X receptor family with the closest relationship to P2X$\sb4.$PhDBiological SciencesMolecular biologyNeurosciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/130767/2/9811131.pd