Characterization of myocardial extracellular ATP receptors by photoaffinity labelling and functional assays.
Extracellular ATP receptors in rat ventricular myocytes were investigated through intact cell photolabelling followed by protein isolation. 8-Azido-ATP (8Az-ATP) was used for labelling under specific conditions determined by parallel functional studies. In those studies ATP-induced cytosolic Ca2+ transients were irreversibly and specifically inhibited by UV-photolyzed 8Az-ATP, but not by 2-azido-ATP (2Az-ATP), even in the presence of high concentrations of phosphonucleotides not affecting myocardial ATP receptors. Under those conditions background labelling is minimized and radioactive 8Az-ATP specifically labels a band of 45-48 kDa on a SDS gel. Labelling under the above conditions in the presence of ATP gamma S or 2-methylthio-ATP (2-meSATP), which are distinct for two functionally different cardiac ATP receptors, shows two different proteins within the same band consistent with the possible labelling of these two receptors.
Giannattasio
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Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44116.
Powers
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K
Scarpa
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D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002118:Calcium / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011983:Receptors, Purinergic / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D011983:Receptors, Purinergic; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002118:Calcium