Characterization of myocardial extracellular ATP receptors by photoaffinity labelling and functional assays

[摘要]

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 ATPγS 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.

[学科分类] 生物化学/生物物理

[关键词] ATP receptor;Photolabelling;Myocyte;8-Azido-ATP;ATPγS;adenosine-5′-O-(3-thiotriphosphate);2Az-ATP;2-azido-adenosine-5′-triphosphate;8Az-ATP;8-azido-adenosine-5′-triphosphate;Bz-ATP;3′-O-(4-benzoyl)benzoyl-[α-32P]adenosine-5′-triphosphate;BSA;bovine serum albumin;2-meSATP;2-methylthio- adenosine-5′-triphosphate;fura2/AM;fura2-acetoxymethyl ester;GTP;guanosine-5′-triphosphate;SDS;sodium dodecyl sulfate;UTP;uridine-5′-triphosphate