Characterisation of ATP analogues to cross-link and label P2X receptors.
P2X receptors are a distinct family of ATP-gated ion channels with a number of physiological roles ranging from smooth muscle contractility to the regulation of blood clotting. In this study we determined whether the UV light-reactive ATP analogues 2-azido ATP, ATP azidoanilide (ATP-AA) and 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) can be used to label the ATP binding site of P2X1 receptors. These analogues were agonists, and in patch clamp studies evoked inward currents from HEK293 cells stably expressing the P2X1 receptor. Following irradiation in the presence of these compounds subsequent responses to an EC50 concentration of ATP were reduced by >65%. These effects were partially reversed by co-application of ATP or suramin with the photo-reactive ATP analogue at the time of irradiation. In autoradiographic studies radiolabelled 2-azido [gamma32P] ATP and ATP-AA-[gamma32P] cross-linked to P2X1 receptors and this binding was reduced by co-incubation with ATP. These studies demonstrate that photo-reactive ATP analogues can be used to label P2X receptor and may prove useful in elucidating the ATP binding site at this novel class of ATP binding proteins.
Agboh
Kelvin C
KC
Department of Cell Physiology and Pharmacology, University of Leicester, P.O. Box 138, University Road, Leicester LE1 9HN, UK.
Powell
Andrew J
AJ
Evans
Richard J
RJ
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D001386:Azides / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D055592:Biophysical Phenomena; D002461:Cell Line, Transformed; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation / Q000379:methods; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D018408:Patch-Clamp Techniques; D010759:Phosphorus Isotopes / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011827:Radiation; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058476:Receptors, Purinergic P2X2; D014162:Transfection / Q000379:methods
D001386:Azides; C546401:P2RX2 protein, human; D010759:Phosphorus Isotopes; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate