Agboh Kelvin C, Powell Andrew J, Evans Richard J
Department of Cell Physiology and Pharmacology, University of Leicester, P.O. Box 138, University Road, Leicester LE1 9HN, UK.
Neuropharmacology. 2009 Jan;56(1):230-6. doi: 10.1016/j.neuropharm.2008.05.018. Epub 2008 Jul 2.
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.
P2X受体是一类独特的ATP门控离子通道家族，具有多种生理作用，从平滑肌收缩到血液凝固调节。在本研究中，我们确定了紫外线反应性ATP类似物2-叠氮基ATP、ATP叠氮苯胺（ATP-AA）和2',3'-O-(4-苯甲酰苯甲酰基)-ATP（BzATP）是否可用于标记P2X1受体的ATP结合位点。这些类似物是激动剂，在膜片钳研究中，它们能诱发稳定表达P2X1受体的HEK293细胞产生内向电流。在这些化合物存在下进行照射后，对ATP的EC50浓度的后续反应降低了>65%。在照射时，将ATP或苏拉明与光反应性ATP类似物共同应用可部分逆转这些效应。在放射自显影研究中，放射性标记的2-叠氮基[γ32P]ATP和ATP-AA-[γ32P]与P2X1受体交联，并且这种结合会因与ATP共同孵育而减少。这些研究表明，光反应性ATP类似物可用于标记P2X受体，并且可能在阐明这类新型ATP结合蛋白的ATP结合位点方面证明是有用的。
