Lack of specificity of [35S]-ATP?S and [35S]-ADP?S as radioligands for ionotropic and metabotropic P2 receptor binding

Abstract

Adenosine-5'-O-3-thio[ 35 S]triphosphate ([ 35 S]-ATPγS) has been reported to specifically bind several P2X receptor subtypes, including P2X 1 , P2X 2 , P2X 3 , and P2X 4 . Similarly, adenosine-5'-O-2-thio[ 35 S]diphosphate ([ 35 S]-ADPβS) has been reported to label putative P2Y receptors. To address whether these radioligands selectively label P2 receptors, the functional activity of various P2 ligands was compared with their ability to compete for [ 35 S]-ATPγS and [ 35 S]-ADPβS binding to cell membrane preparations from rat brain, HEK293 cells, and to native and P2X 4 transfected 1321N1 astrocytoma cells. [ 35 S]-ATPγS (0.2 nM) and [ 35 S]-ADPβS (0.1 nM) displayed a high percentage of specific binding to membranes prepared from 1321N1 human astrocytoma cells, which were found to be devoid of detectable P2X and P2Y functional activity. [ 35 S]-ATPγS and [ 35 S]-ADPβS also exhibited equivalent high percentages of specific binding to HEK293 cell membranes, which endogenously express the P2Y 1 and P2Y 2 receptor subtypes, to 1321N1 cells stably transfected with the human P2X 4 receptor, and to rat brain membranes, which have previously been shown to contain both P2X and P2Y receptor subtypes. The potency order of P2 agonists to compete for radioligand binding to these cell membrane preparations was significantly different from the functional rank order potencies determined in HEK293 cells and 1321N1 cells expressing the P2X 4 receptor, as measured by cytosolic calcium flux. These data indicate that [ 35 S]-ATPγS and [ 35 S]-ADPβS appear to bind sites that do not correspond to known functional P2 receptor subtypes. The apparent lack of specificity of these radioligands for labeling P2 receptors is similar to that reported for other radiolabeled nucleotides and illustrates the need for caution in interpreting the apparent pharmacology of native P2 receptors on the basis of binding data alone.