ATP analogues with modified phosphate chains and their selectivity for rat P2X 2 and P2X 2/3 receptors

1. Heteromeric P2X 2/3 receptors are much more sensitive than homomeric P2X 2 receptors to αβ-methylene-ATP, and this ATP analogue is widely used to discriminate the two receptors on sensory neurons and other cells. 2. We sought to determine the structural basis for this selectivity by synthesising ADP and ATP analogues in which the αß and/or βγ oxygen atoms were replaced by other moieties (including -CH 2-, -CHF-, -CHCl-, -CHBr-, -CF2-, -CCl 2-, -CBr2-, -CHSO 3-, -CHPO 3-, -CFPO 3-, -CClPO 3-, -CH 2-CH 2-, -C≡C-, -NH-, -CHCOOH-). 3. We tested their actions as agonists or antagonists by whole-cell recording from human embryonic kidney cells expressing P2X 2 subunits alone (homomeric P2X 2 receptors), or cells expressing both P2X 2 and P2X 3 subunits, in which the current through heteromeric P2X 2/3 receptors was isolated. 4. ADP analogues had no agonist or antagonist effect at either P2X 2 or P2X 2/3 receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X 2 receptors, except βγ-difluoromethylene-ATP, which was a weak agonist. 5. At P2X 2/3 receptors, βγ-imido-ATP, βγ-methylene-ATP, and βγ-acetylene-ATP were weak agonists, whereas αβ, βγ- and βγ,γδ-bismethylene-AP 4 were potent full agonists. βγ-Carboxymethylene-ATP and βγ-chlorophosphonomethylene-ATP were weak antagonists at P2X 2/3 receptors (IC 50 about 10 μM). 6. The results indicate (a) that the homomeric P2X 2 receptor presents very stringent structural requirements with respect to its activation by ATP; (b) that the heteromeric P2X 2/3 receptor is much more tolerant of αβ and βγ substitution; and (c) that a P2X 2/3-selective antagonist can be obtained by introduction of additional negativity at the βγ-methylene.