Pflugers Arch - Eur J Physiol (2006) 452: 513–537 DOI 10.1007/s00424-006-0070-9 INVITED REVIEW Joel R. Gever . Debra A. Cockayne . Michael P. Dillon . Geoffrey Burnstock . Anthony P. D. W. Ford Pharmacology of P2X channels Received: 6 March 2006 / Accepted: 8 March 2006 / Published online: 29 April 2006 # Springer-Verlag 2006 Abstract Significant progress in understanding the phar- channels into human trials, confirms the medicinal macological characteristics and physiological importance exploitability of this novel target family and provides of homomeric and heteromeric P2X channels has been hope that safe and effective medicines for the treatment achieved in recent years. P2X channels, gated by ATP and of disorders involving P2X channels may be identified in most likely trimerically assembled from seven known P2X the near future. subunits, are present in a broad distribution of tissues and are thought to play an important role in a variety of Keywords P2X . Purinergic . ATP . Ion channel . physiological functions, including peripheral and central Antagonist neuronal transmission, smooth muscle contraction, and inflammation. The known homomeric and heteromeric P2X channels can be distinguished from each other on the Introduction basis of pharmacological differences when expressed recombinantly in cell lines, but whether this pharmacolo- Receptors activated by adenosine 5′-triphosphate (ATP), gical classification holds true in native cells and in vivo is and related di- and tri-phosphate nucleotides, were less well-established. Nevertheless, several potent and originally named P2 receptors to differentiate them from selective P2X antagonists have been discovered in recent P1 receptors, activated most potently by adenosine [35]. In years and shown to be efficacious in various animal models 1985, Burnstock and Kennedy further proposed dividing including those for visceral organ function, chronic P2 receptors into P2X and P2Y receptor families, initially inflammatory and neuropathic pain, and inflammation. on the basis of differences in agonist and antagonist The recent advancement of drug candidates targeting P2X potencies and, later, on the basis of differences in receptor structure and signal transduction mechanism [1, 41]. Accordingly, it is now widely accepted that the terms J. R. Gever (*) . A. P. D. W. Ford P2X and P2Y describe ligand-gated ion channels and G Department of Biochemical Pharmacology, protein-coupled receptors, respectively [91, 247]. Roche Palo Alto, Our understanding of P2X channels emerged gradually 3431 Hillview Avenue, Palo Alto, CA 94304, USA at first from pharmacological investigations of native e-mail: [email protected] excitable tissues, and then exploded with great interest after their molecular cloning and characterization in the mid- D. A. Cockayne . A. P. D. W. Ford 1990s (Fig. 1). Seven P2X receptor subunits have been Department of Neuroscience, Roche Palo Alto, identified that share less than 50% identity and range in 3431 Hillview Avenue, Palo Alto, CA 94304, USA length from 379 to 595 amino acids. P2X receptor subunits share a similar structural topology consisting of two M. P. Dillon transmembrane domains connected by a large extracellular Department of Medicinal Chemistry, loop containing the putative ATP binding site, and Roche Palo Alto, 3431 Hillview Avenue, intracellular N and C termini of various lengths [24, 78, Palo Alto, CA 94304, USA 154, 165, 226, 254, 290, 300, 305]. In the last decade, the subunit composition of functional P2X channels has been G. Burnstock elucidated, especially in recombinant systems, along with Autonomic Neuroscience Centre, Royal Free and University College Medical School, an understanding of their biophysical characteristics, such Rowland Hill Street, as ion selectivity, permeability, and kinetics of activation London, NW3 2PF, UK and inactivation. Data from a variety of experimental 514 techniques, including chemical cross-linking followed by native polyacrylamide gel electrophoresis (PAGE), muta- genesis, and atomic force and electron microscopy, support the idea that P2X channels exist as homomeric and heteromeric trimers [4, 10, 151, 215, 227]. These channels are selectively permeable to cations (pCa2+ approximately twofold to fivefold greater than pNa+ and pK+)[30, 81, 192, 300], and different trimers display unique pharmacological properties [28, 172, 188, 192, 228, 245, 292]. Significant progress has also been made in ascribing functions to various mammalian P2X subtypes in both physiological and pathological settings, in virtually every cell type and organ system [42]. Despite these advances, progress has been less impres- sive in certain regards. First, in many tissues and cells it remains to be established which homomeric or heteromeric form(s) of P2X channels transmit ionotropic responses to ATP, a discrepancy that may be attributable to the failure of recombinant expression systems to fully elaborate the characteristics of native P2X channels. Secondly, there remains a paucity of potent and selective pharmacological tools. Agonists that can selectively activate distinct members of this family have not been found, and with the exception of two notable family members, progress has been slower than perhaps anticipated in identifying selec- tive inhibitors. Thus, exploration of therapeutic potential remains still very superficial. The focus of this review is on the pharmacology of P2X receptors, with the aim of reviewing each reasonably established channel trimer, and a goal of capturing a) pharmacological characteristics that reflect the greatest distinctiveness and b) properties that have been identified more recently (over the last 3–5 years). The reader should be aware that many recognized properties of P2X receptors are based on data from recombinant channels, expressed heterologously in either oocytes or mammalian cells, and the degree to which these properties deviate from the functional characteristics of native channels is not entirely clear. A second caveat is that as a general guiding rule, robust pharmacological classification depends heavily on the determination of ‘constants’ that are derived under condi- tions closely approximating thermodynamic equilibrium. However, the nature of P2X channels, especially varying rates of desensitization, makes it very difficult (if not impossible) to ensure thermodynamic equilibrium has been established. Accordingly, a review of the literature will reveal many “dependent” variables—EC50 and IC50 esti- mates—dependent on the experimental conditions em- ployed. In many cases, because of the difficulty or impossibility in attaining steady-state conditions (e.g., in standard electrophysiological or calcium flux studies), or in clearly establishing “simple, reversible competition”, one Fig. 1 Timeline of the discovery of P2 receptors and the highlights of essentially cannot estimate equilibrium dissociation con- their pharmacological characterization. References used to construct stants. This means that a clear fingerprint cannot yet be timeline: [2, 11, 20, 24, 34, 35, 41, 51, 63, 73, 75, 88, 96, 106, 126, established for many of the P2X channels, and until truly 144, 146, 164, 184, 192, 209, 218, 264, 285, 300, 309] selective antagonists are developed, it will probably remain a challenge. The arrival of novel antagonists will provide a using radioligand binding approaches. Until then, one must greater opportunity to study channels under conditions that remain cautious when claiming unequivocal characteriza- more closely approximate true equilibrium—for example, tions based on agonist EC50 or antagonist IC50 estimates. 515 Homomeric P2X1 channels [122]. Conversely, transgenic mice overexpressing human P2X1 protein subunits in the megakaryocytic cell lineage Key messages exhibit hypersensitive platelet responses in vitro, and increased mortality in a model of systemic thromboembo- lism [234]. Taken together, these data suggest that P2X1 1. P2X1 channels are predominantly expressed in smooth channels may play an important role in platelet physiology muscle and platelets where they regulate smooth muscle and hemostasis. contractility and various prothrombotic functions. 2. Pharmacologically, P2X1 is almost identical to P2X3 in β γ terms of agonist and kinetic properties. However, , - Activation MeATP has a higher potency for P2X1 versus P2X3. 3. Many P2X1 selective antagonists are available but Two defining characteristics of the homomeric P2X1 drug-likeness is low. The only non-acidic small channel are its rapid desensitization kinetics and its molecule P2X1 antagonist is RO-1 (see Recent sensitivity to activation by α,β-MeATP [80, 300]. In advances). cells expressing recombinant rat or human P2X1, α,β- MeATP is generally less potent than ATP and 2-(methylthio) Localization and function ATP (2-MeSATP) (pEC50≈6–7), and somewhat more potent than adenosine 5′-O-(3-thiotriphosphate) (ATP-γ-S) The gene encoding the P2X1 protein subunit was first (pEC50≈5.5) [14, 80, 292, 300, 301]. These characteristics cloned from rat vas deferens [300], and although P2X1 are shared by the homomeric P2X3 channel, and, therefore, messenger ribonucleic acid (mRNA) and protein have a cannot be used to uniquely define P2X1. However, beta, fairly broad tissue distribution, most notable is its dense gamma-methylene ATP (β,γ-MeATP) is reported to be localization within the smooth muscle lining a variety of equipotent to α,β-MeATP at P2X1, but approximately 30- hollow organs including the urinary bladder, intestines, to 50-fold