Profiling at recombinant homomeric and heteromeric rat P2X receptors identifies the suramin analogue NF449 as a highly potent P2X₁ receptor antagonist

Abstract

P2X receptors are cation channels gated by extracellular ATP and related nucleotides. Because of the widespread distribution of P2X receptors and the high subtype diversity, potent and selective antagonists are needed to dissect their roles in intact tissues. Based on suramin as a lead compound, several derivates have been described that block recombinant P2X receptors with orders of magnitude higher potency than suramin. Here we characterized the suramin analogue 4,4′,4″,4‴-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) with respect to its potency to antagonize ATP or αβ-methyleneadenosine 5′-trisphosphate-induced inward currents of homomeric rat P2X₁–P2X₄ receptors or heteromeric P2X₁₊₅ and P2X₂₊₃ receptors, respectively. NF449 most potently blocked P2X₁ and P2X_1 +5 receptors with IC₅₀ values of 0.3 nM and 0.7 nM, respectively. Three to four orders of magnitude higher NF449 concentrations were required to block homomeric P2X₃ or heteromeric P2X₂₊₃ receptors (IC₅₀ 1.8 and 0.3 μM, respectively). NF449 was least potent at homomeric P2X₂ receptors (IC₅₀ 47 μM) and homomeric P2X₄ receptors (IC₅₀ > 300 μM). Altogether, these results characterize NF449 as the so far most potent and selective antagonist of receptors incorporating the P2X₁ subunit such as the P2X₁ homomer and the P2X₁₊₅ heteromer.

Introduction

ATP and other endogenous purine and pyrimidine nucleotides induce neuronal and non-neuronal responses by acting on two types of cell surface receptors, the G protein-coupled metabotropic P2Y receptors and the ligand-gated P2X receptor cation channels (North, 2002, Burnstock, 2004). P2X receptors are involved in a wide variety of essential cellular processes ranging from smooth muscle contraction, fast excitatory neurotransmission and nociception to interleukin-1 secretion from macrophages. Seven receptor subunits (P2X₁–P2X₇) have been identified in vertebrates, without considering the various isoforms originating from alternative splicing. Like other ligand-gated ion channels, P2X receptors are organized as oligomers. Both biochemical and electrophysiological studies indicate that a trimeric subunit organization is inherent to all functional homomeric and heteromeric P2X receptors (Nicke et al., 1998, Jiang et al., 2003, Aschrafi et al., 2004). In the Xenopus oocyte expression system, homomeric P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, and P2X₇ receptors and heteromeric P2X₂₊₃ and P2X₁₊₅ receptors have been fully characterized with respect to kinetics and pharmacological profiles of agonists (North, 2002). P2X₆ subunits are unique in that they form homomeric channels only in a small subset of stably transfected HEK293 cells (Jones et al., 2004), but neither in transiently transfected HEK293 or COS cells nor in Xenopus oocytes (North, 2002).

The widespread and partially overlapping occurrence of P2X receptors and the high subtype diversity arising from homo- and heteropolymerization makes it difficult to determine which subtype mediates a particular response in native tissues. A panel of potent and subtype-selective antagonists is therefore needed to dissect the physiological and pathophysiological role of each subtype. Over the last several years, significant progress has been made in identifying potent and selective P2X receptor antagonists. These include trinitrophenyl-ATP (TNP-ATP), which potently antagonizes P2X₁, P2X₃, and P2X₂₊₃ receptors (Virginio et al., 1998), diinosine pentaphosphate (IP₅I), a P2X₁-selective antagonist (King et al., 1999), and the Abbott compound A-317491, a selective non-nucleotide antagonist of P2X₃ and P2X₂₊₃ receptors (Jarvis et al., 2002).

Another group of antagonists has its origin in suramin, which is a non-selective P2 receptor antagonist (Dunn and Blakeley, 1988, Ralevic and Burnstock, 1998, Lambrecht et al., 2002). Using suramin as a lead compound, derivatives such as NF023 (8,8′-(carbonylbis(imino-3,1-phenylene carbonylimino)(bis(1,3,5-naphthalenetrisulfonic acid)) and NF279 (8,8′-(carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino))bis(1,3,5-naphthalenetrisulfonic acid)) have been identified as potent P2X₁ receptor-selective blockers (Soto et al., 1999, Klapperstück et al., 2000, Rettinger et al., 2000). Most recently, we introduced the suramin derivative NF449 (4,4′,4″,4‴-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid) as the most potent P2X₁ receptor antagonist yet described (Kassack et al., 2004), but its selectivity profile has so far only been characterized in intact tissues and at two recombinant P2X receptor subtypes, P2X₁ and P2X₇ (Braun et al., 2001, Hülsmann et al., 2003, Kassack et al., 2004). The objective of the present study was to evaluate the antagonistic potency of NF449 for the recombinant homomeric and heteromeric receptor subtypes P2X₁–P2X₄, P2X₁₊₅, and P2X₂₊₃. Our results establish NF449 as an extremely potent antagonist of receptors incorporating the rP2X₁ subunit such as the rP2X₁ homomer and the rP2X₁₊₅ heteromer.