P2X RECEPTOR (P2XR) SIGNALING
Identification and Cloning of P2XRs
In 1972 Burnstock advanced the concept of purinergic signaling, hypothesizing that extracellular ATP acts as a signaling molecule, such as neurotransmitters adrenaline and acetylcholine, based on early studies on excitable tissues by Szent-Györgyi and Drury, Holton et al. as well as his own work.1 Before long, surface-located membrane receptors called purinoreceptors including adenosine-activated P1 and ATP-activated P2 receptor classes were described for the first time as receptors that mediate purinergic signaling in tissues.2 In 1985, a new classification system (using functional criteria, such as structure, pharmacology, and mechanism of action) for purinergic receptors was proposed, wherein P2 receptors were divided into ionotropic P2X and metabotropic P2Y receptors.3 In the mid 1990s, P2X1 and P2X2 were the first P2X family members identified by cDNA cloning in rat species.4,5 Hitherto, the P2XR gene family comprises seven distinct genes P2RX1-7, each encoding for P2X1–7 protein subunits and found in various mammalian species including human and rodents.6
Molecular Structure of P2XRs
P2XRs are nonselective ligand-gated ion channels that are activated by extracellular ATP. Each P2X channel is formed by the hetero-/oligomeric assembly of three P2X protein subunits.7 This long-debated minimal trimeric stoichiometry was recently confirmed by a study describing the solved crystal structure of zebrafish P2XR4 ion channel in closed state.8 The basic P2X subunit is a protein 379 (P2X6) to 595 (P2X7) amino acids long, with the following structural topology: intracellular N and C-termini, two transmembrane spanning regions that delimits a large ectodomain.9 This extracellular loop contains the so-called ATP-binding domain of 10 conserved cysteine residues forming disulfide bonds, and several phosphorylation, glycosylation, and palmitoylation sites likely involved channel activity modulation.6 Biochemical and biophysical studies in heterologous expression systems, such as Xenopus oocytes or mammalian HEK293 and 1321N1 cell lines, have shown that all P2XRs can form both heteromeric and homomeric channels. Accordingly, all seven homomeric channels: P2X1–7 and, at least, a dozen heteromeric channels have been identified and pharmacologically characterized.9–13 However, additional functional evidences from studies in native tissues as well as genetically modified mouse models are needed to determine or confirm the existence and physiological relevance of the combinations identified in biochemical studies. This is well illustrated by the debate surrounding the existence of native homomeric P2X6 receptors,14,15 although functional highly glycosylated P2X6 receptors have been described upon transient transfection of HEK293 cells with recombinant rat P2X6 receptor cDNA.11 Also, unknown are the factors influencing the stoichiometry of each partner P2X subunit for a given heteromeric channel.13,14
Activation Mechanism of P2XRs
The basic activation mechanism of P2XR involves conformational changes of the ion channel from a ‘closed’ to an ‘open’ state, upon binding of (at least) three ATP molecules.7 These structural changes lead to the opening of a pore with an increased permeability to Ca2+, Na+, K+, and Cl− ions. The changes in P2X ion channel permeability trigger a fast membrane depolarization, which is followed by the generation of cellular Ca2+ (and to a less extent Na+) influxes that activate still poorly understood downstream intracellular coupling mechanisms. P2XR are characterized by different pharmacological properties. First, the potency of ATP ligand for P2XR is ranging from nanomolars (e.g., P2X1) to millimolars (e.g., P2X7).6,16 Second, prolonged exposure to ATP leads to a desensitization phase of P2XRs that varies from fast (ms for P2X1) to fairly sustained (more than 20 seconds for P2X2–4 and P2X7).6 Third, pharmacokinetics of P2XRs can be distinguished using various synthetic ATP analogs that act as agonists (e.g., α,β-MeATP; ATPγS) or antagonists (e.g., TNP-ATP; PPADS)15,17. For instance, both P2X1 and P2X3 receptors are selectively activated by α,β-MeATP and inhibited by TNP-ATP. Finally, P2XR channel activity can be affected by physiological parameters, such as extracellular pH, divalent ions (e.g., Zn2+, Cu2+, Hg2+, Ni2+ or Cd2+) and interactions with other membrane receptors. For example, the activation of the P2X2 receptor is potentiated by Zn2+ and blocked by Ca2+.15
Now that the reader is hopefully more familiar with the P2XR pharmacology, the following section will recapitulate the existing data about the liver distribution of these receptors.