Contributions of P2X3 homomeric and heteromeric channels to acute and chronic pain

ATP acts as a fast neurotransmitter by activating a family of ligand-gated ion channels, the P2X receptors. Functional homomeric (P2X(3)) and heteromeric (P2X(2/3)) receptors are highly localised on primary sensory afferent neurons that transmit nociceptive sensory information. Activation of these P2X(3)containing channels may provide a specific mechanism whereby ATP, released via synaptic transmission or by cellular injury, elicits pain. The physiological relevance of the pro-nociceptive actions of ATP is supported by data demonstrating that the exogenous peripheral or spinal administration of ATP and other P2X receptor agonists elicits nociceptive behaviour and increases sensitivity to noxious stimuli in both humans and laboratory animals. The nociceptive effects of P2X receptor agonists are also enhanced in the presence of inflammatory mediators. Both permanent (P2X(3) gene knockout) and transient (P2X(3) antisense) receptor gene disruption studies in laboratory rodents have provided hypoalgesic phenotypes, further supporting a role for P2X(3) subunits in contributing to the expression of pain. More recently, the acute systemic administration of a highly selective non-nucleotide P2X(3) antagonist, A317491, has been shown to fully block specific types of chronic inflammatory and neuropathic pain in animal models in the absence of cardiovascular and CNS side effects associated with other analgesic compounds. Therefore, both genetic and pharmacological approaches have provided converging evidence that activation of P2X(3)-containing channels is an important mediator of persistent nociceptive signalling. The available data also indicate potential discrete roles for homomeric P2X(3) and heteromeric P2X(2/3) receptor activation in acute and chronic pain.     

P2X receptors and nociception.

The potential importance for nociception of P2X receptors, the ionotropic receptors activated by ATP, is underscored by the variety of pain states in which this endogenous ligand can be released. Several important findings have been made recently indicating that P2X receptors can be involved in pain mechanisms both centrally and in the periphery. The roles of ATP at these two sites and the P2X receptor subtypes involved appear to be different. In the periphery, ATP can be released as a result of tissue injury, visceral distension, or sympathetic activation and can excite nociceptive primary afferents by acting at homomeric P2X(3) or heteromeric P2X(2/3) receptors. Centrally, ATP released from central afferent terminals or second order neurons can modulate neurotransmitter release or postsynaptically activate neurons involved in central nociceptive transmission, with P2X(2), P2X(4), P2X(6), and some other receptors being potentially involved. Evidence from in vivo studies suggests that peripheral ATPergic mechanisms are most important under conditions of acute tissue injury and inflammation whereas the relevance of central mechanisms appears to be more limited. Furthermore, the release of ATP and P2X receptor-mediated afferent activation appear to have been implicated in visceral and neuropathic pain; the importance of the ATPergic component in these states needs to be investigated further. Thus, peripheral P2X receptors, and homomeric P2X(3) and/or heteromeric P2X(2/3) receptors in particular, constitute attractive targets for analgesic drugs. The development of selective antagonists of these receptors, suitable for a systemic in vivo use although apparently difficult, may prove a useful strategy to generate analgesics with a novel mechanism of action.     

ATP receptors in pain

Extracellular ATP has been known to activate sensory neurons via the ATP-gated ion channels P2X receptors, leading to the proposal that the P2X receptors may play a role in signal transduction of pain from the peripheral site to the spinal cord in vivo. P2X3 receptors are expressed in capsaicin-sensitive small-sized dorsal root ganglion (DRG) neurons, and they are involved in the generation of rapidly desensitizing inward current and evoking nocifensive behavior and thermal hyperalgesia. Heteromeric P2X2/3 (P2X2 and P2X3) receptor is expressed in capsaicin-insensitive primary afferent fibers, and its activation leads to the generation of slow desensitizing currents and induction of mechanical allodynia. In addition, accumulating information suggests the involvement of G protein-coupled ATP receptors in the modulation of the generation and transmission of pain.     

Selective modulation of ligand-gated P2X purinoceptor channels by acute hypoxia is mediated by reactive oxygen species

Purinergic excitatory synapses use ATP to mediate fast synaptic transmission via activation of P2X receptor cation channels, and this response can be altered by acute hypoxia. This study examined the effect of acute hypoxia on cloned homo- and heteromeric P2X2 and P2X3 receptors expressed in human embryonic kidney 293 cells. In cells expressing homomeric P2X2 receptors, perfusion of 5 microM ATP (EC25) induced an inward whole-cell current that showed little desensitization during repeated exposures under continuously normoxic conditions. Exposure to a hypoxic ATP solution (pO2, 25-40 mm Hg) significantly reduced the whole-cell current to 49% of normoxic control. This hypoxic inhibition of P2X2-mediated inward current was maintained across all potentials when a voltage-step protocol was applied. In contrast, currents mediated by homomeric P2X3 receptors or heteromeric P2X(2/3) receptors were insensitive to an acute hypoxic challenge. One mechanism whereby hypoxia may modulate P2X2 channels is via the production of reactive oxygen species (ROS). H2O2 (1.8 mM) reversibly reduced homomeric P2X2 whole-cell currents to 38% of control. Furthermore, H2O2 attenuated the effect of hypoxia on homomeric P2X2 whole-cell currents. Inhibitors of the mitochondrial electron transport chain that reduce (rotenone and myxothiazol) or increase (antimycin A) the production of ROS altered the magnitude of P2X2-mediated currents. In summary, this is the first report indicating that acute hypoxia is able to regulate the activity of any ligand-gated ion channel. Furthermore, our data show that acute hypoxia selectively modulates the P2X2 receptor and that the response of P2X2 receptor subunits to hypoxia is mediated through the mitochondrial production of ROS.     

Selective potentiation of homomeric P2X2 ionotropic ATP receptors by a fast non-genomic action of progesterone

P2X receptors are ligand-gated ion channels activated by ATP that are widely expressed in the organism and regulate many physiological functions. We have studied the effect of progesterone (PROG) on native P2X receptors present in rat dorsal root ganglion (DRG) neurons and on recombinant P2X receptors expressed in HEK293 cells or Xenopus laevis oocytes. The effects of PROG were observed and already maximal during the first coapplication with ATP and did not need any preincubation of the cells with PROG, indicating a fast mechanism of action. In DRG neurons, PROG rapidly and reversibly potentiated submaximal but not saturating plateau-like currents evoked by ATP, but had no effect on the currents activated by α,β-methylene ATP, an agonist of homomeric or heteromeric receptors containing P2X1 or P2X3 subunits. In cells expressing homomeric P2X2 receptors, responses to submaximal ATP, were systematically potentiated by PROG in a dose-dependent manner with a threshold between 1 and 10 nM. PROG had no effect on ATP currents carried by homomeric P2X1, P2X3, and P2X4 receptors or by heteromeric P2X1/5 and P2X2/3 receptors. We conclude that PROG selectively potentiates homomeric P2X2 receptors and, in contrast with dehydroepiandrosterone (DHEA), discriminates between homomeric and heteromeric P2X2-containing receptors. This might have important physiological implications since the P2X2 subunit is the most widely distributed P2X subunit in the organism. Moreover, DHEA and PROG might be useful tools to clarify the distribution and the role of native homo- and heteromeric P2X2 receptors.     

ATP receptors in pain sensation

We reported that activation of P2X2/3 heteromeric channels in A delta-DRG neurons causes tactile allodynia and activation of P2X3 in C-fiber causes nocifensive behavior. We also found that tactile allodynia under the chronic pain state requires an activation of P2X4 ionotropic ATP receptor and p38 mitogen-activated protein kinase in spinal microglia.     

ATP analogues with modified phosphate chains and their selectivity for rat P2X2 and P2X2/3 receptors

1. Heteromeric P2X2/3 receptors are much more sensitive than homomeric P2X2 receptors to alphabeta-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 alphabeta and/or betagamma oxygen atoms were replaced by other moieties (including -CH2-, -CHF-, -CHCl-, -CHBr-, -CF2-, -CCl2-, -CBr2-, -CHSO3-, -CHPO3-, -CFPO3-, -CClPO3-, -CH2-CH2-, C triple bond C, -NH-, -CHCOOH-). 3. We tested their actions as agonists or antagonists by whole-cell recording from human embryonic kidney cells expressing P2X2 subunits alone (homomeric P2X2 receptors), or cells expressing both P2X2 and P2X3 subunits, in which the current through heteromeric P2X2/3 receptors was isolated. 4. ADP analogues had no agonist or antagonist effect at either P2X2 or P2X2/3 receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X2 receptors, except betagamma-difluoromethylene-ATP, which was a weak agonist. 5. At P2X2/3 receptors, betagamma-imido-ATP, betagamma-methylene-ATP, and betagamma-acetylene-ATP were weak agonists, whereas alphabeta,betagamma- and betagamma,gammadelta-bismethylene-AP4 were potent full agonists. betagamma-Carboxymethylene-ATP and betagamma-chlorophosphonomethylene-ATP were weak antagonists at P2X2/3 receptors (IC50 about 10 microm). 6. The results indicate (a). that the homomeric P2X2 receptor presents very stringent structural requirements with respect to its activation by ATP; (b). that the heteromeric P2X2/3 receptor is much more tolerant of alphabeta and betagamma substitution; and (c). that a P2X2/3-selective antagonist can be obtained by introduction of additional negativity at the betagamma-methylene.     

Enteric P2X receptors as potential targets for drug treatment of the irritable bowel syndrome

The irritable bowel syndrome (IBS) is a gastrointestinal motility disorder affecting millions of patients. IBS symptoms include diarrhea, constipation and pain. The etiology of IBS is due partly to changes in the function of nerves supplying the gastrointestinal tract, immune system activation and to psychological factors. P2X receptors are multimeric ATP-gated cation channels expressed by neuronal and non-neuronal cells. Sensory nerve endings in the gastrointestinal tract express P2X receptors. ATP released from gastrointestinal cells activates P2X receptors on sensory nerve endings to stimulate motor reflexes and to transmit nociceptive signals. Antagonists acting at P2X receptors on sensory nerves could attenuate abdominal pain in IBS patients. Primary afferent neurons intrinsic to the gut, and enteric motor- and interneurons express P2X receptors. These neurons participate in motor reflexes. Agonists acting at enteric P2X receptors may enhance gastrointestinal propulsion and secretion, and these drugs could be useful for treating constipation-predominant IBS. Antagonists acting at enteric P2X receptors would decrease propulsion and secretion and they might be useful for treating diarrhea-predominant IBS. Current knowledge of P2X receptor distribution and function in the gut of laboratory animals provides a rational basis for further exploration of the therapeutic potential for drugs acting at P2X receptors in IBS patients. However, more information about P2X receptor distribution and function in the human gastrointestinal tract is needed. Data on the distribution and function of P2X receptors on gastrointestinal immune cells would also provide insights into the therapeutic potential of P2X receptor agents in IBS.     

ATP- and adenosine-mediated signaling in the central nervous system: chronic pain and microglia: involvement of the ATP receptor P2X4

We have been studying the role of ATP receptors in pain and already reported that activation of P2X(2/3) heteromeric channel/receptor in primary sensory neurons causes acutely tactile allodynia, one hallmark of neuropathic pain. We report here that tactile allodynia under the chronic pain state requires an activation of the P2X(4) ionotropic ATP receptor and p38 mitogen-activated protein kinase (MAPK) in spinal cord microglia. Two weeks after L5 spinal nerve injury, rats displayed a marked mechanical allodynia. In the rats, activated microglia were detected in the injured side of the dorsal horn and the level of the dually-phosphorylated active form of p38MAPK (phospho-p38MAPK) in these microglia was increased. Moreover, intraspinal administration of a p38MAPK inhibitor, SB203580, suppressed the allodynia. We also found that the expression level of P2X(4) was increased strikingly in spinal cord microgila after nerve injury and that pharmacological blockade or inhibition of the expression of P2X(4) reversed the allodynia. Taken together, our results demonstrate that activation of P2X(4) or p38MAPK in spinal cord microglia is necessary for tactile allodynia after nerve injury.     

Gastrointestinal afferents as targets of novel drugs for the treatment of functional bowel disorders and visceral pain.

An intricate surveillance network consisting of enteroendocrine cells, immune cells and sensory nerve fibres monitors the luminal and interstitial environment in the alimentary canal. Functional bowel disorders are characterized by persistent alterations in digestive regulation and gastrointestinal discomfort and pain. Visceral hyperalgesia may arise from an exaggerated sensitivity of peripheral afferent nerve fibres and/or a distorted processing and representation of gut signals in the brain. Novel strategies to treat these sensory bowel disorders are therefore targeted at primary afferent nerve fibres. These neurons express a number of molecular traits including transmitters, receptors and ion channels that are specific to them and whose number and/or behaviour may be altered in chronic visceral pain. The targets under consideration comprise vanilloid receptor ion channels, acid-sensing ion channels, sensory neuron-specific Na(+) channels, P2X(3) purinoceptors, 5-hydroxytryptamine (5-HT), 5-HT(3) and 5-HT(4) receptors, cholecystokinin CCK(1) receptors, bradykinin and prostaglandin receptors, glutamate receptors, tachykinin and calcitonin gene-related peptide receptors as well as peripheral opioid and cannabinoid receptors. The utility of sensory neuron-targeting drugs in functional bowel disorders will critically depend on the compounds' selectivity of action for afferent versus enteric or central neurons.     

Effects of A-317491, a novel and selective P2X3/P2X2/3 receptor antagonist, on neuropathic, inflammatory and chemogenic nociception following intrathecal and intraplantar administration

We have recently reported that systemic delivery of A-317491, the first non-nucleotide antagonist that has high affinity and selectivity for blocking P2X3 homomeric and P2X2/3 heteromeric channels, is antinociceptive in rat models of chronic inflammatory and neuropathic pain. In an effort to further evaluate the role of P2X3/P2X2/3 receptors in nociceptive transmission, A-317491 was administered either intrathecally or into the hindpaw of a rat in several models of acute and chronic nociception. Intraplantar (ED50=300 nmol) and intrathecal (ED50=30 nmol) injections of A-317491 produced dose-related antinociception in the CFA model of chronic thermal hyperalgesia. Administration of A-317491 by either route was much less effective to reduce thermal hyperalgesia in the carrageenan model of acute inflammatory hyperalgesia. Intrathecal, but not intraplantar, delivery of A-317491 attenuated mechanical allodynia in both the chronic constriction injury and L5-L6 nerve ligation models of neuropathy (ED50=10 nmol for both models). Intrathecal injections of A-317491 did not impede locomotor performance. Both routes of injection were effective in reducing the number of nocifensive events triggered by the injection of formalin into a hindpaw. Nocifensive behaviors were significantly reduced in both the first and second phases of the formalin assay (intrathecal ED50=10 nmol, intraplantar ED50>300 nmol). Nocifensive behaviors induced by the P2X receptor agonist alpha,beta-meATP were also significantly reduced by intraplantar injection of A-317491. These data indicate that both spinal and peripheral P2X3/P2X2/3 receptors have significant contributions to nociception in several animal models of nerve or tissue injury. Intrathecal administration of A-317491 appears to be more effective than intraplantar administration to reduce tactile allodynia following peripheral nerve injury.     

Purines and pain mechanisms: recent developments

Purines, such as adenosine and ATP, are endogenous ligands involved in modulating pain transmission and pain hypersensitivity by acting on P1 and P2 purinoceptors, respectively, at sites both in peripheral tissues and in the central nervous system. For P1 (adenosine) receptors, clinical studies in humans, as well as experimental animal studies, have demonstrated that activation of the A1 subtype reduces both inflammatory and neuropathic types of chronic pain. For P2 receptors, there is a growing body of evidence indicating that multiple receptor subtypes are differentially involved in pain processing. The most well-known of the P2 receptors is the P2X3 subtype, which is found in primary sensory neurons. Inhibition of P2X3 receptors is effective in reducing pain behaviors in animal models of chronic inflammatory and neuropathic pain. Recently, the P2X, subtype has been implicated in nerve-injury-induced pain hypersensitivity. There is also emerging evidence for roles for P2Y, and P2Y2 receptors, subtypes of G protein-coupled P2 receptors, in pain hypersensitivity. Thus, multiple subtypes of purinoceptors are potential molecular targets for development of new pharmacological agents for the treatment of pain.     

Potential therapeutic targets for ATP-gated P2X receptor ion channels

P2X receptors make up a novel family of ligand-gated ion channels that are activated by binding of extracellular ATP. These receptors can form a number of homomeric and heteromeric ion channels, which are widely distributed throughout the human body. They are thought to play an important role in many cellular processes, including synaptic transmission and thrombocyte aggregation. These ion channels are also involved in the pathology of several disease states, including chronic inflammation and neuropathic pain, and thus are the potential targets for drug development. The recent discovery of potent and highly selective antagonists for P2X(7) receptors, through the use of high-throughput screening, has helped to further understand the P2X receptor pharmacology and provided new evidence that P2X(7) receptors play a specific role in chronic pain states. In this review, we discuss how the P2X family of ion channels has distinguished itself as a potential new drug target. We are optimistic that safe and effective candidate drugs will be suitable for progression into clinical development.     

Activation of adenosine and P2Y receptors by ATP in human peripheral nerve

Receptors for ATP in the peripheral nervous system may contribute to the transduction of sensory, including nociceptive, stimuli and are candidates in the pathogenesis of neuropathic pain. In a complex neural tissue, such as the human peripheral nerve trunk, ATP may activate P2X, P2Y, and adenosine receptors present on various cell types. Experiments were performed on segments of isolated human sural nerves. The experimental set-up enabled simultaneous recording of C fiber excitability, intracellular Ca(2+) ([Ca(2+)](i)) and extracellular K(+) activity (aK(e)). The increase in excitability of unmyelinated fibers seen during bath application of both ATP and adenosine was reversed to a reduction in axonal excitability in the presence of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolol[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), an antagonist of adenosine A2 receptors. The pharmacological profile of the axonal subexcitability indicates the presence and activation of adenosine A1 receptors. Intracellular Ca(2+) transients were observed during bath application of ATP but not of adenosine and were blocked by 2'-deoxy- N(6)-methyladenosine 3',5'-bisphosphate (MRS 2179), an antagonist at P2Y(1) receptors. K(+)-sensitive microelectrodes were used to search for a possible activation of P2X receptors by ATP. In isolated rat vagus nerve, activation of P2X receptors by alpha,beta-methylene-adenosine 5'-triphosphate (alpha,beta-meATP) and by diadenosine pentaphosphate (Ap5A) resulted in a rapid, transient rise in the extracellular K(+) activity. In contrast, in human nerve, application of P2X receptor agonists did not result in a detectable elevation of aK(e). The data suggest that ATP-induced changes in axonal excitability and of [Ca(2+)](i) result from activation of adenosine A2, A1 and P2Y nucleotide receptors in human nerve; a contribution of P2X receptors was not found with the methods used. It is suggested that antagonists of A2 receptors might suppress enhanced activity in human nociceptive afferent nerve fibers under conditions in which ATP and/or adenosine is released into the trunk of a human peripheral nerve.     

Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP-ATP and enhancement by the P2X(3) allosteric modulator, cibacron blue

1. Exogenous ATP produces acute and localized pain in humans, and P2X receptor agonists elicit acute nociceptive behaviours in rodents following intradermal administration to the hindpaw. The predominant localization of P2X(3) mRNA in sensory neurones has led to the hypothesis that activation of P2X(3) and/or P2X(2/3) receptors contributes to nociception. 2. The local administration of the P2X receptor agonist, BzATP (100--1000 nmol paw(-1), s.c.) into the rat hindpaw produced an acute (<15 min) paw flinching response that was similar to that observed in the acute phase of the formalin (5%) test. 3. The co-administration of the potent P2X receptor antagonist, TNP-ATP (30--300 nmol paw(-1)), but not an inactive analogue, TNP-AMP, with BzATP into the rat hindpaw attenuated BzATP-induced nociception. Similarly, co-administration of TNP-ATP, but not TNP-AMP, with 5% formalin reduced both acute and persistent nociception in this test. 4. Co-administration of cibacron blue (30 and 100 nmol paw(-1)), a selective allosteric enhancer of P2X(3) and P2X(2/3) receptor activation, with BzATP (30 and 100 nmol paw(-1)) into the rat hindpaw produced significantly greater nociception as compared to the algogenic effects of BzATP alone. Intradermal co-administration of cibacron blue (30 and 100 nmol paw(-1)) with formalin (1 and 2.5%) into the rat hindpaw also produced significantly greater nociceptive behaviour as compared to formalin alone. 5. The ability of TNP-ATP and cibacron blue to respectively attenuate and enhance nociceptive responses elicited by exogenous BzATP and formalin provide further support for the hypothesis that activation of peripheral P2X(3) containing channels contributes specifically to both acute and persistent nociception in the rat.     

Purinoceptors as therapeutic targets for lower urinary tract dysfunction

Lower urinary tract symptoms (LUTS) are present in many common urological syndromes. However, their current suboptimal management by muscarinic and alpha(1)-adrenoceptor antagonists leaves a significant opportunity for the discovery and development of superior medicines. As potential targets for such therapeutics, purinoceptors have emerged over the last two decades from investigations that have established a prominent role for ATP in the regulation of urinary bladder function under normal and pathophysiological conditions. In particular, evidence suggests that ATP signaling via P2X(1) receptors participates in the efferent control of detrusor smooth muscle excitability, and that this function may be heightened in disease and aging. ATP also appears to be involved in bladder sensation, via activation of P2X(3) and P2X(2/3) receptors on sensory afferent neurons, both within the bladder itself and possibly at central synapses. Such findings are based on results from classical pharmacological and localization studies in non-human and human tissues, knockout mice, and studies using recently identified pharmacological antagonists--some of which possess attributes that offer the potential for optimization into candidate drug molecules. Based on recent advances in this field, it is clearly possible that the development of selective antagonists for these receptors will occur that could lead to therapies offering better relief of sensory and motor symptoms for patients, while minimizing the systemic side effects that limit current medicines.     

Targeting cell surface trafficking of pain-facilitating receptors to treat chronic pain conditions

Introduction: Chronic pain conditions are serious clinical concerns. Its genesis is closely associated with sensitization of nociceptive primary sensory neurons (nociceptors) and dorsal horn neurons by various pain mediators produced during inflammation and tissue injury. Growing evidence showed that increasing cell surface trafficking of pain-facilitating receptors is an important mechanism underlying the peripheral and central sensitization.

Areas covered: We summarized the progress of this area over the past decade by showing that inflammation, tissue damage or pain mediators facilitate cell surface trafficking of pain-facilitating receptors such as transient receptor potential vanilloid-1, transient receptor potential ankyrin-1, voltage-gated sodium channel 1.8, P2X3 and EP4 in primary sensory neurons, GluR1 and GluR2 of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, NR1 and NR2 of N-methyl-d-aspartate receptors and acid-sensing ion channels 1 in dorsal horn neurons and P2X4 in spinal microglia. The anti-allodynic effects of gabapentin was mediated by blocking surface trafficking of α2δ1 and α2δ2 subunits of voltage-gated calcium channels in primary sensory and dorsal horn neurons.

Expert opinion: Pain mediators stimulate forward surface trafficking of their own and/or other pain-facilitating receptors to amplify pain intensity and duration. Enhancing surface abundance of pain-facilitating receptors in nociceptors and dorsal horn neurons is an important mechanism underpinning chronic pain states. Targeting specific trafficking events of pain-facilitating receptors may open a novel therapeutic avenue to more efficiently treat chronic pain conditions.     

Go it alone no more--P2X7 joins the society of heteromeric ATP-gated receptor channels

P2X receptors (P2XR) function as ATP-gated nonselective ion channels permeable to Na+, K+, and Ca2+, and they are expressed in a wide range of excitable, epithelial/endothelial, and immune effector cell types. The channels are trimeric complexes composed of protein subunits encoded by seven different P2XR genes expressed in mammalian and other vertebrate genomes. Current genetic, biochemical, and/or physiological evidence indicates that the extended family of functional P2X receptors includes six homomeric channels composed of P2X1, P2X2, P2X3, P2X4, P2X5, or P2X7 subunits and six heteromeric channels that involve subunit pairings of P2X1/P2X2, P2X1/P2X4, P2X1/P2X5, P2X2/P2X3, P2X2/P2X6, or P2X4/P2X6. Thus, all P2XR subtypes--with the salient exception of P2X7R--have previously been implicated in the assembly of heteromeric ATP-gated ion channels that can comprise unique pharmacological targets in different tissues. The assumed "go-it alone" function of the P2X7R has important implications because agents that target this particular receptor have been proposed as useful therapeutics in various autoinflammatory diseases or amelioration of inflammatory pain. However, this assumption and the interpretations based on it now require reevaluation in light of a new report in this issue of Molecular Pharmacology (p. 1447) that provides convincing biochemical and electrophysiological evidence for the existence of P2X4/P2X7 heteromeric receptors.     

Differential expression of P2X receptors on neurons from different parasympathetic ganglia

Whole-cell patch clamp recording and immunohistochemistry were used to investigate the expression of P2X receptors on rat parasympathetic ganglion neurons of the otic, sphenopalatine, submandibular, intracardiac and paratracheal ganglia. Neurons from all five ganglia responded to ATP with a rapidly activating, sustained inward current. Neurons of intracardiac and paratracheal ganglia were insensitive to alphabeta-meATP, while all neurons in the otic and some neurons of sphenopalatine and submandibular ganglia responded. Lowering pH potentiated ATP responses in neurons from all five ganglia. Co-application of Zn(2+) potentiated ATP responses in intracardiac, paratracheal and submandibular ganglion neurons. Immunohistochemistry revealed strong and specific staining for the P2X(2) subunit in all five ganglia and strong P2X(3) staining in otic, sphenopalatine and submandibular ganglia. In conclusion, there is heterogeneity in P2X receptor expression in different parasympathetic ganglia of the rat, but the predominant receptor subtypes involved appear to be homomeric P2X(2) and heteromeric P2X(2/3).     

Properties of native P2X receptors in rat trigeminal mesencephalic nucleus neurones: lack of correlation with known, heterologously expressed P2X receptors

Trigeminal mesencephalic nucleus (MNV) neurones express functional P2X receptors. In order to determine the molecular identity of the P2X receptors in this nucleus we have used whole cell patch clamp recording of P2X receptor-mediated currents to determine the pharmacological properties of the receptors, and have compared them with those of cloned P2X receptor subunits. The purine nucleotides ATP (300 microM), ATP-gamma-S (30 microM) and alphabetameATP (300 microM) evoked inward currents in all MNV neurones whereas alphabetameADP (300 microM) did not. betagammame-L-ATP (300 microM) evoked only a small ( approximately 20 pA) current in 3 out of 6 MNV neurones. The P2X receptor antagonist TNP-ATP (10 nM-10 microM) and raised extracellular Ca(2+) (8 and 30 mM) reduced, but did not abolish, the current evoked by ATP-gamma-S. The current remaining in TNP-ATP was insensitive to blockade by raised Ca(2+). These properties suggest that MNV neurones do not express homomeric P2X(3), P2X(4) or P2X(6) receptors. Whilst the TNP-ATP-insensitive ATP-gamma-S-evoked current has many characteristics similar to both homomeric P2X(2) and P2X(5) receptors, its insensitivity to blockade by raised Ca(2+) is difficult to reconcile with the receptor being a P2X(2) or P2X(5) homomeric channel. More likely, the receptor is a heteromer that comprises either or both of these subunits. The TNP-ATP-sensitive component of the ATP-gamma-S-evoked current is dissimilar to known cloned homomeric or heteromeric P2X receptors.