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Pharmacological insights into the role of P2X4 receptors in behavioural regulation: lessons from ivermectin.

ABSTRACT: Purinergic ionotropic P2X receptors are a family of cation-permeable channels that bind extracellular adenosine 5'-triphosphate. In particular, convergent lines of evidence have recently highlighted P2X(4) receptors as a potentially critical target in the regulation of multiple nervous and behavioural functions, including pain, neuroendocrine regulation and hippocampal plasticity. Nevertheless, the role of the P2X(4) receptor in behavioural organization remains poorly investigated. To study the effects of P2X(4) activation, we tested the acute effects of its potent positive allosteric modulator ivermectin (IVM, 2.5-10 mg/kg i.p.) on a broad set of paradigms capturing complementary aspects of perceptual, emotional and cognitive regulation in mice. In a novel open field, IVM did not induce significant changes in locomotor activity, but increased the time spent in the peripheral zone. In contrast, IVM produced anxiolytic-like effects in the elevated plus maze and marble burying tasks, as well as depression-like behaviours in the tail-suspension and forced swim tests. The agent induced no significant behavioural changes in the conditioned place preference test and in the novel object recognition task. Finally, the drug induced a dose-dependent decrease in sensorimotor gating, as assessed by pre-pulse inhibition (PPI) of the acoustic startle reflex. In P2X(4) knockout mice, the effects of IVM in the open field and elevated plus maze were similar to those observed in wild type mice; conversely, the drug significantly increased startle amplitude and failed to reduce PPI. Taken together, these results suggest that P2X(4) receptors may play a role in the regulation of sensorimotor gating.

SUBMITTER: Bortolato M

PROVIDER: S-EPMC3593990 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Pharmacological insights into the role of P2X4 receptors in behavioural regulation: lessons from ivermectin.

Bortolato Marco M Yardley Megan M MM Khoja Sheraz S Godar Sean C SC Asatryan Liana L Finn Deborah A DA Alkana Ronald L RL Louie Stan G SG Davies Daryl L DL

The international journal of neuropsychopharmacology 20120917 5

Purinergic ionotropic P2X receptors are a family of cation-permeable channels that bind extracellular adenosine 5'-triphosphate. In particular, convergent lines of evidence have recently highlighted P2X(4) receptors as a potentially critical target in the regulation of multiple nervous and behavioural functions, including pain, neuroendocrine regulation and hippocampal plasticity. Nevertheless, the role of the P2X(4) receptor in behavioural organization remains poorly investigated. To study the ...[more]

PMID: 23174033

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Project description:Protein allosteric modulation is a pillar of metabolic regulatory mechanisms; this concept has been extended to include ion channel regulation. P2XRs are ligand-gated channels activated by extracellular ATP, sensitive to trace metals and other chemicals. By combining in silico calculations with electrophysiological recordings, we investigated the molecular basis of P2X4R modulation by Zn(II) and ivermectin, an antiparasite drug currently used in veterinary medicine. To this aim, docking studies, molecular dynamics simulations and non-bonded energy calculations for the P2X4R in the apo and holo states or in the presence of ivermectin and/or Zn(II) were accomplished. Based on the crystallized Danio rerio P2X4R, the rat P2X4R, P2X2R, and P2X7R structures were modeled, to determine ivermectin binding localization. Calculations revealed that its allosteric site is restricted to transmembrane domains of the P2X4R; the role of Y42 and W46 plus S341 and non-polar residues were revealed as essential, and are not present in the homologous P2X2R or P2X7R transmembrane domains. This finding was confirmed by preferential binding conformations and electrophysiological data, revealing P2X4R modulator specificity. Zn(II) acts in the P2X4R extracellular domain neighboring the SS3 bridge. Molecular dynamics in the different P2X4R states revealed allosterism-induced stability. Pore and lateral fenestration measurements of the P2X4R showed conformational changes in the presence of both modulators compatible with a larger opening of the extracellular vestibule. Electrophysiological studies demonstrated additive effects in the ATP-gated currents by joint applications of ivermectin plus Zn(II). The C132A P2X4R mutant was insensitive to Zn(II); but IVM caused a 4.9 ± 0.7-fold increase in the ATP-evoked currents. Likewise, the simultaneous application of both modulators elicited a 7.1 ± 1.7-fold increase in the ATP-gated current. Moreover, the C126A P2X4R mutant evoked similar ATP-gated currents comparable to those of wild-type P2X4R. Finally, a P2X4/2R chimera did not respond to IVM but Zn(II) elicited a 2.7 ± 0.6-fold increase in the ATP-gated current. The application of IVM plus Zn(II) evoked a 2.7 ± 0.9-fold increase in the ATP-gated currents. In summary, allosteric modulators caused additive ATP-gated currents; consistent with lateral fenestration enlargement. Energy calculations demonstrated a favorable transition of the holo receptor state following both allosteric modulators binding, as expected for allosteric interactions.

Project description:Purinergic P2X4 receptors (P2X4Rs) belong to the P2X superfamily of ion channels regulated by ATP. We recently demonstrated that P2X4R knockout (KO) mice exhibited deficits in sensorimotor gating, social interaction, and ethanol drinking behavior. Dopamine (DA) dysfunction may underlie these behavioral changes, but there is no direct evidence for P2X4Rs' role in DA neurotransmission. To test this hypothesis, we measured markers of DA function and dependent behaviors in P2X4R KO mice. P2X4R KO mice exhibited altered density of pre-synaptic markers including tyrosine hydroxylase, dopamine transporter; post-synaptic markers including dopamine receptors and phosphorylation of downstream targets including dopamine and cyclic-AMP regulated phosphoprotein of 32 kDa and cyclic-AMP-response element binding protein in different parts of the striatum. Ivermectin, an allosteric modulator of P2X4Rs, significantly affected dopamine and cyclic AMP regulated phosphoprotein of 32 kDa and extracellular regulated kinase1/2 phosphorylation in the striatum. Sensorimotor gating deficits in P2X4R KO mice were rescued by DA antagonists. Using the 6-hydroxydopamine model of DA depletion, P2X4R KO mice exhibited an attenuated levodopa (L-DOPA)-induced motor behavior, whereas ivermectin enhanced this behavior. Collectively, these findings identified an important role for P2X4Rs in maintaining DA homeostasis and illustrate how this association is important for CNS functions including motor control and sensorimotor gating. We propose that P2X4 receptors (P2X4Rs) regulate dopamine (DA) homeostasis and associated behaviors. Pre-synaptic and post-synaptic DA markers were significantly altered in the dorsal and ventral striatum of P2X4R KO mice, implicating altered DA neurotransmission. Sensorimotor gating deficits in P2X4R KO mice were rescued by DA antagonists. Ivermectin (IVM), a positive modulator of P2X4Rs, enhanced levodopa (L-DOPA)-induced motor behavior. These studies highlight potential interactions between P2X4Rs and DA system.

Project description:P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current-voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs undergo exocytosis. Finally, the use of P2X4-pHluorin123 showed that the modulator ivermectin did not increase the PM fraction of P2X4 receptors and acted allosterically to potentiate P2X4 receptor responses. Collectively, our data suggest that P2X4-pHluorin123 represents a useful optical probe to quantitatively explore P2X4 receptor distribution, trafficking, and up-regulation.

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Pharmacological insights into the role of P2X4 receptors in behavioural regulation: lessons from ivermectin.

Publications

Pharmacological insights into the role of P2X4 receptors in behavioural regulation: lessons from ivermectin.

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