Article

Protein kinase C regulation of P2X3 receptors is unlikely to involvedirect receptor phosphorylation

Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 02/2007; 1773(2):166-175. DOI: 10.1016/j.bbamcr.2006.09.020

ABSTRACT P2X receptors (P2XR) act as ligand-gated, cation-selective ion channels. A common characteristic of all seven P2X family members is a conserved consensus sequence for protein kinase C (PKC)-mediated phosphorylation in the intracellular N-terminus of the receptor. Activation of PKC has been shown to enhance currents through P2X3R, however the molecular mechanism of this potentiation has not been elucidated. In the present study we show that activation of PKC can enhance adenosine triphosphate (ATP)-mediated Ca2+ signals ∼2.5-fold in a DT-40 3KO cell culture system (P2 receptor null) transiently overexpressing P2X3R. ATP-activated cation currents were also directly studied using whole cell patch clamp techniques in HEK-293 cells, a null background for ionotropic P2XR. PKC activation resulted in a ∼8.5-fold enhancement of ATP-activated current in HEK-293 cells transfected with P2X3R cDNA, but had no effect on currents through either P2X4R- or P2X7R-transfected cells. P2X3R-transfected HEK-293 cells were metabolically labeled with 32PO4− and following treatment with phorbol-12-myristate-13-acetate (PMA) and subsequent immunoprecipitation, there was no incorporation of 32PO4− in bands corresponding to P2X3R. Similarly, in vitro phosphorylation experiments, utilizing purified PKC catalytic subunits failed to establish phosphorylation of either P2X3R or P2X3R-EGFP. These data indicate that PKC activation can enhance both the Ca2+ signal as well as the cation current through P2X3R, however it appears that the regulation is unlikely to be a result of direct phosphorylation of the receptor.

0 Followers
 · 
45 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Nociceptors are peripheral sensory neurones which respond to painful (noxious) stimuli. The terminals of nociceptors, which have a high threshold to stimulation in their native state, undergo a process known as sensitisation, or lowering of threshold, following injury or inflammation. Amongst sensory receptors, sensitisation is a property unique to nociceptors. A shift in the stimulus-response function of nociceptors renders them more sensitive, resulting in both a reduction in the activation threshold, such that previously non-noxious stimuli are perceived as noxious (allodynia) and an increased response to suprathreshold stimuli (hyperalgesia). Sensitisation protects us from harm and is essential for survival, but it can be disabling in conditions of chronic inflammation. This review focuses on three stages in sensitisation: 1) Inflammatory mediators, which are released from damaged resident cells and from others that invade in response to inflammation, and include bradykinin, prostaglandins, serotonin, low pH, ATP, neurotrophins, nitric oxide and cytokines; 2) Intracellular signalling molecules which are important in transmitting the actions of inflammatory mediators and include protein kinase A and C, Src kinase, mitogen-activated protein kinases and the membrane lipid PIP2; and 3) Ion channel targets of intracellular signalling which ultimately cause sensitisation and include the temperaturesensitive transient receptor potential channels, acid-sensitive ion channels, purinoceptor-gated channels, and the voltagesensitive sodium, potassium, calcium and HCN channels.
    The Open Pain Journal 01/2010; ISSN: 1876-3863(1):82-96. DOI:10.2174/1876386301003010082
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Purine and pyrimidine nucleotides are extracellular signaling molecules in the central nervous system (CNS) leaving the intracellular space of various CNS cell types via nonexocytotic mechanisms. In addition, ATP is a neuro-and gliotransmitter released by exocytosis from neurons and neuroglia. These nucleotides activate P2 receptors of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) types. In mammalians, seven P2X and eight P2Y receptor subunits occur; three P2X subtypes form homomeric or heteromeric P2X receptors. P2Y subtypes may also hetero-oligomerize with each other as well as with other G protein-coupled receptors. P2X receptors are able to physically associate with various types of ligand-gated ion channels and thereby to interact with them. The P2 receptor homomers or heteromers exhibit specific sensitivities against pharmacological ligands and have preferential functional roles. They may be situated at both presynaptic (nerve terminals) and postsynaptic (somatodendritic) sites of neurons, where they modulate either transmitter release or the postsynaptic sensitivity to neurotransmitters. P2 receptors exist at neuroglia (e.g., astrocytes, oligodendrocytes) and microglia in the CNS. The neuroglial P2 receptors subserve the neuron-glia cross talk especially via their end-feets projecting to neighboring synapses. In addition, glial networks are able to communicate through coordinated oscillations of their intracellular Ca(2+) over considerable distances. P2 receptors are involved in the physiological regulation of CNS functions as well as in its pathophysiological dysregulation. Normal (motivation, reward, embryonic and postnatal development, neuroregeneration) and abnormal regulatory mechanisms (pain, neuroinflammation, neurodegeneration, epilepsy) are important examples for the significance of P2 receptor-mediated/modulated processes.
    Advances in pharmacology (San Diego, Calif.) 01/2011; 61:441-93. DOI:10.1016/B978-0-12-385526-8.00014-X
  • [Show abstract] [Hide abstract]
    ABSTRACT: Estrogen has been reported to affect pain perception, although the underlying mechanisms remain unclear. In this investigation, pain behavior testing, patch clamp recording and immunohistochemistry were used on rats and transgenic mice to determine which estrogen receptors and the related signaling pathway are involved in the rapid modulation of estrogen on P2X3 receptor-mediated events. The results showed that 17β-estradiol rapidly inhibited pain induced by α,β-methylene ATP (α,β-me-ATP), a P2X1 and P2X3 receptor agonist in ovariectomized rats and normal rats in diestrus. The estrogen receptor-α (ERα) agonist 4,49,499-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) and GPR30 receptor agonist G-1 mimicked the estrogen effect, while the ERβ agonist diarylpropionitrile (DPN) had no effect. In cultured rat dorsal root ganglion (DRG) neurons, PPT and G-1 but not DPN significantly attenuated α,β-meATP-mediated currents, with the dose-response curve of these currents shifted to the right. The inhibitory effect of 17β-estradiol on P2X3 currents was blocked by G-15, a selective antagonist to the GPR30 estrogen receptor. 17β-estradiol lacked this effect in DRG neurons from ERα knockout mice while partly remained in those from ERβ knockout mice. The P2X3 and GPR30 receptors were co-expressed in the rat DRG neurons. Further, the ERK1/2 inhibitor U0126 reversed the inhibitory effect of 17β-estradiol on α,β-me-ATP-induced pain and of PPT or G-1 on P2X3 receptor-mediated currents. The cyclic AMP-protein kinase A (PKA) agonist forskolin, but not the protein kinase C agonist phorbol-12-myristate-13-acetate (PMA) mimicked the estrogen inhibitory effect on P2X3 receptor currents, which was blocked by another ERK1/2 inhibitor, PD98059. These results suggest that estrogen regulates P2X3-mediated peripheral pain by acting on ERα and GPR30 receptors expressed in primary afferent neurons, which probably involves the intracellular cAMP-PKA-ERK1/2 pathway.
    Endocrinology 04/2013; 154(7). DOI:10.1210/en.2012-2119 · 4.64 Impact Factor

Preview

Download
0 Downloads
Available from