Protein kinase C regulation of P2X3 receptors is unlikely to involve direct receptor phosphorylation. Biochim Biophys Acta

University of Rochester, Rochester, New York, United States
Biochimica et Biophysica Acta (Impact Factor: 4.66). 02/2007; 1773(2):166-175. DOI: 10.1016/j.bbamcr.2006.09.020


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.

Full-text preview

Available from:
  • Source
    • "Recombinant rat P2X3 receptors expressed in HEK cells Newly synthesized adenine derivatives 21e28, together with the precursors 17 and 19, were tested for their biological activity on P2X3 receptors with the patch clamp recording technique. For initial screening, we used rat or human P2X3 receptors transfected into HEK 293T cells, which do not constitutively express them [26] [27]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Ligands that selectively block P2X3 receptors localized on nociceptive sensory fibres may be useful for the treatment of chronic pain conditions including neuropathic pain, migraine, and inflammatory pain. With the aim at exploring the suitability of adenine moiety as a scaffold for the development of antagonists of this receptor, a series of 9-benzyl-2-aminoadenine derivatives were designed and synthesized. These new compounds were functionally evaluated at rat or human P2X3 receptors expressed in human embryonic kidney (HEK) cells and on native P2X3 receptors from mouse trigeminal ganglion sensory neurons using patch clamp recording under voltage clamp configuration. The new molecules behaved as P2X3 antagonists, as they rapidly and reversibly inhibited (IC50 in the low micromolar range) the membrane currents induced via P2X3 receptor activation by the full agonist α,β-methyleneATP. Introduction of a small lipophilic methyl substituent at the 6-amino group enhanced the activity, in comparison to the corresponding unsubstituted derivative, resulting in the 9-(5-iodo-2-isopropyl-4-methoxybenzyl)-N(6)-methyl-9H-purine-2,6-diamine (24), which appears to be a good antagonist on recombinant and native P2X3 receptors with IC50 = 1.74 ± 0.21 μM.
    Full-text · Article · Apr 2013 · European Journal of Medicinal Chemistry
  • Source
    • "In the case of conflicting evidence a combination of symbols is given. For calcium and potassium channels the affected channel subtypes are mentioned when known, or marked as NI (not identified) if currents were measured without clear identification of subtypes PKA PKC Src MAPK PIP2 Inflammation TRPV1 ↑[115] [155] ↑ [154] [156] [252] ↑[72] [73] [74] [153] [158] ↑ [74] [78] ↑[158] [253] [254] [255] [256] ↓[257] ↑[258] TRPV4 ↑ [129] ↑[129] [259] ↑[260] [261] ∅ ∅ ↑[262] [263] TRPM8 ↓[177] [264] ↓[114] [177] ∅ ∅ ↑[143] [178] [265] ↔[266] TRPA1 ↑ [170] ∅ ∅ ↑ [266] ↑[267] ↓[171] [268] ↑[266] [269] ASIC ∅ ↑[197] [270] ∅ ↑[77] ∅ ↑ [271] [272] P2X [33] ↑indirect [33] [202] [273] ↓[274] ∅ ∅ ↑ P2X1 [275] ↑[276] [277] Nav1.7 ↑[278] ↓ [221] ↑ [221] ∅ ∅ ∅ ↑[208] [209] [279] Nav1.8 ↑[221] [230] [280] ↑[230] [280] [281] ↓[221] ∅ ∅ ∅ ↑ [208] [209] [279] Nav1.9 ∅ ↑[230] [282] ∅ ∅ ∅ ↑[279] ↔ [208] Potassium ↑ Kv1.2 [283] Kir1.1 [284] Kir2.1 [285] ↓ NI [233] K2P [286] "
    [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.
    Full-text · Article · Jan 2010 · The Open Pain Journal
  • Source
    • "Furthermore, other studies reveal distinct regulation of P2X3 and TRPV1 receptor expression in sensory neurons by acute and chronic NGF as well as by serotonin [32]. The proposal of indirect modulation of the P2X3 receptor [4] but direct phosphorylation of the TRPV1 receptor by protein kinase C [2] [24] may contribute to this divergent adrenergic regulation of the two receptors by nerve injury. Since nerve injury models were developed, there has been considerable emphasis placed on enhanced sensory afferent signaling produced by evoked stimuli. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Local application of alphabetaMeATP (ligand for P2X3 receptors) and capsaicin (ligand for TRPV1 receptors) to the rat hindpaw produces pain behaviors (flinching) which are enhanced by noradrenaline (NA). In this study, we have examined the effect of nerve injury on adrenergic regulation of P2X3 and TRPV1 receptors by administering alphabetaMeATP and capsaicin, alone and in combination with NA, into the lateral and medial hindpaw in the spared nerve injury (SNI) model; this allows for an exploration of the role of injured and uninjured afferents in their effects on nociceptive signaling using a behavioral model. Following lateral hindpaw injections (sural sensory field), effects of NA and alphabetaMeATP, both alone and in combination, were increased following SNI, but no such effects were seen following medial hindpaw injections (saphenous sensory field). Following lateral hindpaw injections, the effect of capsaicin alone was unaltered following SNI, but the effect of NA/capsaicin was reduced; this latter effect was not seen following medial hindpaw injections. At the lateral site, prazosin (alpha1-adrenergic receptor antagonist) inhibited the effect of NA/alphabetaMeATP following SNI, but neither prazosin nor GF109203X (protein kinase C inhibitor) inhibited the effect of NA/capsaicin following SNI. These results demonstrate: (a) an enhanced adrenergic regulation of P2X3 receptor activity at lateral sites following SNI where signaling afferents are directly influenced by injured neurons; (b) differential effects on adrenergic regulation of TRPV1 receptors under the same conditions; (c) lack of such changes when agents are administered into medial sites following SNI.
    Full-text · Article · Sep 2008 · Neuroscience Letters
Show more