Content uploaded by Aswini Gnanansekaran
Author content
All content in this area was uploaded by Aswini Gnanansekaran on Jun 20, 2018
Content may be subject to copyright.
A preview of the PDF is not available
Calcium/calmodulin‐dependent serine protein kinase (CASK) is a new intracellular modulator of P2X3 receptors
Journal of Neurochemistry
Abstract and Figures
AbstractATP‐gated P2X3 receptors of sensory ganglion neurons are important transducers of painful stimuli and are modulated by extracellular algogenic substances, via changes in the receptor phosphorylation state. The present study investigated the role of calcium/calmodulin‐dependent serine protein kinase (CASK) in interacting and controlling P2X3 receptor expression and function in mouse trigeminal ganglia. Most ganglion neurons in situ or in culture co‐expressed P2X3 and CASK. CASK was immunoprecipitated with P2X3 receptors from trigeminal ganglia and from P2X3/CASK‐cotransfected human embryonic kidney (HEK) cells. Recombinant P2X3/CASK expression in HEK cells increased serine phosphorylation of P2X3 receptors, typically associated with receptor upregulation. CASK deletion mutants also enhanced P2X3 subunit expression. After silencing CASK, cell surface P2X3 receptor expression was decreased, which is consistent with depressed P2X3 currents. The reduction in P2X3 expression levels was reversed by the proteasomal inhibitor MG‐132. Moreover, neuronal CASK/P2X3 interaction was up‐regulated by nerve growth factor (NGF) signaling and down‐regulated by P2X3 agonist‐induced desensitization. These data suggest a novel interaction between CASK and P2X3 receptors with positive outcome for receptor stability and function. As CASK‐mediated control of P2X3 receptors was dependent on the receptor activation state, CASK represents an intracellular gateway to regulate purinergic nociceptive signaling.
Content may be subject to copyright.
... Our previous work has demonstrated that, in sensory neurons, calcium/calmodulin-dependent serine kinase (CASK) dynamically associates with P2X3 receptors at rest, while the complex dissociates upon P2X3 channel opening (Gnanasekaran et al. 2013). Interestingly, CASK controls spontaneous synaptic release and synaptic strength of neuronal networks (Atasoy et al. 2007) and CASK genetic defects in humans are associated with cognitive disorders (Najm et al. 2008). ...
... All treatment protocols were approved by the SISSA ethical committee and in accordance with the European Union guidelines. TG were dissociated and cultured in F12 medium with 10% fetal bovine serum (FBS) and used 24 h after plating as previously described (Gnanasekaran et al. 2013). HEK-293T cells (2.5 9 10 4 cells/mL) were transfected with pcDNA3-P2X3, pcDNA3-CASK-myc, or EGFP encoding plasmids as previously described (Gnanasekaran et al. 2013). ...
... TG were dissociated and cultured in F12 medium with 10% fetal bovine serum (FBS) and used 24 h after plating as previously described (Gnanasekaran et al. 2013). HEK-293T cells (2.5 9 10 4 cells/mL) were transfected with pcDNA3-P2X3, pcDNA3-CASK-myc, or EGFP encoding plasmids as previously described (Gnanasekaran et al. 2013). Transfection efficiency was evaluated with western blot experiments. ...
P2X3 receptors, gated by extracellular ATP , are expressed by sensory neurons and are involved in peripheral nociception and pain sensitization. The ability of P2X3 receptors to transduce extracellular stimuli into neuronal signals critically depends on the dynamic molecular partnership with the calcium/calmodulin‐dependent serine protein kinase ( CASK ). The present work used trigeminal sensory neurons to study the impact that activation of P2X3 receptors (evoked by the agonist α,β‐me ATP ) has on the release of endogenous ATP and how CASK modulates this phenomenon. P2X3 receptor function was followed by ATP efflux via Pannexin1 (Panx1) hemichannels, a mechanism that was blocked by the P2X3 receptor antagonist A‐317491, and by P2X3 silencing. ATP efflux was enhanced by nerve growth factor, a treatment known to potentiate P2X3 receptor function. Basal ATP efflux was not controlled by CASK , and carbenoxolone or Pannexin silencing reduced ATP release upon P2X3 receptor function. CASK ‐controlled ATP efflux followed P2X3 receptor activity, but not depolarization‐evoked ATP release. Molecular biology experiments showed that CASK was essential for the transactivation of Panx1 upon P2X3 receptor activation. These data suggest that P2X3 receptor function controls a new type of feed‐forward purinergic signaling on surrounding cells, with consequences at peripheral and spinal cord level. Thus, P2X3 receptor‐mediated ATP efflux may be considered for the future development of pharmacological strategies aimed at containing neuronal sensitization.
image P2X3 receptors are involved in sensory transduction and associate to CASK. We have studied in primary sensory neurons the molecular mechanisms downstream P2X3 receptor activation, namely ATP release and partnership with CASK or Panx1. Our data suggest that CASK and P2X3 receptors are part of an ATP keeper complex, with important feed‐forward consequences at peripheral and central level.
... After rinsing with tap water sections were dehydrated in 100% ethanol, followed by xylol and embedded with DPX (Permount). [61][62][63][64][65][66][67] rabbit anti-P2X2 GeneTex, GTX10266 1:500 [19] rabbit anti-P2X3 (H-60) Santa Cruz, sc-25694 1:50 [68][69][70][71][72][73] goat anti-P2X4 (N-15) Santa Cruz, sc-15187 1:50 [61][62][63][64][65] goat anti-P2X5 (N-16) Santa Cruz, sc-15191 1:25 [19] rabbit anti-P2X6 LSBio, LS-C94426 1:100 [19] goat anti-P2X7 (L-20) Santa Cruz, sc-15200 1:50 [74][75][76][77][78] rabbit anti-P2Y 1 Santa Cruz, sc-20123 1:50 [79][80][81] rabbit anti-P2Y 2 NovusBio, NB110-39032 1:100 [19,82,83] rabbit anti-P2Y 4 GeneTex, GTX87199 1:750 [17] rabbit anti-P2Y 6 Alomone labs, APR-011 1:100 [84,85] rabbit anti-P2Y 11 GeneTex, GTX108241 1:300 [19,86] rabbit anti-P2Y 12 (P-14) Santa Cruz, sc-27152 1:50 [79,87,88] rabbit anti-P2Y 13 LSBio, LS-C145104 1:250 [19] rabbit anti-P2Y 14 LSBio, LS-C120603 1:250 [19] goat anti-rabbit Vector Laboratories, BA-1000 1:500 [89] rabbit anti-goat Vector Laboratories, BA-5000 1:500 [90] ...
ATP and other nucleotides are important glio-/neurotransmitters in the central nervous system. They bind to purinergic P2X and P2Y receptors that are ubiquitously expressed in various brain regions modulating various physiological and pathophysiological processes. P2X receptors are ligand-gated ion channels mediating excitatory postsynaptic responses whereas P2Y receptors are G protein-coupled receptors mediating slow synaptic transmission. A variety of P2X and P2Y subtypes with distinct neuroanatomical localization provide the basis for a high diversity in their function. There is increasing evidence that P2 receptor signaling plays a prominent role in learning and memory and thus, in hippocampal neuronal plasticity. Learning and memory are time-of-day-dependent. Moreover, extracellular ATP shows a diurnal rhythm in rodents. However, it is not known whether P2 receptors have a temporal variation in the hippocampus. This study provides a detailed systematic analysis on spatial and temporal distribution of P2 in the mouse hippocampus. We found distinct spatial and temporal distribution patterns of the P2 receptors in different hippocampal layers. The temporal distribution of P2 receptors can be segregated into two large time domains, the early to mid-day and the mid to late night. This study provides an important basis for understanding dynamic P2 purinergic signaling in the hippocampal glia/neuronal network.
... In case of our third predicted target, i.e. the mRNA of the Cask gene, we could not find significant change either in its gene or protein expression due to hypercholesterolemia. The Cask gene encodes a calcium/ calmodulin-dependent serine protein kinase that has been described to play a role in neurons in regulating purinergic nociceptive signaling 47 and neuronal growth 48 . Although, we found here that the rodent heart expresses Cask, Cask linked miRNAs (i.e. ...
Little is known about the molecular mechanism including microRNAs (miRNA) in hypercholesterolemia-induced cardiac dysfunction. We aimed to explore novel hypercholesterolemia-induced pathway alterations in the heart by an unbiased approach based on miRNA omics, target prediction and validation. With miRNA microarray we identified forty-seven upregulated and ten downregulated miRNAs in hypercholesterolemic rat hearts compared to the normocholesterolemic group. Eleven mRNAs with at least 4 interacting upregulated miRNAs were selected by a network theoretical approach, out of which 3 mRNAs (beta-2 adrenergic receptor [Adrb2], calcineurin B type 1 [Ppp3r1] and calcium/calmodulin-dependent serine protein kinase [Cask]) were validated with qRT-PCR and Western blot. In hypercholesterolemic hearts, the expression of Adrb2 mRNA was significantly decreased. ADRB2 and PPP3R1 protein were significantly downregulated in hypercholesterolemic hearts. The direct interaction of Adrb2 with upregulated miRNAs was demonstrated by luciferase reporter assay. Gene ontology analysis revealed that the majority of the predicted mRNA changes may contribute to the hypercholesterolemia-induced cardiac dysfunction. In summary, the present unbiased target prediction approach based on global cardiac miRNA expression profiling revealed for the first time in the literature that both the mRNA and protein product of Adrb2 and PPP3R1 protein are decreased in the hypercholesterolemic heart.
... It has been reported that protons open ASIC3 by accelerating the release of Ca 2+ from a high-affinity binding site on the extracellular side of the pore 43,44 . P2X3Rs are regulated by an intracellular calcium/calmodulin-dependent serine protein kinase (CASK) which phosphorylates and in consequence upregulates this receptor, resulting in larger current responses to agonists 45 . P2X3Rs can be phosphorylated by an ecto-protein kinase as well 46 . ...
Two subclasses of acid-sensing ion channels (ASIC3) and of ATP-sensitive P2X receptors (P2X3Rs) show a partially overlapping expression in sensory neurons. Here we report that both recombinant and native receptors interact with each other in multiple ways. Current measurements with the patch-clamp technique prove that ASIC3 stimulation strongly inhibits the P2X3R current partly by a Ca2+-dependent mechanism. The proton-binding site is critical for this effect and the two receptor channels appear to switch their ionic permeabilities during activation. Co-immunoprecipation proves the close association of the two protein structures. BN-PAGE and SDS-PAGE analysis is also best reconciled with the view that ASIC3 and P2X3Rs form a multiprotein structure. Finally, in vivo measurements in rats reveal the summation of pH and purinergically induced pain. In conclusion, the receptor subunits do not appear to form a heteromeric channel, but tightly associate with each other to form a protein complex, mediating unidirectional inhibition. Two subclasses of ligand-gated ion channels (ASIC3 and P2X3) are both present at sensory neurons and might be therefore subject to receptor crosstalk. Here authors use electrophysiology, biochemistry and co-immunoprecipitation to show that the two ion channels interact and affect P2X3 currents.
... The relatively late recognition of the various subclasses of purinergic receptors in the 70s, and the subsequent definition of four subtypes of adenosine and multiple purine receptors (7 ion channel, 8 G-protein coupled; Burnstock 2015) has meant the regular appearance of studies addressing receptor assembly, function and signalling. There is impressive recent work utilizing functional receptor mutants (Sundukova et al. 2012;Jindrichova et al. 2015), and contributions on receptor internalization (Vacca et al. 2009;Haas et al. 2014) and phosphorylation (Roberts et al. 2012;Gnanasekaran et al. 2013). ...
This ‘Past to Future’ Review as part of the 60th anniversary year of the Journal of Neurochemistry focuses on synaptic transmission and associated signalling, and seeks to identify seminal progress in neurochemistry over the last 10 years which has advanced our understanding of neuronal communication in brain. The approach adopted analyses neurotransmitters on a case by case basis (i.e. amino acids, monoamines, acetylcholine, neuropeptides, ATP /purines and gasotransmitters) to highlight novel findings that have changed the way we view each type of transmitter, to explore commonalities and interactions, and to note how new insights have changed the way we view the biology of degenerative, psychiatric and behavioural conditions. Across all transmitter systems there was remarkable growth in the identification of targets likely to provide therapeutic benefit and which undoubtedly was driven by the elucidation of circuit function and new vistas of synaptic signalling. There has been an increasing trend to relate signalling to disease, notably for Alzheimer's and Parkinson's disease and related conditions, and which has occurred for each transmitter family. Forebrain circuitry and tonic excitatory control have been the centre of great attention yielding novel findings that will impact upon cognitive, emotional and addictive behaviours. Other impressive insights focus on gasotransmitters integrating activity as volume transmitters. Exciting developments in how serotonin, cholinergic, l ‐glutamate, galanin and adenosine receptors and their associated signalling can be beneficially targeted should underpin the development of new therapies. Clearly integrated, multifaceted neurochemistry has changed the way we view synaptic signalling and its relevance to pathobiology.
image Highlighted are important advances in synaptic signalling over the last decade in the Journal of Neurochemistry . Across all transmitter systems elucidation of circuit function, and notably molecular insights, have underpinned remarkable growth in the identification of targets likely to provide therapeutic benefit in neuropathologies. Another commonality was wide interest in forebrain circuitry and its tonic excitatory control. Increasingly observations relate to signalling in disease and behavioural conditions.
This article is part of the 60th Anniversary special issue .
The mammalian cochlea undergoes a highly dynamic process of growth and innervation during development. This process includes spiral ganglion neuron (SGN) branch refinement, a process whereby Type I SGNs undergo a phase of "debranching" before forming unramified synaptic contacts with inner hair cells. Using Sox2CreERT2 and R26RtdTomato as a strategy to genetically label individual SGNs in mice of both sexes, we report on both a time course of SGN branch refinement and a role for P2rx3 in this process. P2rx3 is an ionotropic ATP receptor that was recently implicated in outer hair cell spontaneous activity and Type II SGN synapse development (Ceriani et al., 2019), but its function in Type I SGN development is unknown. Here, we demonstrate that P2rx3 is expressed by Type I SGNs and hair cells during developmental periods that coincide with SGN branching refinement. P2rx3 null mice show SGNs with more complex branching patterns on their peripheral synaptic terminals and near their cell bodies around the time of birth. Loss of P2rx3 does not appear to confer general changes in axon outgrowth or hair cell formation, and alterations in branching complexity appear to mostly recover by postnatal day (P)6. However, when we examined the distribution of Type I SGN subtypes using antibodies that bind Calb2, Calb1, and Pou4f1, we found that P2rx3 null mice showed an increased proportion of SGNs that express Calb2. These data suggest P2rx3 may be necessary for normal Type I SGN differentiation in addition to serving a role in branch refinement.
It is now well established that antibodies have numerous potential benefits when developed as therapeutics. Here, we evaluate the technical challenges of raising antibodies to membrane-spanning proteins together with enabling technologies that may facilitate the discovery of antibody therapeutics to ion channels. Additionally, we discuss the potential targeting opportunities in the anti-ion channel antibody landscape, along with a number of case studies where functional antibodies that target ion channels have been reported. Antibodies currently in development and progressing towards the clinic are highlighted.
P2X3 receptors are ion channels expressed by autonomic and sensory nerves and specialised in transducing extracellular ATP signals. Structural data, together with functional and biochemical studies, suggest that conformational changes of P2X3 receptors upon agonist binding influence downstream intracellular molecular mechanisms relevant for neuronal responses. Activity of P2X3 receptors is implicated in pain, itch, asthma, cardiovascular dysfunction and other pathologies. The study of these receptors has therefore a large potential in the field of drug development and interdisciplinary efforts could clarify molecular mechanisms controlling P2X3 receptor function in different physiological or pathological contexts.
P2X receptors are cation-selective channels that are activated by the binding of extracellular ATP. They have a high permeability to Ca2 +, Na⁺, and K⁺ and are expressed widely throughout the nervous, immune, cardiovascular, skeletal, gastrointestinal, respiratory, and endocrine systems. Seven mammalian subtypes of P2X receptor subunits have been identified, P2X1–7, and those that function as homotrimeric receptors (P2X1, 2, 3, 4, and 7) are targeted to lipid rafts, although they show limited resistance to solubilization by Triton X-100. Recent crystal structures of P2X3 and P2X4 receptors have provided considerable high-resolution information about the architecture of this family of receptors and yet the molecular details of how they are regulated by cholesterol are unknown. Currents mediated by the P2X1–4 receptors are either inhibited or relatively insensitive to cholesterol depletion, but there is no clear evidence to support the direct binding of cholesterol to these receptors. In contrast, the activity of the low-affinity, proinflammatory P2X7 receptor is potentiated by cholesterol depletion and regions within the proximal C-terminus play an important role in coupling changes in cholesterol to the gating of the pore. Based upon our understanding of the lipid signaling events that are triggered downstream of P2X7 receptor activation, a change in the levels of cholesterol may contribute to the sensitization of receptor currents and the dilation of the pore that occurs following prolonged, high-level stimulation. This chapter focuses on the regulation of P2X7 receptor signaling by cholesterol and our current understanding of the mechanisms that underlie this.
Neurexins are neuronal cell surface proteins with hundreds of isoforms. In yeast two-hybrid screens for intracellular molecules interacting with different neurexins, we identified a single interacting protein called CASK. CASK is composed of an N-terminal Ca2+, calmodulin-dependent protein kinase sequence and a C-terminal region that is similar to the intercellular junction proteins dlg-A, PSD95/SAP90, SAP97, Z01, and Z02 and that contains DHR-, SH3-, and guanylate kinase domains. CASK is enriched in brain in synaptic plasma membranes but is also detectable at low levels in all tissues tested. The cytoplasmic domains of all three neurexins bind CASK in a salt-labile interaction. In neurexin I, this interaction is dependent on the C-terminal three residues. Thus, CASK is a membrane-associated protein that combines domains found in Ca2+ - activated protein kinases and in proteins specific for intercellular junctions, suggesting that it may be a signaling molecule operating at the plasma membrane, possibly in conjunction with neurexins.
CASK, the rat homolog of a gene (LIN-2) required for vulval differentiation in Caenorhabditis elegans, is expressed in mammalian brain, but its function in neurons is unknown. CASK is distributed in a punctate somatodendritic
pattern in neurons. By immunogold EM, CASK protein is concentrated in synapses, but is also present at nonsynaptic membranes
and in intracellular compartments. This immunolocalization is consistent with biochemical studies showing the presence of
CASK in soluble and synaptosomal membrane fractions and its enrichment in postsynaptic density fractions of rat brain. By
yeast two-hybrid screening, a specific interaction was identified between the PDZ domain of CASK and the COOH terminal tail
of syndecan-2, a cell surface heparan sulfate proteoglycan (HSPG). The interaction was confirmed by coimmunoprecipitation
from heterologous cells. In brain, syndecan-2 localizes specifically at synaptic junctions where it shows overlapping distribution
with CASK, consistent with an interaction between these proteins in synapses. Cell surface HSPGs can bind to extracellular
matrix proteins, and are required for the action of various heparin-binding polypeptide growth/differentiation factors.
The synaptic localization of CASK and syndecan suggests a potential role for these proteins in adhesion and signaling at
neuronal synapses.
ATP-gated P2X3 receptors are expressed by nociceptive neurons and participate in transduction of pain. Responsiveness of P2X3 receptors is strongly reduced at low temperatures, suggesting a role for these receptors in analgesic effects of cooling. Since sustained responsiveness depends on receptor trafficking to the plasma membrane, we employed total internal reflection fluorescence (TIRF) microscopy to highlight perimembrane pool of DsRed-tagged P2X3 receptors and studied the effects of temperature on perimembrane turnover of P2X3-DsRed. Patch-clamp recordings confirmed membrane expression of functional, rapidly desensitizing P2X3-DsRed receptors. By combining TIRF microscopy with the technique of fluorescence recovery after photobleaching (FRAP), we measured the rate of perimembrane turnover of P2X3-DsRed receptors expressed in hippocampal neurons. At room temperature, the P2X3-DsRed perimembrane turnover as measured by TIRF-FRAP had a time constant of ∼2 min. At 29°C, receptor turnover was strongly accelerated (0.6 min), yielding an extremely high temperature dependence coefficient Q(10) ∼4.5. In comparison, AMPA receptor turnover measured with TIRF-FRAP was only moderately sensitive to temperature (Q(10) ∼1.5). The traffic inhibitor Brefeldin A selectively decelerated P2X3-DsRed receptor turnover at 29°C, but had no effect at 21°C (Q(10) ∼1.0). This indicates that receptor traffic to plasma membrane is the key temperature-sensitive component of P2X3 turnover. The selective inhibitor of the RhoA kinase Y27632 significantly decreased the temperature dependence of P2X3-DsRed receptor turnover (Q(10) ∼2.0). In summary, the RhoA kinase-dependent membrane trafficking of P2X3 receptors to plasma membrane has an exceptionally high sensitivity to temperature. These findings suggest an important role of P2X3 receptor turnover in hypothermia-associated analgesia.
Astrocytes are a class of neural cells that control homeostasis at all levels of the central and peripheral nervous system. There is a bidirectional neuron-glia interaction via a number of extracellular signaling molecules, glutamate and ATP being the most widespread. ATP activates ionotropic P2X and metabotropic P2Y receptors, which operate in both neurons and astrocytes. Morphological, biochemical, and functional evidence indicates the expression of astroglial P2X(1/5) heteromeric and P2X(7) homomeric receptors, which mediate physiological and pathophysiological responses. Activation of P2X(1/5) receptors triggers rapid increase of intracellular Na(+) that initiates immediate cellular reactions, such as the depression of the glutamate transporter to keep high glutamate concentrations in the synaptic cleft, the activation of the local lactate shuttle to supply energy substrate to pre- and postsynaptic neuronal structures, and the reversal of the Na(+)/Ca(2+) exchange resulting in additional Ca(2+) entry. The consequences of P2X(7) receptor activation are mostly but not exclusively mediated by the entry of Ca(2+) and result in reorganization of the cytoskeleton, inflammation, apoptosis/necrosis, and proliferation, usually at a prolonged time scale. Thus, astroglia detect by P2X(1/5) and P2X(7) receptors both physiological concentrations of ATP secreted from presynaptic nerve terminals and also much higher concentrations of ATP attained under pathological conditions.
The strong expression of ATP-gated P2X3 receptors by a subpopulation of sensory neurons indicates the important role of these membrane proteins in nociceptive signaling in health and disease, especially when the latter is accompanied by chronic pain syndromes. Molecular and cell biology studies have shown that these receptors exist mainly as trimeric homomers, and, in part, as heteromers (assembly of two P2X3 subunits with one P2X2). Recent investigations have suggested distinct molecular determinants responsible for agonist binding and channel opening for transmembrane flux of sodium, calcium and potassium ions. Trimeric P2X3 receptors are rapidly activated by ATP and can be strongly desensitized in the continuous presence of the agonist. Thus, the factors controlling the degree of desensitization and the time necessary to recover from it are essential elements to determine how efficiently and how often the P2X3 receptor can signal pain. Endogenous substances, widely thought to be involved in triggering pain especially in pathological conditions, can potently modulate the expression and function of P2X3 receptors, with differential changes in response amplitude, desensitization and recovery. Hence, studying P2X3 receptors can lead not only to the design of novel antagonists as analgesics, but also to identify intracellular interactors that may be targeted to downregulate P2X3 receptors. Strong facilitation of P2X3 receptor function is induced by enodogenous substances like the neuropeptide calcitonin gene-related peptide and the neurotrophins nerve growth factor and brain-derived neurotrophic factor. These substances possess distinct mechanisms of action on P2X3 receptors, generally attributable to discrete phosphorylation of N- or C-terminal P2X3 domains.
J. Neurochem. (2012) 122, 557–567.
ATP-activated P2X3 receptors of sensory ganglion neurons contribute to pain transduction and are involved in chronic pain signaling. Although highly homologous (97%) in rat and human species, it is unclear whether P2X3 receptors have identical function. Studying human and rat P2X3 receptors expressed in patch-clamped human embryonic kidney (HEK) cells, we investigated the role of non-conserved tyrosine residues in the C-terminal domain (rat tyrosine-393 and human tyrosine-376) as key determinants of receptor function. In comparison with rat P2X3 receptors, human P2X3 receptors were more expressed and produced larger responses with slower desensitization and faster recovery. In general, desensitization was closely related to peak current amplitude for rat and human receptors. Downsizing human receptor expression to the same level of the rat one still yielded larger responses retaining slower desensitization and faster recovery. Mutating phenylalanine-376 into tyrosine in the rat receptor did not change current amplitude; yet, it retarded desensitization onset, demonstrating how this residue was important to functionally link these two receptor states. Conversely, removing tyrosine from position 376 strongly down-regulated human receptor function. The different topology of tyrosine residues in the C-terminal domain has contrasting functional consequences and is sufficient to account for species-specific properties of this pain-transducing channel.
Experimental evidence is presented to support the hypothesis that purinergic mechanosensory transduction can initiate visceral pain in urinary bladder, ureter, gut and uterus. In general, physiological reflexes are mediated via P2X3 and P2X2/3 receptors on low threshold sensory fibres, while these receptors on high threshold sensory fibres mediate pain. Potential therapeutic strategies are considered for the treatment of visceral pain in such conditions as renal colic, interstitial cystitis and inflammatory bowel disease by purinergic agents, including P2X3 and P2X2/3 receptor antagonists that are orally bioavailable and stable in vivo and agents that modulate ATP release and breakdown.
Purinergic neurotransmission, involving release of ATP as an efferent neurotransmitter was first proposed in 1972. Later, ATP was recognised as a cotransmitter in peripheral nerves and more recently as a cotransmitter with glutamate, noradrenaline, GABA, acetylcholine and dopamine in the CNS. Both ATP, together with some of its enzymatic breakdown products (ADP and adenosine) and uracil nucleotides are now recognised to act via P2X ion channels and P1 and P2Y G protein-coupled receptors, which are widely expressed in the brain. They mediate both fast signalling in neurotransmission and neuromodulation and long-term (trophic) signalling in cell proliferation, differentiation and death. Purinergic signalling is prominent in neurone-glial cell interactions. In this review we discuss first the evidence implicating purinergic signalling in normal behaviour, including learning and memory, sleep and arousal, locomotor activity and exploration, feeding behaviour and mood and motivation. Then we turn to the involvement of P1 and P2 receptors in pathological brain function; firstly in trauma, ischemia and stroke, then in neurodegenerative diseases, including Alzheimer's, Parkinson's and Huntington's, as well as multiple sclerosis and amyotrophic lateral sclerosis. Finally, the role of purinergic signalling in neuropsychiatric diseases (including schizophrenia), epilepsy, migraine, cognitive impairment and neuropathic pain will be considered.
The highly polarized morphology and complex geometry of neurons is determined to a great extent by the structural and functional organization of the secretory pathway. It is intuitive to propose that the spatial arrangement of secretory organelles and their dynamic behavior impinge on protein trafficking and neuronal function, but these phenomena and their consequences are not well delineated. Here we analyze the architecture and motility of the archetypal endoplasmic reticulum (ER), and their relationship to the microtubule cytoskeleton and post-translational modifications of tubulin. We also review the dynamics of the ER in axons, dendrites and spines, and discuss the role of ER dynamics on protein mobility and trafficking in neurons.