Amyloid-ß Impairs Dendritic Trafficking of Golgi-Like Organelles in the Early Phase Preceding Neurite Atrophy: Rescue by Mirtazapine.

Neurite atrophy with loss of neuronal polarity is a pathological hallmark of Alzheimer's disease (AD) and other neurological disorders. While there is substantial agreement that disruption of intracellular vesicle trafficking is associated with axonal pathology in AD, comparatively less is known regarding its role in dendritic atrophy. This is a significant gap of knowledge because, unlike axons, dendrites are endowed with the complete endomembrane system comprising endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC), Golgi apparatus, post-Golgi vesicles, and a recycling-degradative route. In this study, using live-imaging of pGOLT-expressing vesicles, indicative of Golgi outposts and satellites, we investigate how amyloid-ß (Aß) oligomers affect the trafficking of Golgi-like organelles in the different dendritic compartments of cultured rat hippocampal neurons. We found that short-term (4 h) treatment with Aß led to a decrease in anterograde trafficking of Golgi vesicles in dendrites of both resting and stimulated (with 50 mM KCl) neurons. We also characterized the ability of mirtazapine, a noradrenergic and specific serotonergic tetracyclic antidepressant (NaSSA), to rescue Golgi dynamics in dendrites. Mirtazapine treatment (10 µM) increased the number and both anterograde and retrograde motility, reducing the percentage of static Golgi vesicles. Finally, mirtazapine reverted the neurite atrophy induced by 24 h treatment with Aß oligomers, suggesting that this drug is able to counteract the effects of Aß by improving the dendritic trafficking of Golgi-related vesicles.

P2X3 receptors are transducers of sensory signals.

Peripheral stimuli are transduced by specific receptors expressed by sensory neurons and are further processed in the dorsal horn of spinal cord before to be transmitted to the brain. While relative few receptor subtypes mediate the initial depolarisation of sensory neurons, an impressive number of molecules and ion channels integrate these inputs into coded signals. Soluble mediators and ambient conditions further shape these processes, potentially triggering peripheral and central sensitisation, or sensory downregulation. Extracellular ATP is a major signaling molecule that acts via purinergic receptors and is a powerful modulator of cell communication as well as a neurotransmitter at peripheral/central synapses. In particular, ATP-mediated signals are transduced by P2X3 receptors expressed mainly by peripheral sensory neurons. Recent evidence suggests that P2X3 receptor function not only induces neuron depolarisation and firing with consequent neurotransmitter release, but it also triggers intracellular molecular changes that amplify purinergic signaling with important consequences.

Author Correction: The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel.

The originally published version of this article contained an error in the name of the author Flóra Gölöncsér, which was incorrectly given as Flóra Göröncsér. This has now been corrected in both the PDF and HTML versions of the article.

The ASIC3/P2X3 cognate receptor is a pain-relevant and ligand-gated cationic channel.

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.

In situ imaging reveals properties of purinergic signalling in trigeminal sensory ganglia in vitro.

Chronic pain is supported by sterile inflammation that induces sensitisation of sensory neurons to ambient stimuli including extracellular ATP acting on purinergic P2X receptors. The development of in vitro methods for drug screening would be useful to investigate cell crosstalk and plasticity mechanisms occurring during neuronal sensitisation and sterile neuroinflammation. Thus, we studied, at single-cell level, membrane pore dilation based on the uptake of a fluorescent probe following sustained ATP-gated P2X receptor function in neurons and non-neuronal cells of trigeminal ganglion cultures from wild-type (WT) and R192Q CaV2.1 knock-in (KI) mice, a model of familial hemiplegic migraine type 1 characterised by neuronal sensitisation and higher release of soluble mediators. In WT cultures, pore responses were mainly evoked by ATP rather than benzoyl-ATP (BzATP) and partly inhibited by the P2X antagonist TNP-ATP. P2X7 receptors were expressed in trigeminal ganglia mainly by non-neuronal cells. In contrast, KI cultures showed higher expression of P2X7 receptors, stronger responses to BzATP, an effect largely prevented by prior administration of CaV2.1 blocker ω-agatoxin IVA, small interfering RNA (siRNA)-based silencing of P2X7 receptors or the P2X7 antagonist A-804598. No cell toxicity was detected with the protocols. Calcitonin gene-related peptide (CGRP), a well-known migraine mediator, potentiated BzATP-evoked membrane permeability in WT as well as R192Q KI cultures, demonstrating its modulatory role on trigeminal sensory ganglia. Our results show an advantageous experimental approach to dissect pharmacological properties potentially relevant to chronic pain and suggest that CGRP is a soluble mediator influencing purinergic P2X pore dilation and regulating inflammatory responses.

ATP-Gated P2X3 Receptors Are Specialised Sensors of the Extracellular Environment.

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.

The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex.

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,ß-meATP) 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. 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.

Brain natriuretic peptide constitutively downregulates P2X3 receptors by controlling their phosphorylation state and membrane localization.

BACKGROUND: ATP-gated P2X3 receptors are important transducers of nociceptive stimuli and are almost exclusively expressed by sensory ganglion neurons. In mouse trigeminal ganglion (TG), P2X3 receptor function is unexpectedly enhanced by pharmacological block of natriuretic peptide receptor-A (NPR-A), outlining a potential inhibitory role of endogenous natriuretic peptides in nociception mediated by P2X3 receptors. Lack of change in P2X3 protein expression indicates a complex modulation whose mechanisms for downregulating P2X3 receptor function remain unclear. RESULTS: To clarify this process in mouse TG cultures, we suppressed NPR-A signaling with either siRNA of the endogenous agonist BNP, or the NPR-A blocker anantin. Thus, we investigated changes in P2X3 receptor distribution in the lipid raft membrane compartment, their phosphorylation state, as well as their function with patch clamping. Delayed onset of P2X3 desensitization was one mechanism for the anantin-induced enhancement of P2X3 activity. Anantin application caused preferential P2X3 receptor redistribution to the lipid raft compartment and decreased P2X3 serine phosphorylation, two phenomena that were not interdependent. An inhibitor of cGMP-dependent protein kinase and siRNA-mediated knockdown of BNP mimicked the effect of anantin. CONCLUSIONS: We demonstrated that in mouse trigeminal neurons endogenous BNP acts on NPR-A receptors to determine constitutive depression of P2X3 receptor function. Tonic inhibition of P2X3 receptor activity by BNP/NPR-A/PKG pathways occurs via two distinct mechanisms: P2X3 serine phosphorylation and receptor redistribution to non-raft membrane compartments. This novel mechanism of receptor control might be a target for future studies aiming at decreasing dysregulated P2X3 receptor activity in chronic pain.

P2X receptors, sensory neurons and pain.

Pain represents a very large social and clinical problem since the current treatment provides insufficient pain relief. Plasticity of pain receptors together with sensitisation of sensory neurons, and the role of soluble mediators released from non-neuronal cells render difficult to understand the spatial and temporal scale of pain development, neuronal responses and disease progression. In pathological conditions, ATP is one of the most powerful mediators that activates P2X receptors that behave as sensitive ATP-detectors, such as neuronal P2X3 receptor subtypes and P2X4 and P2X7 receptors expressed on non-neuronal cells. Dissecting the molecular mechanisms occurring in sensory neurons and in accessory cells allows to design appropriate tissue- and cell- targeted approaches to treat chronic pain.

ATP P2X3 receptors and neuronal sensitization.

Increasing evidence indicates the importance of extracellular adenosine triphosphate (ATP) in the modulation of neuronal function. In particular, fine control of ATP release and the selective and discrete ATP receptor operation are crucial elements of the crosstalk between neuronal and non-neuronal cells in the peripheral and central nervous systems. In peripheral neurons, ATP signaling gives an important contribution to neuronal sensitization, especially that involved in neuropathic pain. Among other subtypes, P2X3 receptors expressed on sensory neurons are sensitive even to nanomolar concentrations of extracellular ATP, and therefore are important transducers of pain stimuli. P2X3 receptor function is highly sensitive to soluble factors like neuropeptides and neurotrophins, and is controlled by transduction mechanisms, protein-protein interactions and discrete membrane compartmentalization. More recent findings have demonstrated that P2X3 receptors interact with the synaptic scaffold protein calcium/calmodulin-dependent serine protein kinase (CASK) in a state dependent fashion, indicating that CASK plays a crucial role in the modulation of P2X3 receptor stability and efficiency. Activation of P2X3 receptors within CASK/P2X3 complex has important consequences for neuronal plasticity and possibly for the release of neuromodulators and neurotransmitters. Better understanding of the interactome machinery of P2X3 receptors and their integration with other receptors and channels on neuronal surface membranes, is proposed to be essential to unveil the process of neuronal sensitization and related, abnormal pain signaling.

Mutated CaV2.1 channels dysregulate CASK/P2X3 signaling in mouse trigeminal sensory neurons of R192Q Cacna1a knock-in mice.

BACKGROUND: ATP-gated P2X3 receptors of sensory ganglion neurons are important transducers of pain as they adapt their expression and function in response to acute and chronic nociceptive signals. The present study investigated the role of calcium/calmodulin-dependent serine protein kinase (CASK) in controlling P2X3 receptor expression and function in trigeminal ganglia from Cacna1a R192Q-mutated knock-in (KI) mice, a genetic model for familial hemiplegic migraine type-1. RESULTS: KI ganglion neurons showed more abundant CASK/P2X3 receptor complex at membrane level, a result that likely originated from gain-of-function effects of R192Q-mutated CaV2.1 channels and downstream enhanced CaMKII activity. The selective CaV2.1 channel blocker ω-Agatoxin IVA and the CaMKII inhibitor KN-93 were sufficient to return CASK/P2X3 co-expression to WT levels. After CASK silencing, P2X3 receptor expression was decreased in both WT and KI ganglia, supporting the role of CASK in P2X3 receptor stabilization. This process was functionally observed as reduced P2X3 receptor currents. CONCLUSIONS: We propose that, in trigeminal sensory neurons, the CASK/P2X3 complex has a dynamic nature depending on intracellular calcium and related signaling, that are enhanced in a transgenic mouse model of genetic hemiplegic migraine.

Calcium/calmodulin-dependent serine protein kinase (CASK) is a new intracellular modulator of P2X3 receptors.

ATP-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.

TNFα levels and macrophages expression reflect an inflammatory potential of trigeminal ganglia in a mouse model of familial hemiplegic migraine.

Latent changes in trigeminal ganglion structure and function resembling inflammatory conditions may predispose to acute attacks of migraine pain. Here, we investigated whether, in trigeminal sensory ganglia, cytokines such as TNFα might contribute to a local inflammatory phenotype of a transgenic knock-in (KI) mouse model of familial hemiplegic migraine type-1 (FHM-1). To this end, macrophage occurrence and cytokine expression in trigeminal ganglia were compared between wild type (WT) and R192Q mutant Ca(V)2.1 Ca(2+) channel (R192Q KI) mice, a genetic model of FHM-1. Cellular and molecular characterization was performed using a combination of confocal immunohistochemistry and cytokine assays. With respect to WT, R192Q KI trigeminal ganglia were enriched in activated macrophages as suggested by their morphology and immunoreactivity to the markers Iba1, CD11b, and ED1. R192Q KI trigeminal ganglia constitutively expressed higher mRNA levels of IL1ß, IL6, IL10 and TNFα cytokines and the MCP-1 chemokine. Consistent with the report that TNFα is a major factor to sensitize trigeminal ganglia, we observed that, following an inflammatory reaction evoked by LPS injection, TNFα expression and macrophage occurrence were significantly higher in R192Q KI ganglia with respect to WT ganglia. Our data suggest that, in KI trigeminal ganglia, the complex cellular and molecular environment could support a new tissue phenotype compatible with a neuroinflammatory profile. We propose that, in FHM patients, this condition might contribute to trigeminal pain pathophysiology through release of soluble mediators, including TNFα, that may modulate the crosstalk between sensory neurons and resident glia, underlying the process of neuronal sensitisation.

Effects of LPS on P2X3 receptors of trigeminal sensory neurons and macrophages from mice expressing the R192Q Cacna1a gene mutation of familial hemiplegic migraine-1.

A knockin (KI) mouse model with the R192Q missense mutation in the Cacna1a gene commonly detected in familial hemiplegic migraine was used to study whether trigeminal ganglia showed a basal inflammatory profile that could be further enhanced by the lipopolysaccharide (LPS) toxin. Adenosine-5'-triphosphate (ATP)-gated purinergic ionotropic receptor 3 (P2X3) currents expressed by the large majority of trigeminal sensory neurons were taken as functional readout. Cultured R192Q KI trigeminal ganglia showed higher number of active macrophages, basal release of tumor necrosis factor alpha (TNFα), and larger P2X3 receptor currents with respect to wild type (WT) cells. After 5 h application of LPS in vitro, both WT and R192Q KI cultures demonstrated significant increase in macrophage activation, very large rise in TNFα mRNA content, and ambient protein levels together with fall in TNFα precursor, suggesting potent release of this inflammatory mediator. Notwithstanding the unchanged expression of P2X3 receptor protein in WT or R192Q KI cultures, LPS evoked a large rise in WT neuronal currents that recovered faster from desensitization. Basal R192Q KI currents were larger than WT ones and could not be further augmented by LPS. These data suggest that KI cultures had a basal neuroinflammatory profile that might facilitate the release of endogenous mediators (including ATP) to activate constitutively hyperfunctional P2X3 receptors and amplify nociceptive signaling by trigeminal sensory neurons.

Functional crosstalk in culture between macrophages and trigeminal sensory neurons of a mouse genetic model of migraine.

BACKGROUND: Enhanced activity of trigeminal ganglion neurons is thought to underlie neuronal sensitization facilitating the onset of chronic pain attacks, including migraine. Recurrent headache attacks might establish a chronic neuroinflammatory ganglion profile contributing to the hypersensitive phenotype. Since it is difficult to study this process in vivo, we investigated functional crosstalk between macrophages and sensory neurons in primary cultures from trigeminal sensory ganglia of wild-type (WT) or knock-in (KI) mice expressing the Cacna1a gene mutation (R192Q) found in familial hemiplegic migraine-type 1. After studying the number and morphology of resident macrophages in culture, the consequences of adding host macrophages on macrophage phagocytosis and membrane currents mediated by pain-transducing P2X3 receptors on sensory neurons were examined. RESULTS: KI ganglion cultures constitutively contained a larger number of active macrophages, although no difference in P2X3 receptor expression was found. Co-culturing WT or KI ganglia with host macrophages (active as much as resident cells) strongly stimulated single cell phagocytosis. The same protocol had no effect on P2X3 receptor expression in WT or KI co-cultures, but it largely enhanced WT neuron currents that grew to the high amplitude constitutively seen for KI neurons. No further potentiation of KI neuronal currents was observed. CONCLUSIONS: Trigeminal ganglion cultures from a genetic mouse model of migraine showed basal macrophage activation together with enhanced neuronal currents mediated by P2X3 receptors. This phenotype could be replicated in WT cultures by adding host macrophages, indicating an important functional crosstalk between macrophages and sensory neurons.

Regulation of P2X3 receptor structure and function.

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 endogenous 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.

Reactive oxygen species contribute to the promotion of the ATP-mediated proliferation of mouse skeletal myoblasts.

Reactive oxygen species (ROS) and extracellular adenosine 5'-triphosphate (ATP) participate in autocrine and paracrine regulation in skeletal muscle. However, the link between these two signaling systems is not well established. Here, we studied cell proliferation as a possible consequence of the trophic effect of ATP in cultured skeletal mouse myoblasts and we tested the possibility that low concentrations of ROS represent the intermediate signaling molecule mediating this effect. Exposure to 10 µM ATP increased proliferation of mouse myoblasts by ~20%. ATP also induced intracellular Ca(2+) oscillations, which were independent of extracellular Ca(2+). Both effects of ATP were prevented by suramin, a broad-spectrum purinergic P2 receptor antagonist. In contrast, the adenosine receptor blocker CGS-15943 did not modify the ATP-mediated effects. Consistent with this, adenosine per se did not change myoblast growth, indicating the direct action of ATP via P2 receptor activation. The proliferative effect of ATP was prevented after depletion of hydrogen peroxide (H(2)O(2)) by the peroxidase enzyme catalase. Low-micromolar concentrations of exogenous H(2)O(2) mimicked the stimulatory effect of ATP on myoblast growth. DCF imaging revealed ATP-induced catalase and DPI-sensitive ROS production in myoblasts. In conclusion, our results indicate that extracellular ATP controls mouse myoblast proliferation via induction of ROS generation.

Functional differences between ATP-gated human and rat P2X3 receptors are caused by critical residues of the intracellular C-terminal domain.

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.

P2X3 receptor expression by HEK cells conditions their survival.

On nociceptive neurons, one important mechanism to generate pain signals is the activation of P2X3 receptors, which are membrane proteins gated by extracellular ATP. In this work, we have studied the recovery of recombinant P2X3 receptor expression in human embryonic kidney (HEK) cells. Our data demonstrated that HEK cells were not permissive for stable P2X3 expression, since the significant time-dependent cell loss. In vivo treatment with P2X3 receptor antagonist limited the effect. The expression of a single P2X3 point mutant Y393A, also largely accelerated cell death. We suggest the requirements of a permissive intracellular molecular machinery for appropriate receptor expression. The present report suggests that despite HEK cells are often used as recombinant expression system for the study a variety of receptors function, they represent a limiting permissive environment for P2X3 receptors.

Lipid rafts control P2X3 receptor distribution and function in trigeminal sensory neurons of a transgenic migraine mouse model.

BACKGROUND: A genetic knock-in mouse model expressing the R192Q mutation of the α1-subunit of the Ca(V)2.1 channels frequently found in patients with familial hemiplegic migraine shows functional upregulation of ATP-sensitive P2X3 receptors of trigeminal sensory neurons that transduce nociceptive inputs to the brainstem. In an attempt to understand the basic mechanisms linked to the upregulation of P2X3 receptor activity, we investigated the influence of the lipid domain of these trigeminal sensory neurons on receptor compartmentalization and function. RESULTS: Knock-in neurons were strongly enriched with lipid rafts containing a larger fraction of P2X3 receptors at membrane level. Pretreatment with the Ca(V)2.1 channel blocker ω-agatoxin significantly decreased the lipid raft content of KI membranes. After pharmacologically disrupting the cholesterol component of lipid rafts, P2X3 receptors became confined to non-raft compartments and lost their functional potentiation typically observed in KI neurons with whole-cell patch-clamp recording. Following cholesterol depletion, all P2X3 receptor currents decayed more rapidly and showed delayed recovery indicating that alteration of the lipid raft milieu reduced the effectiveness of P2X3 receptor signalling and changed their desensitization process. Kinetic modeling could reproduce the observed data when slower receptor activation was simulated and entry into desensitization was presumed to be faster. CONCLUSIONS: The more abundant lipid raft compartment of knock-in neurons was enriched in P2X3 receptors that exhibited stronger functional responses. These results suggest that the membrane microenvironment of trigeminal sensory neurons is an important factor in determining sensitization of P2X3 receptors and could contribute to a migraine phenotype by enhancing ATP-mediated responses.