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Background: Cultured sensory neurons are a common experimental model to elucidate the molecular mechanisms of pain transduction typically involving activation of ATP-sensitive P2X or capsaicin-sensitive TRPV1 receptors. This applies also to trigeminal ganglion neurons that convey pain inputs from head tissues. Little is, however, known about the plasticity of these receptors on trigeminal neurons in culture, grown without adding the neurotrophin NGF which per se is a powerful algogen. The characteristics of such receptors after short-term culture were compared with those of ganglia. Furthermore, their modulation by chronically-applied serotonin or NGF was investigated.

The molecular mechanisms of migraine pain are incompletely understood, although migraine mediators such as NGF and calcitonin gene-related peptide (CGRP) are believed to play an algogenic role. Although NGF block is proposed as a novel analgesic approach, its consequences on nociceptive purinergic P2X receptors of trigeminal ganglion neurons remain unknown. We investigated whether neutralizing NGF might change the function of P2X 3 receptors natively coexpressed with NGF receptors on cultured mouse trigeminal neurons. Treatment with an NGF antibody (24 h) decreased P2X 3 receptor-mediated currents and Ca 2ϩ transients, an effect opposite to exogenously applied NGF. Recovery from receptor desensitization was delayed by anti-NGF treatment without changing desensitization onset. NGF neutralization was associated with decreased threonine phosphorylation of P2X 3 subunits, presumably accounting for their reduced responses and slower recovery. Anti-NGF treatment could also increase the residual current typical of heteromeric P2X 2/3 receptors, consistent with enhanced membrane location of P2X 2 subunits. This possibility was confirmed with cross-linking and immunoprecipitation studies. NGF neutralization also led to increased P2X 2e splicing variant at mRNA and membrane protein levels. These data suggest that NGF controlled plasticity of P2X 3 subunits and their membrane assembly with P2X 2 subunits. Despite anti-NGF treatment, CGRP could still enhance P2X 3 receptor activity, indicating separate NGF-or CGRP-mediated mechanisms to upregulate P2X 3 receptors. In an in vivo model of mouse trigeminal pain, anti-NGF pretreatment suppressed responses evoked by P2X 3 receptor activation. Our findings outline the important contribution by NGF signaling to nociception of trigeminal sensory neurons, which could be counteracted by anti-NGF pretreatment.

Background: We examined the role of membrane anchoring of voltage-gated calcium channel α2δ subunits.Results: We used a truncated α2δ-1 construct (α2δ-1ΔC-term), which still increases CaV2.1/β1b currents, despite being mainly secreted.Conclusion: The effect of α2δ-1ΔC-term on calcium currents does not involve secretion and subsequent re-binding to the plasma membrane.Significance: C-terminal membrane anchoring of α2δ is not essential for calcium current enhancement.

On sensory neurons, sensitization of P2X 3 receptors gated by extracellular ATP contributes to chronic pain. We explored the possibility that receptor sensitization may arise from down-regulation of an intracellular signal negatively controlling receptor function. In view of the structural modeling between the Src region phosphorylated by the C-terminal Src inhibitory kinase (Csk) and the intracellular C terminus domain of the P2X 3 receptor, we investigated how Csk might regulate receptor activity. Using HEK cells and the in vitro kinase assay, we observed that Csk directly phosphorylated the tyrosine 393 residue of the P2X 3 receptor and strongly inhibited receptor currents. On mouse trigeminal sensory neurons, the role of Csk was tightly controlled by the extracellular level of nerve growth factor, a known algogen. Furthermore, silencing endogenous Csk in HEK or trigeminal cells potentiated P2X 3 receptor responses, confirming constitutive Csk-mediated inhibition. The present study provides the first demonstration of an original molecular mechanism responsible for negative control over P2X 3 receptor function and outlines a potential new target for trigeminal pain suppression.ATP-activated P2X 3 receptors are expressed almost exclusively by mammalian sensory neurons to play an important role in the transduction of painful stimuli to the central nervous system (1). Activation of P2X 3 receptors by ATP released during acute and chronic pain is thought to send nociceptive signals to central pain-related networks (2). In view of the multitude of environmental stimuli normally reaching sensory terminals, the question then arises how inappropriate activation of P2X 3 receptors is normally prevented. This process may contribute to suppression of continuous pain sensation in conjunction with central synaptic inhibition.The molecular pathways triggered by algogenic substances and responsible for modulating P2X 3 receptor structure and function remain incompletely understood. This topic is of particular interest because it can provide original clues for novel approaches related to treat pain. The nerve growth factor, NGF, 2 is one of the most powerful endogenous substances which elicit pain and inflammation via the tyrosine kinase receptor TrkA (3). This neurotrophin stimulates an intracellular cascade that elicits PKC-dependent P2X 3 receptor phosphorylation with ensuing facilitation of receptor currents. Conversely, suppression of NGF signaling powerfully down-regulates P2X 3 receptor function (4). These observations are consistent with the raised NGF levels in acute or inflammatory pain conditions (3). The molecular mechanisms underlying these effects remain, however, unclear.A dynamic balance between tyrosine phosphorylation and dephosphorylation is a major factor controlling the activity of many neurotransmitter receptors (5). TrkA stimulation activates intracellular signaling including Src tyrosine kinases (6) that, in neurons, are important modulators of ligand-gated receptors like nicotinic (7), NMDA receptors ...

On nociceptive neurons, one important mechanism to generate pain signals is the activation of P2X 3 receptors, which are membrane proteins gated by extracellular ATP. In the presence of the agonist, P2X 3 receptors rapidly desensitize and then recover slowly. One unique property of P2X 3 receptors is the recovery acceleration by extracellular Ca 2؉ that can play the role of the gainsetter of receptor function only when P2X 3 receptors are desensitized. To study negatively charged sites potentially responsible for this action of Ca 2؉ , we mutated 15 non-conserved aspartate or glutamate residues in the P2X 3 receptor ectodomain with alanine and expressed such mutated receptors in human embryonic kidney cells studied with patch clamping. Unlike most mutants, D266A (P2X 3 receptor numbering) desensitized very slowly, indicating that this residue is important for generating desensitization. Recovery appeared structurally distinct from desensitization because E111A and D266A had a much faster recovery and D220A and D289A had a much slower one despite their standard desensitization. Furthermore, E161A, E187A, or E270A mutants showed lessened sensitivity to the action of extracellular Ca 2؉ , suggesting that these determinants were important for the effect of this cation on desensitization recovery. This study is the first report identifying several negative residues in the P2X 3 receptor ectodomain differentially contributing to the general process of receptor desensitization. At least one residue was important to enable the development of rapid desensitization, whereas others controlled recovery from it or the facilitating action of Ca 2؉ . Thus, these findings outline diverse potential molecular targets to modulate P2X 3 receptor function in relation to its functional state.P2X 3 receptors of nociceptive sensory neurons transduce the action of extracellular ATP into painful signals especially during chronic pain states (1). Similar to other ligand-gated ionotropic receptors, P2X 3 receptors undergo rapid, full desensitization in the continuous presence of their agonist (2). However, a distinctive property of P2X 3 receptors is the prompt re-attainment of function in high extracellular Ca 2ϩ solution that operates by facilitating recovery from desensitization (3, 4).Thus, Ca 2ϩ can exert a profound, rapid action on the ability of P2X 3 receptors to transmit sensory inputs to the central nervous system. However, the precise sites mediating the effect of Ca 2ϩ remain unknown and carry considerable interest for any attempts to manipulate transduction of pain signals.The large family of ionotropic ATP receptors (P2X 1-7 ) shares a similar topology that comprises two transmembrane domains joined by one large extracellular loop with 10 disulfide bonds and intracellular N-and C-terminal regions (2, 5, 6). The facilitating action of extracellular Ca 2ϩ is exclusively produced on desensitized P2X 3 receptors, perhaps via extracellular sites (3, 4). To identify the receptor region involved in this action and the amino acid sit...

As auxiliary subunits of voltage-gated Ca 2ϩ channels, the ␣ 2 ␦ proteins modulate membrane trafficking of the channels and their localization to specific presynaptic sites. Following nerve injury, upregulation of the ␣ 2 ␦-1 subunit in sensory dorsal root ganglion neurons contributes to the generation of chronic pain states; however, very little is known about the underlying molecular mechanisms. Here we show that the increased expression of ␣ 2 ␦-1 in rat sensory neurons leads to prolonged Ca 2ϩ responses evoked by membrane depolarization. This mechanism is coupled to Ca V 2.2 channel-mediated responses, as it is blocked by a -conotoxin GVIA application. Once initiated, the prolonged Ca 2ϩ transients are not dependent on extracellular Ca 2ϩ and do not require Ca 2ϩ release from the endoplasmic reticulum. The selective inhibition of mitochondrial Ca 2ϩ uptake demonstrates that ␣ 2 ␦-1-mediated prolonged Ca 2ϩ signals are buffered by mitochondria, preferentially activated by Ca 2ϩ influx through Ca V 2.2 channels. Thus, by controlling channel abundance at the plasma membrane, the ␣ 2 ␦-1 subunit has a major impact on the organization of depolarization-induced intracellular Ca 2ϩ signaling in dorsal root ganglion neurons.

Calcium signaling resulting from depolarization of neurons can trigger changes in transcription, and this response has been called excitation-transcription (E-T) coupling. In neurons, voltage-gated and ligand-gated calcium-permeable channels contribute to the increase in intracellular calcium. It appears that calcium signals mediated by specific voltage-gated calcium channels may have distinct roles in E-T coupling.