The upregulation of α2δ-1 subunit modulates activity-dependent Ca2+ signals in sensory neurons.

As auxiliary subunits of voltage-gated Ca(2+) channels, the α2δ proteins modulate membrane trafficking of the channels and their localization to specific presynaptic sites. Following nerve injury, upregulation of the α2δ-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 α2δ-1 in rat sensory neurons leads to prolonged Ca(2+) responses evoked by membrane depolarization. This mechanism is coupled to CaV2.2 channel-mediated responses, as it is blocked by a ω-conotoxin GVIA application. Once initiated, the prolonged Ca(2+) transients are not dependent on extracellular Ca(2+) and do not require Ca(2+) release from the endoplasmic reticulum. The selective inhibition of mitochondrial Ca(2+) uptake demonstrates that α2δ-1-mediated prolonged Ca(2+) signals are buffered by mitochondria, preferentially activated by Ca(2+) influx through CaV2.2 channels. Thus, by controlling channel abundance at the plasma membrane, the α2δ-1 subunit has a major impact on the organization of depolarization-induced intracellular Ca(2+) signaling in dorsal root ganglion neurons.

A CaV2.1 N-terminal fragment relieves the dominant-negative inhibition by an Episodic ataxia 2 mutant.

Episodic ataxia 2 (EA2) is an autosomal dominant disorder caused by mutations in the gene CACNA1A that encodes the pore-forming CaV2.1 calcium channel subunit. The majority of EA2 mutations reported so far are nonsense or deletion/insertion mutations predicted to form truncated proteins. Heterologous expression of wild-type CaV2.1, together with truncated constructs that mimic EA2 mutants, significantly suppressed wild-type calcium channel function, indicating that the truncated protein produces a dominant-negative effect (Jouvenceau et al., 2001; Page et al., 2004). A similar finding has been shown for CaV2.2 (Raghib et al., 2001). We show here that a highly conserved sequence in the cytoplasmic N-terminus is involved in this process, for both CaV2.1 and CaV2.2 channels. Additionally, we were able to interfere with the suppressive effect of an EA2 construct by mutating key N-terminal residues within it. We postulate that the N-terminus of the truncated channel plays an essential part in its interaction with the full-length CaV2.1, which prevents the correct folding of the wild-type channel. In agreement with this, we were able to disrupt the interaction between EA2 and the full length channel by co-expressing a free N-terminal peptide.

Calcium currents are enhanced by α2δ-1 lacking its membrane anchor.

The accessory α(2)δ subunits of voltage-gated calcium channels are membrane-anchored proteins, which are highly glycosylated, possess multiple disulfide bonds, and are post-translationally cleaved into α(2) and δ. All α(2)δ subunits have a C-terminal hydrophobic, potentially trans-membrane domain and were described as type I transmembrane proteins, but we found evidence that they can be glycosylphosphatidylinositol-anchored. To probe further the function of membrane anchoring in α(2)δ subunits, we have now examined the properties of α(2)δ-1 constructs truncated at their putative glycosylphosphatidylinositol anchor site, located before the C-terminal hydrophobic domain (α(2)δ-1ΔC-term). We find that the majority of α(2)δ-1ΔC-term is soluble and secreted into the medium, but unexpectedly, some of the protein remains associated with detergent-resistant membranes, also termed lipid rafts, and is extrinsically bound to the plasma membrane. Furthermore, heterologous co-expression of α(2)δ-1ΔC-term with Ca(V)2.1/ß1b results in a substantial enhancement of the calcium channel currents, albeit less than that produced by wild-type α(2)δ-1. These results call into question the role of membrane anchoring of α(2)δ subunits for calcium current enhancement.

Neutralization of nerve growth factor induces plasticity of ATP-sensitive P2X3 receptors of nociceptive trigeminal ganglion neurons.

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 P2X3 receptors natively coexpressed with NGF receptors on cultured mouse trigeminal neurons. Treatment with an NGF antibody (24 h) decreased P2X3 receptor-mediated currents and Ca2+ 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 P2X3 subunits, presumably accounting for their reduced responses and slower recovery. Anti-NGF treatment could also increase the residual current typical of heteromeric P2X2/3 receptors, consistent with enhanced membrane location of P2X2 subunits. This possibility was confirmed with cross-linking and immunoprecipitation studies. NGF neutralization also led to increased P2X2e splicing variant at mRNA and membrane protein levels. These data suggest that NGF controlled plasticity of P2X3 subunits and their membrane assembly with P2X2 subunits. Despite anti-NGF treatment, CGRP could still enhance P2X3 receptor activity, indicating separate NGF- or CGRP-mediated mechanisms to upregulate P2X3 receptors. In an in vivo model of mouse trigeminal pain, anti-NGF pretreatment suppressed responses evoked by P2X3 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.

Delayed upregulation of ATP P2X3 receptors of trigeminal sensory neurons by calcitonin gene-related peptide.

Recent evidence indicates a key role for the neuropeptide calcitonin gene-related peptide (CGRP) in migraine pain, as demonstrated by the strong analgesic action of CGRP receptor antagonists, although the mechanisms of this effect remain unclear. Most trigeminal nociceptive neurons releasing CGRP also express ATP-activated purinergic P2X3 receptors to transduce pain. To understand whether the CGRP action involves P2X3 receptor modulation, the model of trigeminal nociceptive neurons in culture was used to examine the long-term action of this peptide. Although 79% of CGRP-binding neurons expressed P2X3 receptors, acute application of CGRP did not change P2X3 receptor function. Nevertheless, after 1 h of CGRP treatment, strong enhancement of the amplitude of P2X3 receptor currents was observed together with accelerated recovery from desensitization. Receptor upregulation persisted up to 10 h (despite CGRP washout), was accompanied by increased P2X3 gene transcription, and was fully prevented by the CGRP antagonist CGRP(8-37). Surface biotinylation showed CGRP augmented P2X3 receptor expression, consistent with confocal microscopy data indicating enhanced P2X3 immunoreactivity beneath the neuronal membrane. These results suggest that CGRP stimulated trafficking of P2X3 receptors to the cell-surface membrane. Using pharmacological tools, we demonstrated that this effect of CGRP was dependent on protein kinase A and PKC activation and was prevented by the trafficking inhibitor brefeldin A. Capsaicin-sensitive TRPV1 vanilloid receptors were not upregulated. The present data demonstrate a new form of selective, slow upregulation of nociceptive P2X3 receptors on trigeminal neurons by CGRP. This mechanism might contribute to pain sensitization and represents a model of neuronal plasticity in response to a migraine mediator.

Comparison of P2X and TRPV1 receptors in ganglia or primary culture of trigeminal neurons and their modulation by NGF or serotonin.

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. RESULTS: Rat or mouse neurons in culture mainly belonged to small and medium diameter neurons as observed in sections of trigeminal ganglia. Real time RT-PCR, Western blot analysis and immunocytochemistry showed upregulation of P2X(3) and TRPV1 receptors after 1-4 days in culture (together with their more frequent co-localization), while P2X(2) ones were unchanged. TRPV1 immunoreactivity was, however, lower in mouse ganglia and cultures. Intracellular Ca(2+) imaging and whole-cell patch clamping showed functional P2X and TRPV1 receptors. Neurons exhibited a range of responses to the P2X agonist alpha, beta-methylene-adenosine-5'-triphosphate indicating the presence of homomeric P2X(3) receptors (selectively antagonized by A-317491) and heteromeric P2X(2/3) receptors. The latter were observed in 16 % mouse neurons only. Despite upregulation of receptors in culture, neurons retained the potential for further enhancement of P2X(3) receptors by 24 h NGF treatment. At this time point TRPV1 receptors had lost the facilitation observed after acute NGF application. Conversely, chronically-applied serotonin selectively upregulated TRPV1 receptors rather than P2X(3) receptors. CONCLUSION: Comparing ganglia and cultures offered the advantage of understanding early adaptive changes of nociception-transducing receptors of trigeminal neurons. Culturing did not prevent differential receptor upregulation by algogenic substances like NGF or serotonin, indicating that chronic application led to distinct plastic changes in the molecular mechanisms mediating pain on trigeminal nociceptors.

L-type calcium channels: on the fast track to nuclear signaling.

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.

The C-terminal Src inhibitory kinase (Csk)-mediated tyrosine phosphorylation is a novel molecular mechanism to limit P2X3 receptor function in mouse sensory neurons.

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.

Identification of negative residues in the P2X3 ATP receptor ectodomain as structural determinants for desensitization and the Ca2+-sensing modulatory sites.

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 gain-setter 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.