Familial hemiplegic migraine CaV2.1 channel mutation R192Q enhances ATP-gated P2X3 receptor activity of mouse sensory ganglion neurons mediating trigeminal pain

Background The R192Q mutation of the CACNA1A gene, encoding for the α1 subunit of voltage-gated P/Q Ca2+ channels (Cav2.1), is associated with familial hemiplegic migraine-1. We investigated whether this gain-of-function mutation changed the structure and function of trigeminal neuron P2X3 receptors that are thought to be important contributors to migraine pain. Results Using in vitro trigeminal sensory neurons of a mouse genetic model knockin for the CACNA1A R192Q mutation, we performed patch clamp recording and intracellular Ca2+ imaging that showed how these knockin ganglion neurons generated P2X3 receptor-mediated responses significantly larger than wt neurons. These enhanced effects were reversed by the Cav2.1 blocker ω-agatoxin. We, thus, explored intracellular signalling dependent on kinases and phosphatases to understand the molecular regulation of P2X3 receptors of knockin neurons. In such cells we observed strong activation of CaMKII reversed by ω-agatoxin treatment. The CaMKII inhibitor KN-93 blocked CaMKII phosphorylation and the hyperesponsive P2X3 phenotype. Although no significant difference in membrane expression of knockin receptors was found, serine phosphorylation of knockin P2X3 receptors was constitutively decreased and restored by KN-93. No change in threonine or tyrosine phosphorylation was detected. Finally, pharmacological inhibitors of the phosphatase calcineurin normalized the enhanced P2X3 receptor responses of knockin neurons and increased their serine phosphorylation. Conclusions The present results suggest that the CACNA1A mutation conferred a novel molecular phenotype to P2X3 receptors of trigeminal ganglion neurons via CaMKII-dependent activation of calcineurin that selectively impaired the serine phosphorylation state of such receptors, thus potentiating their effects in transducing trigeminal nociception.


Background
Migraine is a common debilitating neurovascular disorder with complex etiology that is clinically divided into two main subtypes based on the absence or presence of an aura that is characterized by transient visual, sensory and/or speech related neurological symptoms [1]. Most molecular genetic insight in the pathophysiology of migraine comes from studies of a rare monogenic subtype of migraine with aura, called Familial Hemiplegic Migraine type 1 (FHM-1) [2]. FHM-1 is due to mutations in the α1 subunit of voltage-gated Ca V 2.1 (P/Qtype) Ca 2+ channels [3]. Transgenic knock-in (KI) mice carrying a FHM-1 glutamine for arginine (R192Q) mutation revealed increased glutamate release in the cortex that explains their increased susceptibility to cortical spreading depression (CSD) [4,5], the electrophysiological correlate of the human migraine aura [6]. Patch clamp analysis of transfected neurons expressing human R192Q-mutant α1 protein revealed facilitated activation of Ca V 2.1 channels and increased Ca 2+ influx as the underlying molecular mechanism [7]. Whereas there is little doubt that CSD causes the aura, the mechanisms leading migraine headache are less clear. The current view is that the headache is caused by the trigeminovascular system that releases a number of "migraine mediators" activating nociceptive receptors expressed by trigeminal neurons [8]. Previous studies from our group have indicated that migraine mediators (e.g., nerve growth factor and calcitonin gene-related peptide) can persistently sensitize trigeminal sensory neurons [9][10][11] and that enhanced pain perception involves activation of neuronal P2X 3 receptors by extracellular ATP [12]. The role of trigeminal sensory neuron P2X 3 receptors as important contributors to migraine pain has recently been discussed [13][14][15][16].
The present study used R192Q KI mice to investigate peripheral pain mechanisms at the level of trigeminal sensory neurons to explore whether they show evidence of enhanced pain transduction. Since Ca V 2.1 Ca 2+ channels normally are expressed by trigeminal sensory neurons and contribute by approximately 40% to voltagegated Ca 2+ influx [17], we assessed whether the R192Q mutation might introduce a cascade of Ca 2+ -dependent signals controlling expression and function of the main pain receptors P2X 3 and TRPV1 [18][19][20][21]. Although we observed unchanged activity of TRPV1 receptors in mutant mouse sensory neurons, we detected a significant increase in P2X 3 receptor activity that was characterized by combining electrophysiological and molecular biology techniques.

Ca V 2.1 R192Q KI trigeminal neurons show enhanced membrane currents mediated by P2X 3 receptors
Because the large majority of trigeminal ganglion neurons typically express P2X 3 receptors [22] that are implicated in pain transducing mechanisms in migraine [8], we investigated whether the Ca V 2.1 channel α1 subunit R192Q mutation affected P2X 3 receptor function. Fig.  1A shows examples of current traces induced by a 2-s application of the selective P2X 3 receptor agonist α,β-meATP (10 μM) to WT and R192Q KI neurons. On WT neurons, the fast-developing inward current was on average -354 ± 28 pA (n = 154) and then fully desensitized during agonist application, a characteristic typical of currents mediated by P2X 3 receptors [18]. On R192Q KI neurons, the peak amplitude of α,β-meATP-evoked current was, on average, -547 ± 32 pA (n = 183: p = 0.035 from WT) with subsequent full desensitization. Fig. 1B shows average concentration-response plots for WT (n = 18) and R192Q KI (n = 17) neurons tested with α,β-meATP. The R192Q KI plot revealed a significantly larger maximal response compared to WT without changing the α,β-meATP potency (EC 50 = 6.9 ± 1.1 μM for WT; 4.3 ± 0.8 μM for KI) or the Hill coefficient (0.86 ± 0.34 for WT; 1.11 ± 0.34 for KI). Other parameters of P2X 3 receptor function, such as current rise-time (τ on ), desensitization onset (τ fast ), and recovery from desensitization at 30 s interpulse interval, were not significantly different between genotypes (Fig. 1C). Mouse trigeminal neurons usually express a low level of heteromeric P2X 2/3 receptors mediating a sustained inward current following the initial transient peak [22]. In the present study no significant difference in the amplitude or occurrence of heteromeric responses (measured at steady state) was observed between WT and KI neurons (-60 ± 6 pA in WT, n = 53; 34% incidence and -61 ± 8 pA in KI, n = 47; 25% incidence). Fig. 1D,E shows the amplitude distribution of current responses evoked by α,β-meATP (10 μM) in WT and R192Q KI neurons (n = 454 and n = 414, respectively). Because of the heterogeneity of trigeminal sensory neurons in culture, responses were further analyzed in terms of cell capacitance, which reflects the cell size. In view of the distribution of P2X 3 receptor expression [22], we restricted our sampling to neurons that are larger than 15 μm in diameter. Fig. 1E shows that enhanced responses to α,β-meATP were observed in a population of neurons with 15-35 pA/pF current density value that matched the somatic size profile shown in Fig. 1D. This upregulation of P2X 3 receptor function was not due to increased receptor synthesis and/or expression at membrane level as indicated by real time RT-PCR data ( Fig. 2A) and membrane biotinylation results ( Fig. 2B; p > 0.05) performed in accordance with our previous studies [9,10]. Current responses induced by 1 μM capsaicin, a selective agonist of TRPV1 channels, were not significantly different between WT (-38 ± 3 pA; n = 64) and KI (-40 ± 4 pA; n = 80) neurons as exemplified by the middle panel of Fig. 2C. mRNA synthesis and immunohistochemical expression of TRPV1 and P2X 2 receptors was similar between WT and KI (Fig. 2C,D).

Intracellular Ca 2+ transients of Ca V 2.1 R192Q neurons
Without perturbing intracellular Ca 2+ homeostasis with the whole-cell patch clamp method, we tested the Figure 1 Functional characterization and expression of P2X 3 receptors in WT and R192Q KI trigeminal ganglion neurons. A, Representative current traces induced by α,β-meATP (10 μM, 2-s application) on WT and KI neurons. B, Dose-response curves for WT (n = 18, filled circles) and KI (n = 17, open circles) cells. * p = 0.04, ** p = 0.035, ***p = 0.03. Note larger responses of α,β-meATP-mediated currents on KI neurons with respect to WT, as the dose-effect plot is shifted upwards for KI neurons. C, Rise time (left; expressed as τ on calculated on the 10-90% response rise), desensitization onset (middle; expressed as the first time constant, τ fast , of current decay) and recovery from desensitization (right; expressed as% of control amplitude in a paired pulse agonist application) of P2X 3 receptor currents are similar for WT and KI neurons. All responses were evoked by α,β-meATP (10 μM, 2 s). n = 154 for WT and n = 183 for KI. D, Histograms show peak amplitude distribution of α,β-meATP (10 μM)-induced P2X 3 currents for WT (n = 414, open bars) and KI (n = 454, filled bars). E, Histograms show the distribution of current density (i.e., current amplitude normalized with respect to the neuronal capacitance, pA/pF) for WT (n = 414, open bars) and KI (n = 454, filled bars) neurons, indicating significantly higher α,β-meATP-evoked KI responses over a span of 15-35 pA/pF values compared to WT. contribution of P/Q-type channels to intracellular Ca 2+ levels following depolarizing pulses of K + or α,β-meATP. This approach enabled us to test the excitability (in terms of intracellular Ca 2+ transients) of trigeminal sensory neurons [10]. To this end, we compared Ca 2+ transients evoked by these stimuli before and after 30 min application of ω-agatoxin (200 nM) to WT or KI neurons. It is noteworthy that the size of the intracellular Ca 2+ rise induced by α,β-meATP was significantly smaller than the one elicited by K + presumably because the amplitude of neuronal membrane depolarization (and its duration) evoked by K + was much larger and, thus activated a wider population of Ca 2+ channels [10,22]. This finding is corroborated by the data shown in Fig. 3A.
Despite comparable levels of dye loading and similar responses to rather high (50 mM) pulse application of K + (not shown), we consistently found that KI neurons produced significantly (p = 0.005) larger Ca 2+ transients (Fig. 3B) when stimulated with a lower concentration (20 mM) of K + . We also observed that 78% of KI neurons (n = 35/45) were sensitive to ω-agatoxin inhibition (i.e., showed a response decrease of >10%), whereas in the case of WT neurons, only 50% (n = 14/28) were sensitive to this toxin. Fig. 3B indicates that, following application of ω-agatoxin, the depression of Ca 2+ transients induced K + was proportionally similar in WT and KI neurons (27 ± 4%, n = 14; and 30 ± 3%, n = 35, respectively), indicating that the toxin-sensitive contribution to the global Ca 2+ transient was not affected by the Ca V 2.1 mutation that is reported to affect only the activation threshold of P/Q-type channels [4,5].
Immunohistochemical analysis of trigeminal ganglion cultures showed that the number of immunopositive neurons for Ca V 2.1 channels and P2X 3 receptors was similar between genotypes (Fig. 3E). We also investigated (by double immunofluorescence with anti-P2X 3 and anti-Ca V 2.1 antibodies) the fraction of Ca V 2.1 immunoreactive neurons expressing P2X 3 receptors as well: this number amounted to 68 ± 4% for WT and 66 ± 3% for KI (n = 7, p > 0.05) for small-medium neurons (<20 μm), suggesting a large, though not complete, occurrence of protein co-expression. In fact, when we examined the number of P2X 3 immunoreactive neurons that were positive for Ca V 2.1, we found 42 ± 3% and 43 ± 4% for WT and KI, respectively (n = 4). These observations were consistent with a functional interaction between these proteins in a subpopulation of sensory neurons.
High CaMKII activity confers enhanced P2X 3 receptor phenotype to KI trigeminal neurons To discover the mechanisms responsible for the enhanced activity of trigeminal P2X 3 receptors despite no change in their expression levels, we studied whether the molecular regulation of the receptors was changed by the R192Q gain-of-function mutation of Ca V 2.1 channels. Our previous studies have demonstrated that changes in P2X 3 receptor activity could be brought about by pharmacological manipulations affecting the phosphorylation state of the receptor, rather than its expression level [9,23,12]. Thus, it seemed likely that the origin of the augmented P2X 3 receptor activity of KI neurons might reside in a changed phosphorylation state of the intracellular protein domains. It is likely that increased Ca 2+ influx via mutated Ca V 2.1 channels may regulate multiple intracellular Ca 2+ -dependent signalling mechanisms via altering the physiological function of many Ca 2+ binding proteins, amongst which CaMKII is a likely contributor [24,25]. For these reasons, immunofluorescence experiments were performed with antibodies selective for active CaMKII phosphorylated at Thr286 [11] that showed a larger number of immunopositive trigeminal neurons in culture (45 ± 5%), and intact ganglia (36 ± 6%) from KI mice (Fig. 4A). By comparison, CaMKII immunoreactivity was detected only in 15 ± 4 and 9 ± 3% of neurons in cultures or intact ganglia from WT mice (n = 3-5; p = 0.03; see example in Fig. 5A). Previous experiments have confirmed the antibody selectivity by showing that its signal was blocked by the CaMK inhibitor KN-93 in immunofluorence and western blotting [11].
Immunoblotting performed on protein extracts from trigeminal ganglia and cultures further demonstrated that, despite the lack of change in total CaMKII level between WT and KI (n = 4, p > 0.05; Fig. 4B), the active fraction of CaMKII (T286-phosphorylated) was significantly larger in KI than WT ( Fig. 4B; n = 4, p = 0.03). We also observed that the larger activation of CaMKII in KI samples was due to the presence of mutated Ca V 2.1 channel activity, because pretreatment with ωagatoxin (200 nM, 24 h) significantly (p = 0.027; n = 4) reduced the CaMKII signal (Fig. 4B). Increased CaMKII activity in KI neurons was not apparently accompanied by an overactivation of the CREB transcription factor compared with that in WT neurons (Fig. 4C, see [11]).

R192Q KI neurons lack constitutive serine phosphorylation of P2X 3 receptors
The observation of strong CaMKII activation in KI neurons led us to examine the phosphorylation state of their P2X 3 receptors. Hence, we screened immunoprecipitated P2X 3 receptors from WT and KI trigeminal neurons with antibodies against phosphorylated residues. Fig. 5A,B shows that P2X 3 receptor serine-phosphorylation was significantly reduced in extracts of trigeminal cultures (n = 5, p = 0.02) from KI compared with WT. Although significant, this difference was not very large probably because the number of P2X 3 receptor expressing neurons that also expressed Ca V 2.1 channels was less than 50% of the total population whose contribution diluted the overall change (see Fig. 3F). Notwithstanding this issue, the dissimilar level of P2X 3 receptor serinephosphorylation was also found between KI and WT ganglia (n = 3, p = 0.04; data not shown). It is noteworthy that the antibodies we used for studying the phosphorylated aminoacid residues were not sequencespecific and, therefore, did not allow us to identify the consensus sequence of the receptor domain responsible for conferring the upregulated phenotype of KI neurons.
The change in P2X 3 receptor serine phosphorylation of KI neurons was linked to high CaMKII activity because pre-application of the selective CaMKII inhibitor KN-93 (5 μM; 90 min; Fig. 5D) to KI cultures reversed this phenotype and restored a level of serine phosphorylation analogous to the WT one. Conversely, KN-93 had no effect on WT serine P2X 3 phosphorylation probably because of the constitutively low level of CaMKII activity in these cells (see Fig. 4A,B). Patchclamp data validated our findings because, whereas KN-93 did not significantly change membrane currents evoked by α,β-meATP (10 μM) from WT neurons (n = 17), it significantly (p = 0.039) attenuated currents recorded from KI neurons (n = 22; Fig. 5E).
These data suggest that Ca V 2.1 channel-mediated activation of CaMKII exerted a positive effect on P2X 3 receptor current of KI neurons by inhibiting serine phosphorylation.

Serine dephosphorylated P2X 3 receptors of KI neurons show increased function
We have recently observed P2X 3 receptor serine phosphorylation by Cdk5 to be a powerful negative regulator of receptor function [26]. In keeping with the enhanced functional responses of KI neurons, we compared the expression of Cdk5 in WT and KI neurons. The percent of trigeminal neurons expressing Cdk5 was similar in WT and KI (61 ± 2%) cultured neurons (n = 3, p > 0.05). Western blotting of membrane fractions indicated a significant overall decrease of Cdk5 expression in lysates from KI ganglia (0.7 ± 0.025 fold change versus WT; n = 4, p = 0.04; Fig. 6A) together with lower P2X 3 / Cdk5 co-immunoprecipitation (0.8 ± 0.02 fold change in KI versus WT; n = 3, p = 0.03; Fig. 6B) despite analogous levels of total Cdk5 expression (see Fig. 6A), suggesting a different compartmentalisation of this molecules in KI neurons.
The observation of decreased P2X 3 receptor serine phosphorylation in KI neurons (see Fig. 5A) prompted us to consider the potential role of phosphatases contributing to this effect. The balance between phosphorylation and dephosphorylation is a primary means for rapid regulation of a variety of neuronal functions, such as membrane excitability, neurotransmitter release, and receptor function [27]. Multiple families of serine/threonine phosphatases have been identified in the brain, including PP1, PP2A, PP2B, and PP2C families [28][29][30], that are closely linked to intracellular Ca 2+ . Calcineurin (PP2B) is one major Ca 2+ -dependent phosphatase that controls the activity of membrane channels [31]. Although cell-free assays show that CaMKII can inhibit calcineurin [32], in neuronal cell lines both CaMKII and calcineurin are activated by Ca 2+ influx and are necessary for Ca 2+ -dependent signalling pathways [33]. Hence, we tested calcineurin involvement in P2X 3 receptor phosphorylation state and function by incubating trigeminal neurons with the calcineurin inhibitor FK-506 (5 μM, 30 min). This treatment reversed the KI phenotype by decreasing the P2X 3 receptor current (44 ± 5%, n = 20, p = 0.03; Fig. 6C) and in KI increased P2X 3 receptor serine phosphorylation (134 ± 16%, n = 4, p = 0.03; Fig. 6D). To confirm the origin of this effect, we applied the calcineurin inhibitor peptide (100 μM) via the patch pipette during recording from WT or KI neurons. In WT neurons recorded with the peptide inhibitor filled pipettes there was a delayed (6-10 min) rise in α,β-meATP peak currents (on average -427 ± 89 pA; n = 8 vs -269 ± 35; n = 20; p < 0.05), whereas for KI neurons recorded with the same protocol there was late decrease (-231 ± 28; n = 11 vs -477 ± 97, n = 8; p < 0.01). These experiments suggest that calcineurin activity plays an important role in regulating P2X 3 receptor currents. Although calcineurin has been shown to facilitate desensitisation of P2X 3 receptors expressed by oocytes [34], on native P2X 3 receptors of mouse ganglion neurons, application of the calcineurin inhibitor FK-506 did not change the time constant of fast current decay (τ fast ) in either WT or KI neurons ( Table 1).

Discussion
The main finding of the present study is the novel report of the phenotype of trigeminal ganglion neurons from KI mice expressing a Ca V 2.1 α1 channel subunit mutation that was previously found in FHM-1 patients [3]. KI neurons showed enhanced P2X 3 receptor activity with decreased serine phosphorylation. This finding was associated with an increased activation of CaMKII and stronger a Ca V 2.1 (P/Q-type) mediated rise in intracellular Ca 2+ . We envisage that such a phenotype will facilitate trigeminal pain. To the best of our knowledge, our results provide the first demonstration of P2X 3 receptor contribution to an animal model of a neurological disease.

Functional characteristics of the R192Q KI mouse model
ATP-gated P2X 3 and capsaicin-sensitive TRPV1 receptors are representative of the two main classes of pain transducers in trigeminal neurons [22]. We observed potentiation of P2X 3 receptor activity in trigeminal neurons from KI mice without changes in potency or desensitization of these receptors, and with no altered occurrence (or size) of heteromeric P2X 2/3 responses. Likewise, no change in P2X 2 subunit expression was detected. Responses to the TRPV1 receptor agonist capsaicin were also unaffected. Thus, our data indicate a major gain of function of P2X 3 receptors without their concomitant overexpression at the plasma membrane or a larger number of P2X 3 receptor expressing neurons. We set out to study whether such changes in P2X 3 receptor function were indeed attributable to the gain of function of R192Q-mutated Ca V 2.1 Ca 2+ channels in trigeminal neurons.
We showed that a subpopulation of P2X 3 expressing neurons also expressed Ca V 2.1 channels, thus providing ample opportunity for their crosstalk. Indeed, intracellular Ca 2+ imaging showed that KI neurons had stronger Ca 2+ transients evoked by α,β-meATP with a larger component blocked by the Ca V 2.1 channel antagonist ω-agatoxin, indicating that this effect was probably attributable to overactivity of such mutated channels. Furthermore, ω-agatoxin reversed the mutated phenotype of P2X 3 receptors to WT, providing a functional link between overactive Ca V 2.1 and P2X 3 channels. It is, however, noteworthy that in ganglia in situ or in culture conditions, we observed no evidence for a deleterious action by these enhanced channel activities as the number of neurons as well as the size distribution remained similar to that in WT neurons.
Hence, the present data demonstrated a novel gain-offunction produced by the R192Q mutation on P2X 3 receptors of trigeminal ganglion neurons, that complements the synaptic upregulation reported for brain neurons [4,5].

Molecular mechanisms involved in enhanced P2X 3 sensitivity of R192Q KI neurons
Interestingly, KI neurons from intact trigeminal ganglia and in culture showed elevated basal activation of CaM-KII (phosphorylation of its 286 threonine residue [28,35]). CaMKII is a multifunctional kinase involved in the regulation of many cell processes and its action is dependent on intracellular Ca 2+ changes (for review see [25]). Although we did not systematically observe spontaneous Ca 2+ transients in cultured trigeminal neurons, our data suggest that in KI neurons ongoing Ca 2+ signalling was facilitated because ω-agatoxin blocked the enhanced CaMKII activity.
We examined several intracellular targets which could be modulated by the increased CaMKII activity. Regulation of protein phosphorylation state is a common way to modify the function of pain receptors [36]. We observed that, under unchallenged conditions, the basal phosphorylation state of threonine and tyrosine in P2X 3 receptors of KI neurons was unchanged compared with WT neurons. Likewise, there was no change in basal CREB phosphorylation. However, we did observe decreased serine phosphorylation of P2X 3 receptors as the molecular phenotype of Ca V 2.1-immunoreactive neurons in KI ganglia and in culture. This phenotype was strongly dependent on Ca V 2.1 channel activity and CaMKII function, because in KI neurons, both ω-agatoxin and CaMKII inhibitor KN-93 restored the level of serine phosphorylation to that of WT, and returned P2X 3 receptor responses to basal levels. Future experiments are necessary to identify the mechanisms regulating the basal serine phosphorylation of P2X 3 receptors of WT cells.

Molecular mechanisms controlling P2X 3 receptor potentiation of KI neurons
The inverse correlation between P2X 3 serine phosphorylation and P2X 3 activity (i.e., reduced serine phosphorylation associated with enhanced P2X 3 receptor function) suggests that P2X 3 serine dephosphorylation could be an important molecular contributor to receptor operation, particularly in view of the fact that Cdk5 (a kinase associated with negative P2X 3 receptor regulating function [26]) was less associated with P2X 3 receptors in neuronal membranes of KI neurons.
We posited that, in WT neurons (either in situ or in culture), serine phosphorylation likely depended on the balance between kinases and phosphatases, in which kinases were perhaps predominant and exerting a tight regulatory role because of the consistently high basal level of serine phosphorylation we observed. On the contrary, in KI neurons such a regulation was impaired (as shown by decreased serine phosphorylation) and could be restored after treatment with calcineurin inhibitors like FK-506 [37][38][39] or the calcineurin peptide autoinhibitor [40]. This observation indicated that the molecular phenotype likely involved phosphatase overactivity because, when the latter was pharmacologically blocked, endogenous kinase activity readily restored serine phosphorylation. Since FK-506 is also known to inhibit ryanodine receptors to favour intracellular Ca 2+ leakage [39], it was useful to confirm similar observations with the calcineurin peptide autoinhibitor. The precise molecular link between Cdk5 and calcineurin activity in terms of P2X 3 receptor function of trigeminal neurons remains to be clarified. Upstream of this interaction, it is, however, interesting that Cdk5 is reported to downregulate P/Q-type channels [41] since in our KI model we found evidence for gain of function of such channels together with low Cdk5 membrane expression. Furthermore, Cdk5 activity is also negatively associated with activation of neuronal CaMKII [42], in analogy with the data reported in the present study. As far as the relationship between Cdk5 and calcineurin is concerned, these proteins appear to possess opposite actions on a number of intracellular neuronal proteins involved in neurotransmitter release [43,44]. Their contrasting actions are in keeping with the effects observed by us and outline even a potential process to control the release of excitatory neurotransmitter by sensory neurons, a phenomenon which might enhance trigeminal pain sensitization.

The phosphorylation state of P2X 3 receptors is important to control ATP-mediated responses of nociceptive neurons
Potentiation of P2X 3 receptor responses of WT trigeminal neurons observed after application of calcineurin blockers was not accompanied by a change in receptor serine phosphorylation, that remained at its constitutive level. However, calcineurin inhibition enhanced P2X 3 receptor threonine phosphorylation, a process that was previously associated with PKC-dependent potentiation of P2X 3 receptor function especially after stimulation with NGF [9].
Conversely, for KI neurons, calcineurin inhibition was sufficient to reverse the depression of serine phosphorylation to a level associated with impaired receptor function despite enhanced threonine phosphorylation. Thus, our data are consistent with the notion that the phosphorylation state of serine residues is dominant to regulate P2X 3 receptor activity.
It is well established that increased concentrations of intracellular Ca 2+ activate the protein phosphatase calcineurin [45,46] that is enriched in neuronal cells. We posit that the gain-of-function of mutated Ca V 2.1 channels in Ca V 2.1-immunoreactive neurons was accompanied by significant fluctuations in intracellular Ca 2+ sufficient to trigger a multisystem signaling mechanism that probably involved CaMKII and calcineurin. In addition, enhanced influx of Ca 2+ via mutated Ca V 2.1 channels might modulate the activity of various transcription factors [47] and might control neuronal gene expression via calcineurin in distinct ways [48], conferring a new molecular phenotype with distinctive neuronal signalling.
Even though the structure of the P2X 4 receptor in its closed state has been recently reported [49], the numerous serine residues in the N-and C-terminal domains of the P2X 3 receptor make it difficult to predict those responsible for binding and catalytic activity of kinases and phosphatases. Future studies will be necessary to clarify this complex scenario in which multiple pathways control the phosphorylation state of P2X 3 receptors with important and differential outcomes in terms of function and thus, presumably, in terms of pain sensing efficiency.

Conclusions
Could P2X 3 upregulation be an important component of migraine headache and to what extent do R192Q KI mice help our understanding of the disease process? The KI mouse model, carrying a human FHM-1 mutation, through abnormal activation of Ca V 2.1 channels and a CaMKII-dependent pathway, exhibited potentiation of P2X 3 receptor function in trigeminal neurons, thus suggesting the role of P2X 3 receptors in trigeminal pain that requires future investigation especially because of the role of such receptors in sporadic migraine [13][14][15][16]. Because these purinergic nociceptors activate one major pain transducing mechanism of the trigeminovascular system [50], it is attractive to think that strong P2X 3 receptor activation may facilitate ATPdependent migraine headache [13,14]. Furthermore, the disabling headache attacks triggered by FK-506 treatment for immunosuppression in humans [51] might comprise a component due to intracellular Ca 2+ dysregulation and potential enhancement of P2X 3 receptor function. Future studies are necessary to clarify the mechanisms responsible for transforming acute trigeminal pain into the long-lasting chronic headache typical of migraine attacks.

Genetic model
Ca V 2.1 α1 R192Q mutant KI and wild type (WT) littermates were used for in vitro experiments. Genotyping was performed by PCR using primers 5′-TGTCGGGAC GGAGTTTGAC-3′ and 5′-AGACTCACGCACTTGGG ATT-3′ followed by enzyme digestion of PCR products with AlwNI (New England Biolabs, Ipswich, MA, USA) as previously described [5]. Animals were maintained in accordance with the Italian Animal Welfare Act and their use was approved by the Local Authority Veterinary Service.

Culture of mouse trigeminal ganglia
Trigeminal ganglion neurons were obtained from R192Q KI and WT mice (P12-P14) and cultured as described before [22]. Cells were used 24 h after plating. To support the comparison between KI and WT neurons, we ran initial feasibility studies to find out any obvious morphological or immunocytochemical differences between trigeminal neuronal cultures of R192Q KI and WT mice. Fig. 7A, B shows similar in vitro survival of neuronal subpopulations (n = 4, p > 0.05), whose largest fraction had somatic neuronal diameter of approximately 15 μm with analogous distribution between genotypes for both ganglia and cultured ganglion neurons (Fig. 7B) in analogy with our previous observations [22]. We also confirmed that Ca V 2.1 α1 expression as assessed by immunocytochemistry of intact trigeminal ganglia showed unchanged numbers of immunopositive cells in samples of both genotypes (Fig. 7C). Furthermore, Ca V 2.1 α1 western blotting confirmed this finding and revealed similar expression levels in total membrane fractions from trigeminal neurons from intact ganglia or cultured ganglion neurons (Ca V 2.1 α1 protein size: 180 kDa, n = 3, p > 0.05; Fig. 7D). The unchanged Ca V 2.1 expression levels are in line with previous observations in the brains of R192Q KI mice [5].

Protein lysates, immunoprecipitation and immunoblotting
For western blotting and immunoprecipitation experiments, proteins from ganglia or cultures were extracted in TNE buffer (10 mM Tris-HCl at pH 7.5, 150 mM NaCl, 2 mM EDTA, 100 mM NaF, 2% n-octyl β-D-glucopyranoside and 1% NP40) plus protease inhibitors (Sigma). Membrane extracts were performed as described [23]. The following antibodies were used: anti- . To avoid complications in the analysis because of the presence of immunoglobulin heavy chains, a mouse anti-rabbit IgG-HRP conjugated (Jackson Immu-noResearch, Suffolk, UK) was used as secondary antibody. Western blot signals were detected with the enhanced chemiluminescence light system (GE Healthcare, Milano, Italy). For quantification, band density was measured using CorelDraw Photopaint software and ImageJ software. Membrane protein biotinylation and streptavidin pulldown experiments were performed, as described previously [9].

Real time RT-PCR
Total mRNA extraction from trigeminal cultures or ganglia and reverse transcription reactions was performed as described previously [9]. Data were normalized with respect to β-tubulin III housekeeping mRNA contents using primers previously reported [9].

Patch-clamp recording
After 1 day in culture, trigeminal neurons were superfused continuously (2 mL/min) with physiological solution containing (in mM): 152 NaCl, 5 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 glucose, and 10 HEPES (pH adjusted to 7.4 with NaOH). Cells were patch-clamped in the wholecell configuration using pipettes with a resistance of 3-4 MΩ when filled with the following solution (in mM): 140 KCl, 0.5 CaCl 2 , 2 MgCl 2 , 2 Mg 2 ATP 3 , 2 GTP, 10 HEPES, and 10 EGTA (pH adjusted to 7.2 with KOH). Cells were held at -60 mV. Currents were filtered at 1 kHz and acquired by means of a DigiData 1200 Interface and pClamp 8.2 software (Molecular Devices, Sunnyvale, CA, USA). To obtain stable and reproducible P2X 3 receptor currents, its synthetic agonist α,β-methylene-adenosine-5′-triphosphate (α,β-meATP) was applied using a fast superfusion system (Rapid Solution Changer RSC-200; BioLogic Science Instruments, Claix, France). The time for solution exchange was approximately 30 ms. Responses were measured as peak amplitude, while the response rise time was expressed as its time constant (τ on ). Whenever the current response did not decay to baseline prior the end of agonist application, we considered the residual current as indicative of heteromeric P2X 2/3 receptors [21] and measured it to quantify its size and occurrence. To express agonist potency as EC 50 values (concentration producing 50% of the maximum response), dose-response curves were constructed by applying different agonist doses to the same cells, and fitting them with a logistic equation (Origin 6.0; Microcal, Northampton, MA, USA). The onset of desensitization was estimated by calculating the first-time constant of current decay (τ fast ) in accordance with our previous reports [52]. Previous studies have indicated that recovery from desensitization of P2X 3 receptors is a relatively slow process which depends on the agonist used for the test [52,53]. In the case of α,β-meATP, the half-time recovery is typically under 1 min and thus, we use the amplitude of a second response evoked by α,β-meATP 30 s after the first one as a simple index to assess the extent of recovery from desensitization in accordance with former reports [9,22]. Heteromeric responses of native P2X 2/3 receptors were identified on the basis of their residual current (≥5% of peak current) at the end of a 2-s-long agonist application as previously reported [9]. Capsaicin was applied at the standard test dose of 1 μM (for 2 s) to evoke reproducible inward currents. Functional current responses were recorded approximately 10 min after washout of the culture medium. To quantify the effect of a drug on the α,β-meATP-induced current amplitude for a drugtreated neuron, the peak response was expressed as a percentage of the mean peak current amplitude obtained from control neurons from cultures prepared in parallel.

Ca 2+ imaging
Cultured trigeminal neurons were incubated for 40 min at 20-22°C in physiological solution containing Fluo3-AM (5 μM; Invitrogen) followed by a wash for 30 min. Fluorescence emission signals were acquired with a CCD camera (Coolsnap HQ; Roper Scientific, Duluth, GA, USA). We did not observe any difference in dye loading between WT and KI neurons. Data were collected from cells that produced a rapid response to a pulse of high concentration of KCl (i.e., 50 mM for 1 s) to maximize the activation of voltage-dependent Ca 2+ channels: their responsiveness indicated that they were neurons [10]. We could not detect any difference in the amplitude of such responses between WT and KI neurons tested in parallel with sister cultures. For routine testing of the neuronal excitability we used 20 mM K + to stimulate neurons without excessive depolarization and 10 μM α,β-meATP in analogy to patch clamp protocols. The analysis was performed using Metafluor software (Metafluor Imaging Series 6.0; Molecular Devices). Intracellular Ca 2+ transients are expressed as fractional amplitude increase (ΔF/F 0 , where F 0 is the baseline fluorescence level and ΔF is the increment over baseline). To test the effect of ω-agatoxin (200 nM) on Ca 2+ transients, neurons were challenged with a 2-s pulse of α,β-meATP, capsaicin, 20 mM K + , 50 mM K + before and after 30 min application of ω-agatoxin.

Pharmacological inhibitors
Pharmacological blockers were used to support our biochemical and electrophysiological data suggesting a role for certain enzymes and channels in the observed mouse neuron phenotype. In particular, we used ω-agatoxin (200 nM) as a selective inhibitor of P/Q-type Ca 2+ channels [54]: this toxin has been extensively employed to probe the influence of such channels on a variety of in vivo pain models [55,56] including trigeminal neurons [57]. To block activated CaMKII, we used KN-93 (5 μM; see also [11]), an agent widely employed to depress experimental pain [58][59][60]. Conversely, we also applied FK-506 (5 μM), a well known immunosuppressant drug that acts by binding to proteins regulating the Ca 2+ -dependent phosphatase calcineurin [39]. This drug can induce strong, persistent pain in humans [61], including severe headache [51]. We also tested the calcineurin autoinhibitory peptide F2/47 (100 μM; Tocris, Bristol, UK) [40] applied via the intracellular solution in patch clamp studies.

Statistics
Data are expressed as mean ± standard error of the mean (SEM), where n indicates the number of experiments in molecular biology/immunocytochemistry or the number of investigated cells in electrophysiology (unless stated otherwise). Statistical analysis was performed using the Student's t-test, the Mann-Whitney rank sum test or the ANOVA test, when appropriate for parametric or non-parametric data, respectively. A p value of ≤ 0.05 was accepted as indicative of a statistically significant difference.