Mutated Ca V 2.1 Channels Dysregulate CASK/P2X3 Signaling in Mouse Trigeminal Sensory Neurons of R192Q Cacna1a Knock-in Mice

The CASK/P2X3 receptor complex is abundantly expressed in KI ganglia and is modulated by Ca2+ influx

In order to study the effects of CASK on P2X3 receptors expressed in WT and KI ganglia, we first compared CASK/P2X3 complex levels in ganglion extracts. Immunoprecipitation experiments showed that the complex was significantly more abundant in KI than in WT samples (Figure 1A; p=0.038, n=5). A significant increase (n=5, p = 0.005) in CASK associated with cell membrane fractions was observed in KI tissue (Additional file 1: Figure S1A), although total CASK lysate preparations did not show any difference between WT or KI samples (Additional file 1: Figure S1B). Further experiments concerning the specificity of the CASK/P2X3 complex, based on immunoprecipitating CASK first and then performing western blotting with P2X3 antibodies, validated our previous findings [6] and are included in Additional file 2: Figure S2A, B.

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Figure 1

CASK/P2X3 complex in KI trigeminal neurons. A, Example of immunopurified P2X3 from trigeminal ganglia probed with anti-CASK antibodies reveals more abundant CASK/P2X3 complex in KI ganglion cultures than in WT ones. Histograms quantify this effect (n=5, p = 0.038). Ab, indicate signal from unrelated antibody. B, CASK/P2X3 co-immunoprecipitation experiments from ganglion cultures demonstrate that CASK/P2X3 complex is sensitive to ω-Agatoxin IVA treatment (400 nM, overnight; Aga). Note the more intense CASK/P2X3 signal in KI samples as seen in A. P2X3 immunoprecipitation input and β-Tubulin expression shown as gel loading controls. C, CASK/P2X3 complex is sensitive to KN-93 pre-incubation (5 μM, 90 min). D, Examples of anti-phospho-CaMKII T286 Western immunoblots of extracts from WT and KI trigeminal cultures in control conditions and after CASK silencing. β-Tubulin is shown as loading control. Histograms quantify the effect (#, p=0.05; *p=0.03 for WT and p=0.025 for KI; n = 3).

In analogy to its effect on other receptors (e.g. NMDA receptors; [15]), CASK might exert a role in the process of P2X3 receptor export to surface membranes. In fact, pulled-down biotinylated surface P2X3 receptors showed co-purification with intracellular CASK (Additional file 3: Figure S3), supporting the view that CASK/P2X3 complexes are membrane-bound. In these biotinylation experiments, no difference was observed in the levels of surface membrane CASK in WT and KI samples (Additional file 3: Figure S3).

We further explored whether the origin of the stronger CASK/P2X3 association in KI samples could be directly linked to the Ca_V2.1 R192Q gain-of-function and concomitant higher CaMKII activation [9]. To this end, we used selective pharmacological tools to specifically block either Ca_V2.1 channel function or apply inhibitors of downstream CaMKII activity. Trigeminal ganglion cultures were treated with the selective Ca_V2.1 channel blocker ω-Agatoxin IVA (400 nM, 15 h) and then tested for CASK/P2X3 protein interaction by immunoprecipitation. Immunoprecipitation experiments with purified P2X3 receptors and CASK expression (Figure 1B) confirmed that CASK/P2X3 receptor interaction was stronger in KI cultures and that it was significantly reversed by ω-Agatoxin IVA application. Interestingly, CASK/P2X3 complex in WT culture was not affected by the channel blocker suggesting that larger CASK/P2X3 interaction in KI neurons was likely due to the enhanced Ca_V2.1 channel activity.

Likewise, after cultures treatment with the CaMKII inhibitor KN-93 (5 μM, 90 min, Figure 1C), the CASK/P2X3 interaction was significantly inhibited in KI, but not in WT (Figure 1C). In addition, after RNA silencing of CASK expression [6], there was a significant decrease in CaMKII phosphorylation (Figure 1D).

P2X3 expression and function after siCASK in WT and KI ganglion cultures

Our recent findings [6] that showed how siCASK significantly lowered P2X3 expression in trigeminal ganglion cultures, have been further validated in the present study in which no difference between WT and KI cultures was observed as a consequence of siCASK (Figure 2A). To further explore functional consequence of CASK/P2X3 complex in the KI model, patch clamp experiments were carried out (Figure 2B). Sample P2X3 receptor currents elicited by pulse application of the selective agonist α,β-methylene-adenosine-5′-triphosphate (α,β-meATP; 10 μM) were clearly smaller after siCASK silencing, but proportionally similar in WT and KI neurons. As expected [9], KI neuronal currents were constitutively larger than WT ones (Figure 2B).

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Figure 2

Effect of siCASK silencing in KI and WT neurons. A, Example of Western immunoblotting for CASK, P2X3 and β-tubulin, in control and after siCASK silencing. Note the significant decrease in P2X3 receptor expression in both WT and KI samples. Histograms show significant reduction of P2X3 protein expression in silenced samples (*p=0.026 for WT and p=0.04 for KI; n = 5, number of experiments). B, representative examples of currents induced by α,β-meATP (α,β, black bar, 10 μM) from WT or KI trigeminal neurons in control or after siCASK. Histograms show quantification of mean current amplitude values (WT: 230 ± 19 pA or 170 ± 13 pA; KI: 296 ± 92 pA or 225 ± 16 pA for control or siCASK respectively). # p=0.036, *p=0.02 for WT and p=0.019 for KI; n = 15–23 cells.

Tissue culture

Ca_V2.1 R192Q KI and WT mice [8] were euthanized by slowly raising levels of CO₂ in accordance with the Italian Animal Welfare Act and approved by SISSA Ethical Committee. Trigeminal ganglia primary cultures were obtained as previously described [6]. In specific experiments ω-Agatoxin IVA (400 nM; 15 h; Sigma, Milan, Italy) or CaMKII inhibitor KN-93 (5 μM; Sigma) were added to the culture medium for the indicated time [9]. CASK siRNA experiments were performed as previously reported [6].

Western blotting and immunoprecipitation

Protein extracts and immunoprecipitation experiments and membrane protein biotinylation were performed as previously described [6]. P2X3 receptors were solubilized in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 20 mM EDTA, and 1% Triton-X-100 plus protease inhibitors (Roche Products, Welwyn Garden City, UK) as previously reported [3,6]. The non-ionic detergent n-octyl-β-D-glucoside (ODG; [9]) was not used. For western blot quantification, signals expressed in optical density absolute units (AU) were measured with digital imaging system (UVITEC, Cambridge, UK).

Patch clamp recordings

Currents from siRNA-treated cultures (72 h) were recorded under whole cell voltage clamp mode at holding potential of −65 mV after correcting for the liquid junction potential [6]. P2X3 receptor synthetic agonist α,β-meATP (10 μM; Sigma) was applied (2 s) using a fast superfusion system (RSC-200; BioLogic Science Instruments, Claix, France).

Data analysis

Data are presented as mean ± standard error. Statistical significance was evaluated with paired Student's t-test or Mann-Whitney rank sum test using OriginPro 7.5 (OriginLab, Northampton, MA, USA).

Competing interests

The authors state that the content of this article does not create any competing interest.

Authors' contributions

AG carried out biochemical studies and statistical analysis, TB performed further data collection, SH carried out functional studies and statistical analysis, MF, AM provided animal model; EF, MS conceived of the study, EF, AN design and coordination; AG, AM, AN, EF draft the manuscript. All authors read and approved the final manuscript.