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

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This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process which may lead to

differences between this version and the Version of Record. Please cite this article as an

'Accepted Article', doi: 10.1111/jnc.12272

This article is protected by copyright. All rights reserved.

Received Date : 08-Feb-2013

Revised Date : 02-Apr-2013

Accepted Date : 03-Apr-2013

Article type : Original Article

Corresponding author email id: [email protected]

CASK is a new intracellular modulator of P2X3 receptors

Aswini Gnanasekaran1, Mayya Sundukova1, Swathi Hullugundi1, Nicol Birsa1, Giorgio

Bianchini1, Yi-Ping Hseuh3, Andrea Nistri1, and Elsa Fabbretti1,2*

1 Neuroscience Department, International School for Advanced Studies (SISSA), Via Bonomea

265, 34136 Trieste, Italy.

2 Center for biomedical sciences and engineering, University of Nova Gorica, SI-5000, Nova

Gorica, Slovenia.

3 Institute of Molecular Biology, Academia Sinica, 128 Academia Rd. Sec. 2, Taipei 11529,

Taiwan.

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*Corresponding Author: Elsa Fabbretti 2 Center for biomedical sciences and engineering,

University of Nova Gorica, SI-5000, Nova Gorica, Slovenia.

Keywords: pain, ATP, purinergic signaling, DRG

Running title: CASK and P2X3 receptors

Abbreviations

α,β-meATP, α,β-methyleneATP; CaMK, calcium/calmodulin-dependent kinase; CASK-

calcium/calmodulin-dependent serine kinase; τfast, time constant of fast current decay; τslow, time

constant of slow decay of the current; TG, trigeminal ganglia.

Abstract

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 HEK cells. Recombinant P2X3/CASK expression in HEK cells

increased serine phosphorylation of P2X3 receptors, typically associated with receptor

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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 of P2X3 expression levels was reversed by the proteasomal

inhibitor MG-132. Moreover, neuronal CASK/P2X3 interaction was upregulated by NGF

signaling and downregulated 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.

Introduction

P2X3 receptors are ATP-gated membrane proteins, expressed predominantly by sensory neurons,

and are important transducers of peripheral nociceptive signals (Burnstock 2006; Wirkner et al.

2007). Previous studies have indicated that P2X3 receptors undergo rapid activity-dependent

turnover (Xu et al. 2004; Vacca et al. 2009; Pryazhnikov et al. 2011), a process that requires a

complex series of events, including oligomerization of protein subunits as well as post-

translational folding and modification (Royle et al. 2003). Several intracellular kinases modulate

the expression and function of P2X3 receptors of sensory neurons by discrete phosphorylation of

intracellular domains of this receptor (Fabbretti and Nistri, 2012). We have previously shown

that CaMKII and the Ser/Thr kinase Cdk5 are highly involved in this process (Nair et al. 2010 a,

b). Since adaptor and scaffold molecules are likely to participate in the compartmentalized signal

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transduction essential for receptor stabilization and function, we investigated the potential role of

CASK (calcium/calmodulin-dependent serine protein kinase), a membrane protein belonging to

the membrane-associated guanylate kinase (MAGUK) family, in regulation of P2X3 expression

and function. CASK is mainly known for its function as a neuronal scaffolding protein (Cohen et

al. 1998). CASK deficiency impairs synaptic transmission and dendritic spine formation (Lu et

al. 2003; Zordan et al. 2005; Chao et al. 2008; Sun et al. 2009; Slawson et al. 2011) as CASK

expression is essential for receptor trafficking to membrane level (Butz et al. 1998; Jeyifous et al.

2009) and the link between plasma membrane to cytoskeletons (Cohen et al., 1998; Chao et al.,

2008).

We wondered if CASK might have a role also in the regulation of the expression and function of

P2X3 receptors in sensory ganglion neurons. For this purpose, using silencing experiments we

check role of CASK in P2X3 stability and with immunoprecipitation experiments we

investigated the association of CASK and P2X3 in trigeminal sensory ganglia in situ and in

culture. Furthermore, we explored the dynamic nature of the interaction of CASK and P2X3 and

the impact of CASK on P2X3 receptor function.

Materials and Methods

Tissue and culture preparation

Postnatal day 12–15 C57-Black/6Jico mice were fully anesthetized by slowly raising levels of

CO2 and decapitated, a procedure in accordance with the Italian Animal Welfare Act and

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approved by the Ethical Committee of SISSA. The animals were from both sexes and were

obtained from in-house animal facility at SISSA. Trigeminal ganglia were excised and processed

for molecular and cell biology experiments or dissociated (10–20 min at 37°C) in a solution

containing 0.25 mg/ml trypsin, 1 mg/ml collagenase, and 0.2 mg/ml DNase (Sigma, Milan, Italy)

in F-12 medium (Invitrogen, San Diego, CA) to prepare primary cultures. Cultured cells were

used 24 h after plating (Simonetti et al. 2006) unless otherwise indicated. In a set of

experiments, cultures were incubated with a neutralizing anti-NGF antibody (12 ng/ml, 14 h;

Sigma) to suppress endogenous NGF-mediated signaling (D’Arco et al. 2007; 2009), or with

NGF (100 ng/ml, 5 min; Alomone, Jerusalem, Israel) applied immediately before cell lysis.

Cell culture and transfection

HEK-293T cells were obtained from our in-house cell bank and used as previously described

(Nair et al. 2010). For transfection experiments, the following DNA plasmids were used: pEGFP

(Clontech, Mountain View, CA, USA), pcDNA3-P2X3 (rat sequence, NCBI accession number:

CAA62594), pcDNA3-CASK (kindly provided by Dr. Li-Huei Tsai, Cambridge, MA, USA) and

the CASK deletion mutants (Gene Bank Accession number: 447110; Chao et al. 2008) ΔCAMK

(Δ3–323), ΔPDZ (Δ488–568), ΔSH3 (Δ604-665), ΔGK (Δ721-895). In selected experiments,

Myc-tag CASK plasmid (Hsueh et al. 1998) was used. Since in preliminary experiments ΔPDΖ

and ΔSH3 CASK showed limited expression in HEK cells, in this instance we used 10% larger

concentration of plasmid transfection with respect to the standard protocol (Sundukova et al.

2012).

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Immunofluorescence and microscopy

For immunofluorescence staining, paraformaldehyde-fixed ganglia or cultures were

immunostained with antibodies against the P2X3 receptors (dilution 1:200; Alomone), anti-

CASK (MAB5230; dilution 1:100; Millipore Milan Italy), and neuron-specific β-tubulin III

(dilution 1:300; Sigma). DAPI was used for nuclear staining. Immunofluorescence reactions

were visualized using as secondary antibodies AlexaFluor 488, AlexaFluor 546, or Streptavidin

647 (dilution 1:500; Invitrogen). Images were analyzed and quantified with a Zeiss fluorescence

microscope or Leica confocal microscope (Leica Microsystems, Heidelberg, Germany) and

dedicated software. The perimembrane region was defined as the one within 5 µm from the cell

surface. Unless otherwise stated, an average of 500 cells was analyzed in each test, and data are

the mean of three independent experiments.

siCASK Silencing

For siRNA experiments, cultured trigeminal neurons (from 2 mice) were transfected with mouse

CASK siRNA SmartPools (100 nM, Dharmacon RNAi Technology, Lafayette, CO, USA) using

the DharmaFECTTM-1 transfection reagent (Dharmacon). For transfection efficiency control,

cells were transfected with scramble RNA and siGLO RISC-Free siRNA (Dharmacon).

Efficiency of CASK silencing was tested with western immunoblotting as exemplified in

Supplemental Fig. 1. Forty eight hour after silencing, cells were used for protein expression and

patch clamping experiments.

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Protein lysates, immunoprecipitation and immunoblotting

For western blotting (WB) and immunoprecipitation (IP) experiments, proteins from neuronal

cultures were extracted in TNE 1.5X buffer (10 mM Tris-HCl at pH 7.5, 150 mM NaCl, 2 mM

EDTA, 100 mM NaF, 20 mM Na orthovanadate, and 1% Triton-X 100) plus protease inhibitors

(Sigma) (D’Arco et al. 2009). The following antibodies were used: anti-P2X3 (WB, 1:300;

Alomone), anti-P2X3 (H-60, IP, 1:500; Santa Cruz); anti-β-tubulin III (1:2,000; Sigma); anti-β-

actin (1:3,000; Sigma); anti-CASK (C-19 or H-107, 1:500 Santa Cruz, CA, USA); anti-phospho-

Serine antibody (1:600; Millipore) and anti-Myc (9B11; IP, 1:1000; WB, 1:4000; Cell

Signaling). Western blot signals were detected with the enhanced chemiluminescence light

system (GE Healthcare, Milano, Italy). For quantification of intensities of the immunoreactive

protein bands (expressed in optical density absolute units, AU), we used Scion Image software

(NIH, Bethesda, USA) or the digital imaging system UVTEC (Cambridge, UK).

Membrane biotinylation

Membrane protein biotinylation and streptavidin pulldown experiments were performed as

described previously (D’Arco et al. 2007). Briefly, cultures were incubated with 2 mg/ml EZ-

Link Sulfo-NHS-LC-biotin (Thermo Fisher Scientific corp. Germany) for 30 min at 4 ºC in PBS

containing 1mM MgCl2 and CaCl2 (pH 8.0). Cells were quenched in 100 mM ice-cold glycine

for 30 minutes, lysed 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). Pulldown of

biotinylated proteins was obtained with ImmunoPure Immobilized Streptavidin beads (Pierce)

for 2 h at 4°C in a rotator. Streptavidin bead complexes washed twice with PBS with 0.1M CaCl2

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/ MgCl2 and 0.1% Triton-X. Supernatant was eluted by boiling in presence of 0.1M DTT (5 min)

and loaded on SDS gel. Biotinylation experiments resulted free of intracellular protein

contaminants when tested with β-actin.

Patch clamp recordings

As previously described (D’Arco et al. 2009; Sundukova et al. 2012), currents were recorded

from small/medium size mouse trigeminal neurons in culture under whole cell voltage clamp

mode at holding potential of -80 mV. Such cells were continuously superfused at room

temperature with control solution containing (in mM): 152 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10

glucose, 10 HEPES; pH 7.4 adjusted with NaOH. Patch pipettes had a resistance of 3-4 MΩ

when filled with (in mM): 140 KCl, 2 MgCl2, 0.5 CaCl2, 2 ATP-Mg, 2 GTP-Li, 20 HEPES, 5

EGTA; pH 7.2 adjusted with KOH. After establishing whole cell configuration, access resistance

was never >10 MΩ and was routinely compensated by 70%. Data were acquired and analyzed

with the pCLAMP software Clampex 9.2 (Molecular Devices, Palo Alto, CA, USA). The

receptor agonist α,β-methyleneATP (α,β-meATP; Sigma) was applied by rapid solution changer

system (Perfusion Fast-Step System SF-77B, Warmer Instruments, Hamden, CT, USA).

Membrane currents were analyzed in terms of current density (calculated as amplitude divided

by cell slow capacitance) to take into account cell size. Current decay due to receptor

desensitization during agonist application was fitted with either a monoexponential (for 1 µM

and 3 µM doses of α,β-meATP) or biexponential (for 10 µM) function using pCLAMP Clampfit

9.2. Recovery from desensitization was assessed by a paired pulse protocol over 30 s intervals in

accordance with previous reports (Sokolova et al. 2006; Nair et al. 2010).

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Data analysis

All data are presented as mean ± standard error of the mean (S.E.); n is the number of cells.

Statistical significance was evaluated with paired Student’s t-test (for parametric data) or Mann-

Whitney rank sum test (for nonparametric data) using OriginPro 7.5 (Northampton, MA, USA),

p<0.05 was considered statistically significant.

Results

CASK expression in mouse trigeminal ganglion neurons

Fig 1a,b shows immunofluorescence experiments to examine CASK and P2X3 receptor

expression in mouse trigeminal ganglion neurons, which were labeled by neuron-specific β-

tubulin. While CASK/P2X3 expression was observed in the majority of trigeminal neurons, cell

size analysis indicates that most P2X3 and CASK co-localization was found in medium-size

neurons (15-20 µm somatic size; Simonetti et al. 2006), although large-diameter neurons,

typically lacking P2X3 receptors, also expressed CASK (Fig. 1b, right). This expression pattern

was maintained in trigeminal ganglion culture in which CASK signal was found in both neurons

and non-neuronal cells (Fig 1c,d). In our conditions, no CASK nuclear signal was found in

trigeminal ganglia neurons.

CASK modulates the P2X3 protein stability

Lack of selective pharmacological inhibitors of CASK prompted us to use RNA interference to

knock down endogenous CASK (siCASK) in trigeminal ganglion cultures and to look for its

effects on P2X3 receptor expression. Fig. 2a shows that, after siCASK, while expression of total

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P2X3 receptor in whole cell lysates was reduced by about 40 % (see open bar in right

histograms), protein biotinylation experiments showed that an even stronger decrease (by nearly

2/3rd; shaded bar in Fig. 2a, right) was observed for the surface expression of P2X3 receptors. To

check if siCASK-induced P2X3 receptor loss was due to proteasomal degradation, trigeminal

cultures were treated with the proteasome inhibitor MG-132 (30 µM; 4 h; Vacca et al. 2011), that

per se increased P2X3 receptor protein expression already in control basal conditions

(Supplemental Fig. 2a). Fig. 2b indicates that application of MG-132 to trigeminal ganglion

cultures was sufficient to significantly (p<0.04) reverse the effect of siCASK, therefore

preventing siCASK-mediated decrease in P2X3 receptor expression.

We performed confocal line-profile analysis of trigeminal neurons in culture with or without

CASK silencing and immunostained for P2X3 and CASK (Fig. 2c). While in control conditions

the P2X3 receptor signal was uniformly distributed across the cell section (Fig. 2c), resembling

the staining of neurons in intact ganglia (as exemplified in Supplemental Fig. 2b), after CASK

knock down, P2X3 receptor staining was highly reduced especially at the perimembrane region,

in accordance with the decrease in cell surface expression demonstrated in Fig. 2a,b. P2X3

receptor trafficking was explored in siCASK-treated trigeminal neuronal cultures either in

control or in the presence of brefeldin A (BFA), that alters the transport from the endoplasmatic

reticulum to Golgi complex (Valenzuela et al. 2011). These experiments confirmed larger

expression of P2X3 subunits after BFA treatment (5 μg/ml; 30 min; Fabbretti et al. 2006), while

P2X3 expression remained sensitive to siCASK (Supplemental Figure 3).

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Characteristics of P2X3/CASK interaction

As these data suggest that CASK had a role in the stabilization of P2X3 receptors in trigeminal

neurons, we explored whether CASK and P2X3 could be part of a unique macromolecular

complex. To address this issue, P2X3/CASK co-immunoprecipitation experiments were

performed using lysates from intact murine trigeminal ganglia or from cultures. Fig. 3a shows

that CASK signal was clearly detected in immunopurified P2X3 receptors from ganglion tissue

or cultures (control signals for P2X3, CASK and β-tubulin are also shown in Fig. 3a). After

siCASK in trigeminal cultures, the CASK/P2X3 complex was clearly reduced (Fig. 3b). The

P2X3/CASK interaction was also observed in a neuronal free environment, as similar results

were obtained with HEK cells co-transfected with CASK and P2X3 plasmids (Fig. 3c, right

lane). Even endogenous CASK of HEK cells could co-precipitate with the P2X3 protein as

shown in Fig. 3c (middle lane indicated as P2X3/GFP). Similar data were observed with pull

down of either P2X3 or CASK-myc, providing further validation of CASK pull down

experiments (Fig. 3d, e). Interestingly, the expression of recombinant P2X3 receptors was also

affected by silencing endogenous CASK in HEK cells (Supplemental Fig 1, left), thus supporting

a strong role of CASK in the stability of P2X3 receptors (Fig. 2b). After P2X3/CASK co-

expression in HEK cells, immunoprecipitation with anti-P2X3 antibodies and western blot with

an anti-phosphoserine antibody showed significant increment in serine phosphorylation of P2X3

receptors (Fig. 3f), which is known to be associated with receptor function (Nair et al. 2010).

To further explore the role of CASK in the stability of P2X3 receptors, we used a series of

CASK deletion mutants (Chao et al. 2008) for P2X3 receptors co-transfection in HEK cells. Fig.

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4 (left) shows representative immunoblots of CASK and P2X3 expression in cell lysates after

transfection with wildtype CASK or the CASK mutants ΔCAMK, ΔPDΖ, ΔSH3 and ΔGK

(characterized by different molecular weight). We observed that total P2X3 receptor expression

was significantly enhanced by overexpressing either CASK (see also Fig. 3c) or anyone of these

mutants (Fig. 4).

Receptor state-dependent interaction of P2X3 and CASK

We next inquired if the CASK/P2X3 interaction was a dynamic process related to the activity

state of P2X3 receptors at membrane level. We, therefore, tested if enhanced P2X3 receptor

activity by NGF or P2X3 desensitization by sustained application of the selective agonist α,β-

meATP was accompanied by changes in the P2X3/CASK interaction.

To determine whether NGF signaling could modulate P2X3/CASK interaction, trigeminal

ganglion cultures were grown in basal conditions or under NGF deprivation (D’Arco et al. 2007,

2009). The potent algogenic action of NGF-TrkA signaling results in enhanced P2X3 receptor

function via modulation of PKC (D’Arco et al. 2007) and the Csk/Src pathways (D’Arco et al.

2009). Acute application of NGF (100 ng/mL; 5 min) to anti-NGF pre-treated cultures (Fig. 5a)

significantly (p=0.003; n=5) increased CASK/P2X3 co-precipitation, while the anti-NGF

antibody per se induced a modest CASK/P2X3 signal decrease (p=0.03, n=3).

Full desensitization of P2X3 receptors evoked by sustained application of the selective agonist

α,β-meATP (100 μM; 30 s; Sokolova et al 2006) induced a significant (p=0.0027; n= 5) decrease

in P2X3/CASK interaction, an effect prevented by co-application of α,β-meATP together the

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selective P2X3 antagonist A-317491 (10 μM; 20 s; Jarvis et al. 2002; 2004) (Fig. 5b). Similar

results were obtained also in trigeminal ganglion primary cultures (Fig. 5c).

We subsequently studied the action of CASK on the properties of P2X3 receptor activation on

trigeminal neurons in culture. Patch clamping was used to measure membrane currents evoked

by short pulses of α,β-meATP (1-10 μM; 2 s) to minimize receptor desensitization. Fig. 6a

compares sample responses elicited by α,β-meATP in control conditions or after siCASK: in the

latter condition, there was significant depression of P2X3 receptor currents as quantified in Fig.

6b. No significant change in fast desensitization properties was, however, observed as indicated

by similar values for current decay (Fig. 6c) and recovery from desensitization 30 s later (Fig.

6d). CASK silencing did not change cell capacitance (18 ± 1 vs 18 ± 1 pF control; n=31 cells), or

input resistance (278 ± 24 vs 280 ± 30 MΩ control), indicating that this treatment did not modify

basic neuronal properties under resting condition (Fig. 6c,d). Patch clamp experiments were also

performed to study the functional impact of co-expression of P2X3 receptors with CASK

mutants in HEK cells as summarized in Fig. 6e. Thus, significantly larger amplitude of P2X3

receptor currents induced by 2 s pulses of 10 μM α,β-meATP was observed after co-transfection

with WT CASK (~40 %), ΔCAMK (~50 %), ΔPDΖ (~40 %) whereas no significant change vs

control currents was detected after co-expressing P2X3 with ΔSH3 or ΔGK.

Discussion

The principal finding of the present report is the demonstration that the multidomain scaffold

protein CASK is a novel interactor of P2X3 receptors to control their membrane dwelling and

responsiveness. CASK is, therefore, proposed to be a new member of the family of intracellular

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signaling molecules that determine the efficiency of P2X3 receptors on sensory neurons

(Fabbretti and Nistri 2012).

CASK is important for P2X3 protein expression

CASK is molecular scaffold playing a major role in the assembly of multiprotein complexes at

specialized regions of neuronal plasma membranes (Borg et al. 1998; Butz et al. 1998) and is

associated with pre- and post-synaptic proteins and cytoskelethon (Hata et al. 1996; Hsueh et al.

1998; Biederer and Sudhof, 2001; Butz et al. 1998; Jeyifous et al. 2009). Hence, CASK is

reported to be involved in different neuronal functions, including synaptic strength, vesicle

release, and receptor expression and trafficking as well as neuronal development (Olsen et al.

2005): indeed, its genetic ablation is lethal to mice (Atasoy et al. 2007). Our present data

revealed for the first time that, in trigeminal ganglion neurons, CASK was widely expressed by

P2X3-positive neurons, suggesting its potential role in sensory neuron physiology and

nociceptive activity. The association of CASK with P2X3 receptors, confirmed in a recombinant

neuron-free cell system, appeared important for the stability of P2X3 receptors on cell surface, as

indicated by the biotinylation experiments. These results outline a new role of CASK in

modulating the properties of P2X3 receptors and, perhaps, pain transduction.

Characteristics of CASK/P2X3 interaction

After knocking down of CASK, P2X3 receptor expression was decreased, an effect prevented by

the proteasome inhibitor MG-132, suggesting that CASK could stabilize surface P2X3 receptors

normally subjected to a relatively rapid turnover via internalization and proteasomal degradation

(Vacca et al. 2011). In addition, the cell distribution of P2X3 immunoreactivity was altered by

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siCASK, indicating a change in receptor compartmentalization. We further tested the Golgi

export inhibitor BFA (effective in controlling P2X3 receptor sorting; Fabbretti et al. 2006) that

did not change the consequences of siCASK on P2X3 receptor expression. A likely possibility is

that CASK modulates the stability and clustering of P2X3 receptors at membrane levels, where

surface redistribution is a pre-requisite of appropriate receptor function and plasticity.

Furthermore, we report that CASK/P2X3 receptor co-expression in HEK cells led to increased

P2X3 serine phosphorylation, which is known to be a signature for enhanced receptor function

and trafficking (Fabbretti et al. 2006; Mo et al. 2009). These observations accord with the notion

that CASK functions as a molecular scaffold to recruit signaling molecules to the membrane

domain for modulation of signal transduction (Vaccaro and Dente, 2002). Because CASK can

act as an adaptor for various proteins including other kinases (Kaech et al. 1998; Lu et al. 2003),

it is likely that manipulating CASK expression modulated P2X3 receptors through influencing

other kinase anchors or adaptor proteins.

The question then arises how CASK can modulate P2X3 receptor expression. To address this

issue, we explored four CASK mutants missing the CaMK, PDZ, SH3 or GK domains and

observed that P2X3 receptor expression was still enhanced when co-expressed with anyone of

such mutants. In this study, we have not tested the role of the L27 domain, because the epitope of

the CASK antibody is located at this site. Future experiments are necessary to clarify the nature

of the P2X3/CASK complex since it is not ruled out that CASK uses an undefined protein motif

to associate with P2X3.

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We noted that expression of these mutants differentially changed P2X3 receptor mediated

currents. In fact, ΔCaMK or ΔPDZ expression largely enhanced the current amplitude, while this

was not the case of ΔSH3. Thus, against a background of comparable increment in P2X3 subunit

expression by these mutants, the functional data suggest that expression of CASK or the ΔCaMK

or ΔPDZ mutants might have recruited other interactors to modulate the P2X3 receptor function.

This observation strengthens the notion that, in addition to stabilizing P2X3 protein levels,

CASK also modified P2X3 activity. A body of evidence indicates that CAMK has an important

role in P2X3 expression and function in sensory neurons via intracellular calcium increase (Nair

et al. 2010; Simonetti et al. 2008; Xu and Huang, 2004). The present study examined if the

function of CAMK included regulation of the CASK/P2X3 complex: our data suggest that P2X3

receptors expression and function could be controlled by CAMK (Nair et al. 2010; Simonetti et

al. 2008) even with mechanisms independent from its binding to CASK. Due to the multiple

partners of CASK (including CAMK and PDZ) and its heterogeneous roles in neurons (Hsueh,

2011), it is difficult to precisely explain how CASK modulates P2X3 receptor currents. It is

possible that the differential affinity of CASK for multiple intracellular adaptors can modulate

various components of receptor responses comprising clustering, gating or internalization after

agonist binding. Future studies are, therefore, necessary to identify key components in the

P2X3/CASK complex and multiple mechanisms modulating their interaction.

Functional impact of P2X3/CASK interaction

Acute application of NGF, known to upregulate P2X3 receptor currents (D’Arco et al. 2007),

enhanced P2X3/CASK interaction, while NGF deprivation had the opposite effect. Conversely,

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desensitization of P2X3 receptors with a sustained application of large concentrations of α,β-

meATP, led to strong decrease in P2X3/CASK interaction, indicating that prolonged receptor

conformational modifications elicited by the agonist (Sokolova et al., 2006) were sufficient to

perturb the P2X3/CASK domain sites. These results propose that the strength of association

between P2X3 and CASK was dynamically regulated by the receptor state. Patch clamp

experiments on trigeminal neurons after siCASK demonstrated a small, yet significant

depression of P2X3 receptor mediated responses evoked by short pulses of α,β-meATP. This

phenomenon was, however, not associated with a change in receptor desensitization or recovery

from it. Thus, under basal conditions, block of CASK expression likely decreased the amount of

P2X3 receptors (or of unidentified interactors) at membrane level and, therefore, impaired

neuronal functional responses without altering their kinetic properties.

Conclusions

P2X3 receptors are highly regulated by soluble mediators (including ATP itself) and complex

intracellular signaling pathways (Burnstock, 2011; Giniatullin et al. 2008; Illes et al. 2011). This

property offers an array of molecular processes underlying the dysregulation of P2X3-mediated

nociception especially in chronic pain syndromes (Abbracchio et al. 2009; Burnstock, 2012;

Wirkner et al. 2007). Since the present reports shows that CASK has a key role in ensuring P2X3

receptor stability and function, it will be interesting to study whether sensory neurons of chronic

pain models possess enhanced CASK/P2X3 interaction as a mechanism to support this

syndrome.

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Acknowledgments

This work was financially supported by grants from the Telethon Foundation – Italy

(GGP10082), the Cariplo Foundation (2011-0505), the EU Crossborder Cooperation Initiative

(MINA project) managed by the Friuli Venezia Giulia Region Government, the AHA-MOMENT

grant from Slovenian Ministry for Education and Science. We acknowledge the help by Drs.

Manuela Simonetti for preliminary experiments.

Conflict of interest

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

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Figure legends

Fig. 1 P2X3/CASK co-expression in trigeminal ganglion neurons in vivo and in vitro

(a) Trigeminal ganglia neurons immunostained with antibodies against P2X3 receptors (green),

CASK (red). (b) Histograms quantify the percentage of P2X3 and CASK expressing neurons

(left, 100 % is the total number of β-tubulin III positive neurons) and the somatic size

distribution of neurons expressing CASK and P2X3 receptors (right), where open bars represent

the percentage of P2X3 expressing neurons and filled bars the percentage of CASK expressing

neurons (β-tubulin III positive). The database for analysis comprises an average of 1,000 cells.

(c) Example of trigeminal ganglion cultures (24 h) immunostained as in (a). (d) Histograms

quantify the percentage of P2X3/CASK co-expressing neurons (left) and their somatic size

distribution (right) in culture. Data are the mean of three independent experiments. Calibration

bars in (a) and (c): 50 µm.

Fig. 2 CASK silencing reduces P2X3 expression by trigeminal neurons

(a) Example of total lysates and membrane biotinylation western immunoblots of trigeminal

ganglion culture lysates from control or siCASK samples probed with the anti-P2X3 antibody.

Gel loading is shown with β-tubulin signal. In the right panels, the total or surface P2X3 protein

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levels in siCASK cultures were compared to those of cells transfected with scramble siRNA.

n=5. Note significantly lower expression of P2X3 receptors in total lysates (p=0.0028) and

membrane samples (p=0.038) from siCASK cultures. (b) Examples of P2X3 or CASK

immunoblots from trigeminal neuron lysates in control, siCASK or siCASK treated with MG-

132 (30 µM; 4 h) conditions. β-tubulin shown gel loading. Histograms compare P2X3 receptor

expression of control, siCASK or siCASK and MG-132 treated samples. In the latter case, P2X3

receptor expression is significantly larger vs siCASK (n=3 experiments, p=0.035). (c) left,

confocal microscopy images of single cultured trigeminal neurons in control, or after siCASK.

Localization of nuclei is indicated with N. Bar=10 μm. Right, cell line-scan immunostaining

profile for P2X3 or CASK indicates sparse and decreased P2X3 immunostaining after siCASK.

Similar results were obtained in at least three independent experiments (n= 15 cells).

Fig. 3 Characteristics of CASK/P2X3 interactions

(a) Example of P2X3/CASK co-immunoprecipitation experiments from either trigeminal

ganglion tissue or culture. β-Tubulin shows gel loading in different lanes. (b) Similar approach is

shown for P2X3/CASK immunoprecipitation from cultures in control or after siCASK. (c)

Panels show total expression of HEK cell lysates transfected with P2X3 and CASK or GFP. β-

actin is used for gel loading control. NT shows no signal in non-transfected HEK cell lysates.

Histograms quantify increased P2X3/CASK co-immunoprecipitation after CASK over

expression in HEK cells (n=3 experiments, p=0.03). (d) HEK cells transfected with P2X3 and

GFP or P2X3 and CASK are used for P2X3 immunoprecipitation experiments as above.

Different negative control lanes include (from left to right): Ab, antibody alone showing no

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crossreactivity with IgG heavy chains; NT shows no signal in P2X3 immunoprecipitates from

non-transfected HEK cell lysates; no primary, indicating lack of unspecific signal of secondary

antibody crossreactivity. (e) Example of P2X3 pull down with anti-Myc antibody

immunoprecipitation of P2X3/CASK-myc co-transfected lysates. Note stronger expression of

P2X3 protein in the complex when co-transfected with CASK. (f) Example of enhanced P2X3

receptors serine phospshorylation in P2X3/CASK co-transfected HEK cells vs P2X3/GFP alone.

To quantitatively evaluate P2X3 receptor phosphorylated residues, we loaded equal amounts of

P2X3 receptor in different lanes (D’Arco et al 2009). Histograms quantify serine

phosphorylation of P2X3 receptors with or without CASK overexpression in HEK cells (n=3

experiments, p=0.03). Immunoprecipitates of cell extracts with an unrelated IgG antibody (Ab)

were also shown as control.

Fig 4. CASK domains necessary for P2X3 receptor interaction

Left: Western blot of HEK cells lysates co-transfected P2X3 with full length CASK and domain

deletion mutants ΔCAMK, ΔPDΖ, ΔSH3 and ΔGK, as indicated. Transfection with P2X3 only

was used as control (see methods), while β-Actin shows loading control. Note upregulation of

total P2X3 protein expression in co-transfected cells. Right: histogram quantifies the relative

P2X3 expression in different groups, as indicated. n= 4 experiments; p <0.05.

Fig. 5 Dynamic changes in P2X3/CASK association

(a) Left, CASK expression in purified P2X3 receptor samples from trigeminal ganglion cultures

is enhanced by acute NGF application (100 ng/ml, 5 min) following overnight neutralizing NGF

antibody (αNGF; 14 h) application to block endogenous NGF. P2X3 or CASK input is also

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shown. In order to exclude artifact signals due to contamination of heavy chain of anti-NGF

antibody used for the NGF deprivation treatment, an aliquot of an unrelated IgG was also loaded

in first lane as control. Right, Histograms quantify increase in co-precipitation of CASK and

P2X3 receptors by NGF application (n=3 experiments, p=0.03); data of immunoprecipitated

CASK are normalized with respect to total P2X3 expression. (b) Left, Example of effect of

sustained application of α,β-meATP (100 μM, 30 s) on CASK expression by P2X3 receptors

immunoprecipitation from transfected HEK cells. The action of α,β-meATP is blocked by co-

applying the P2X3 antagonist A-317491 (10 μM, 30 s). Right, Histograms quantify the effects of

α,β-meATP exemplified on the left (n=5 experiments, *p=0.027; data expressed as in a). (c),

Immunoblots run with protocol similar to the one in b, show the P2X3/CASK complex in

trigeminal primary cultures after a short α,β-meATP pulse (100 μM, 30 sec). Histograms quantify

the effect (n=3, p>0.05).

Fig. 6 Electrophysiology of P2X3 receptors after siCASK

(a) Examples of neuronal currents elicited by short (2 s) application of α,β-meATP at the doses

indicated above traces. Note smaller responses after siCASK. (b) histograms quantify responses

(expressed as current density) evoked by α,β-meATP that were significantly depressed after

siCASK. Current density: For 1 μM, Control n= 24 vs. siCASK n= 20, (*, p=0.02). For 3 μM,

control n=19 versus n=24 for siRNA (*, p=0.04). For 10 μM, control n=11 versus n=7 for

siRNA (*, p=0.02). After siCASK, no change in the time constant of current decay (c), or current

recovery at 30 s (d), expressed as % of first response following a double pulse application of α,β-

meATP) is observed. (e) histograms quantify effects of wildtype CASK or its mutants ΔCAMK,

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ΔPDΖ, ΔSH3 and ΔGK on currents evoked by short (2s) pulses of α,β-meATP applied to

P2X3/CASK co-transfected HEK cells or P2X3/GFP co-transfected controls cells. CASK

overexpression or CASK mutants ΔCAMK, ΔPDΖ significantly enhanced P2X3 receptor

responses (n=19 for P2X3; n=21, *p=0.03 for CASK WT; n=15, *p= 0,03 for ΔCAMK; n=13,

p=0,73 for ΔSH3; n=13, *p=0,02 for ΔPDΖ; n=9, p=0.07 for ΔGK; data were compared vs

control).

Supplemental Fig 1. Effect of CASK silencing in P2X3 receptor transfected HEK cells.

Example of western blot of P2X3-transfected HEK cells lysates in control and after siCASK,

shows total P2X3 protein expression. Histograms quantify reduced P2X3 expression after

siCASK (n=3 experiments; p=0.04).

Supplemental Fig. 2. P2X3 receptors in trigeminal ganglia

(a), example of P2X3 receptors western blot from trigeminal ganglia culture lysates shows

enhanced P2X3 receptor expression after application of the proteasome inhibitor MG-132 (4 h).

(b), confocal microscopy photographs show similar distribution of P2X3 (green) or CASK (red)

immunoreactivity in intact ganglia.

Supplemental Fig 3. Effect of BFA on P2X3 receptor expression

Lysates from trigeminal neuronal culture in control or after siCASK were incubated with BFA (5

μg/ml; 30 min) and analysed for total P2X3 receptor expression or CASK. Note that, while there

was higher P2X3 expression after BFA, the P2X3 expression was decreased in CASK

knockdown cells as well as BFA treated CASK knockdown cells.

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