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Cell Calcium 54 (2013) 120– 125

Contents lists available at SciVerse ScienceDirect

Cell Calcium

jo u rn al h om epage: www.elsev ier .com/ locate /ceca

RPC1-mediated Ca2+ and Na+ signalling in astroglia: Differential filtering ofxtracellular cations

eno C. Reyesa,b, Alexei Verkhratskyc,d,e, Vladimir Parpuraa,f,∗

Department of Neurobiology, Center for Glial Biology in Medicine, Atomic Force Microscopy & Nanotechnology Laboratories, Civitan International Research Center,velyn F. McKnight Brain Institute, University of Alabama, Birmingham, AL 35294, USADepartment of Psychiatry, Langley Porter Psychiatric Institute, 401 Parnassus Avenue, University of California, San Francisco, San Francisco, CA 94143, USAFaculty of Life Sciences, The University of Manchester, Manchester M13 9PT, UKIKERBASQUE, Basque Foundation for Science, 48011 Bilbao, SpainDepartment of Neurosciences, University of the Basque Country (UPV/EHU), 48940 Leioa, SpainDepartment of Biotechnology, University or Rijeka, 51000 Rijeka, Croatia

a r t i c l e i n f o

rticle history:eceived 7 May 2013eceived in revised form 13 May 2013ccepted 14 May 2013vailable online 12 June 2013

eywords:

a b s t r a c t

Canonical transient receptor potential 1 (TRPC1) plasmalemmal cation channels mediate Ca2+ and Na+

fluxes and control respective cytoplasmic ion signals in rat cortical astrocytes. Mechanical stimulationof astrocytes results in an increase in the levels of cytosolic Ca2+ and Na+ that are in part due to entryof extracellular cations through TRPC1 containing channels. Inhibition of the TRPC1 pore with an anti-body against its selective filter reduced cytosolic Ca2+ accumulation caused by mechanical stimulation.In contrast, this immunological treatment increased the cytosolic Na+ peak accumulation induced by

strocytealciumifferential filteringodiumtore operate calcium entry

mechanical stimulation. We propose that TRPC channels are amenable to changes in selective filtering,as mutations in previous studies and antibody binding in our present study differentially affect the fluxof Ca2+ and Na+. TRPC1 containing channels might represent focal points for co-ordination of Ca2+ andNa+ signalling in astroglia and this can have consequences on Ca2+- and Na+-dependent processes such asregulated exocytosis and lactate production, respectively, which in turn can modulate neuronal synaptictransmission.

. Introduction

Astrocytes, the main homeostatic cells of the central nervousystem (CNS), express multiple plasmalemmal ion channels thatontribute to cytosolic ion signalling, which is central for astroglialomeostatic functions [1–3]. These channels are activated by mul-iple stimuli such as neurotransmitters or mechanical stimulation.his latter mechanism is physiologically relevant for astrocytesince they have remarkable morphological plasticity and mayapidly change their volume. The majority of plasmalemmal ionhannels expressed in astrocytes belong (with obvious exceptionf selective K+ channels) to nonselective cationic channels perme-ble to Ca2+, Na+ and K+. Activation of these channels results in Ca2+

nd Na+ signalling events that control various functional responses

4,5].

Canonical transient receptor potential 1 (TRPC1) channel isne of the seven mammalian TRPC subtypes widely expressed in

∗ Corresponding author at: Department of Neurobiology, 1719 6th Avenue South,IRC 429, University of Alabama, Birmingham, AL 35294, USA. Tel.: +1 205 996 7369.

E-mail address: vlad@uab.edu (V. Parpura).

143-4160/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.ceca.2013.05.005

© 2013 Elsevier Ltd. All rights reserved.

various tissues [6]. TRPC1 is a nonselective cation channel withequal permeability for Ca2+ and Na+ and is different from otherTRPC channels, some of which are highly Ca2+ permeable withPCa/Pmonovalent between 2 and 9 [7]. Astroglial cells express severaltypes of TRPC proteins, which, through heteromeric assembly withthe TRPC1 subunit (this subunit is known to be obligatory for chan-nel formation), form functional channels [8–11]. TRPC channelsin astrocytes are responsible for the generation of store-operatedCa+ entry (SOCE), although the mechanism linking them to theendoplasmic reticulum (ER) Ca2+ store remains elusive. Targetingthe TRPC1 subunit in astrocytes with a blocking antibody or byreducing TRPC1 expression with a silencing RNA treatment sub-stantially reduced the SOCE in cultured astroglial cells [8,10]. TheTRPC1-mediated SOCE contributes to astroglial Ca2+ signalling andit has been shown to modulate Ca2+-dependent vesicular glutamaterelease in cortical astrocytes in response to mechanical stimu-lation [12,13]. The TRPC channel in astrocytes can also providesubstantial Na+ influx that may occur in response to mechanical

stimulation or develop alongside with SOCE following depletion ofthe ER Ca2+ store. This Na+ influx can be instrumental for local Na+

signals that are critical for glial homeostatic response [4,14]. In thisstudy, we characterized TRPC1-mediated Ca2+ and Na+ signalling in

alcium

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−20 mV, 10 ms square pulses at 50 Hz. Once established, cell contactwas maintained for ∼1 s. The strength of the stimulus, expressed as�R/R0 (%), where R0 represents the pipette resistance (2.6–4.8 M�)

R.C. Reyes et al. / Cell C

echanically stimulated cultured astrocytes and demonstrate thata2+ and Na+ fluxes can be dissociated following the treatment with

TRPC1 blocking antibody.

. Materials and methods

.1. Astrocyte cultures

Solitary astrocytes from visual cortices of 1–2-day-oldprague–Dawley rats were maintained in vitro as previouslyescribed [15]. Briefly, visual cortices were dissected and enzy-atically treated with papain (20 IU/ml, 1 h at 37 ◦C) in the

resence of l-cysteine (0.2 mg/ml); digestion was arrested byrypsin inhibitor (10 mg/ml; type II-O; 5 min at room tempera-ure). Tissue was mechanically dissociated and neural cells wereeeded into culture flasks containing culture medium composedf �-minimum essential medium (�-MEM, without phenol red;ife Technologies Corp. InvitrogenTM, Carsbad, CA, USA) supple-ented with foetal bovine serum (10%, v/v; Thermo ScientificyClone, Logan, UT, USA), glucose (20 mM), l-glutamine (2 mM),

odium pyruvate (1 mM), sodium bicarbonate (14 mM), peni-illin (100 IU/ml), and streptomycin (100 �g/ml), pH 7.35. Afterllowing cells to adhere to the bottom of the flasks for 1 h, theyere washed and provided with fresh media. Cells were thenaintained at 37 ◦C in a 95% air/5% CO2 incubator for 5–7 days to

each ∼60% confluency. At that juncture, the cell cultures wereurified for astrocytes using previously described procedure [16].urified astrocytes were detached from the flasks using trypsin10,000 N�-benzoyl-arginine ethyl ester hydrochloride units/ml;igma–Aldrich, St. Louis, MO, USA). After inhibition of trypsinctivity by the addition of complete culture medium, cells wereelleted using centrifugation (100 × g for 10 min), resuspendednd plated onto round (12 mm in diameter) glass coverslipsThermo Fisher Scientific) pre-coated with polyethyleneimine1 mg/ml; Sigma). Purified astrocytes were kept in culture mediumt 37 ◦C in a 95% air/5% CO2 atmosphere incubator for 5–8 daysntil used in experiments. The purity of astrocytic culture (>99%)as confirmed: (i) by indirect immunocytochemistry using anti-

lial fibrillary acidic protein antibody and (ii) by visualizationf the accumulation of a dipeptide, �-Ala-Lys, conjugated to-amino-4-methylcoumarin-3-acetic acid as previously described16]. Astrocytes in our culture system are flat polygonal cells andhus have a simplified morphology compared to astrocytes in situ16,17].

.2. Anti-TRPC1 antibody treatment

Astrocytes grown on coverslips were incubated in external solu-ion (pH 7.35) consisting of sodium chloride (140 mM), potassiumhloride (5 mM), calcium chloride (2 mM), magnesium chloride2 mM), HEPES (10 mM), and glucose (5 mM), with or without0 �g/ml of anti-TRPC1 antibody (cat. No. ACC-010, Alomone labs,erusalem, Israel) for 30 min at room temperature (22–25 ◦C) asescribed previously [10]. Antibody incubation was performedfter loading cells with either the Ca2+ indicator fluo-3 ace-oxymethyl (AM) ester or the Na+ indicator CoroNaTMGreen AMnd de-esterification of the indicators. Antibody was kept in solu-ion during the entire imaging procedure lasting ∼200 s for Ca2+

nd Na+ measurements.

.3. Intracellular Ca2+ imaging

Cytosolic Ca2+ concentration ([Ca2+]i) in the somata of culturedolitary astrocytes were assessed using the Ca2+ indicator fluo-3 asescribed earlier [17]. Briefly, astrocytes were loaded with fluo-3M (10 �g/ml; Life Technologies Corp. InvitrogenTM) in external

54 (2013) 120– 125 121

solution containing pluronic acid (0.025%, w/v) for 30 min at roomtemperature. To allow de-esterification of fluo-3 AM, cells weresubsequently kept in external solution for 30 min at room tem-perature. Coverslips were transferred onto a recording chambermounted on an inverted microscope, and astrocytes were visu-alized with a standard fluorescein isothiocyanate (FITC) filter set(Chroma Technology, Rockingham, VT, USA). Fluorescence inten-sities obtained from the somata of indicator-loaded astrocyteswere corrected (digital subtraction) for the background fluores-cence measured from regions of coverslips containing no cells.Fluorescence data were expressed as �F/F0 (%) with the cell base-line fluorescence (F0) representing the average of the first 5 imagesbefore mechanical stimulation, while �F represents the change influorescence emission. The [Ca2+]i was determined using calibra-tion of fluo-3 as described elsewhere [18].

2.4. Intracellular Na+ imaging

Cytosolic Na+ concentration ([Na+]i) in the somata of cul-tured solitary astrocytes was monitored using the Na+ indicatorCoroNaTMGreen AM (10 �M; Life Technologies Corp. InvitrogenTM)[15,19]. Astrocytes were loaded with the indicator, imaged, anddata were collected and processed as described above for Ca2+

imaging. Because CoroNaTMGreen tends to leak out of the cell, itsintracellular fluorescence intensity substantially decays over time[19]. Consequently, using a linear regression and extrapolation ofthe baseline fluorescence of individual traces, we corrected themfor the leak of the dye. The [Na+]i was determined using calibrationof CoroNaTMGreen as described elsewhere [15].

2.5. Image acquisition and processing

An inverted microscope (TE 300; Nikon, Melville, NY, USA),equipped with differential interference contrast and wide-field flu-orescence illumination, was used in all experiments. Experimentswere performed using a 60× Plan Apo oil-immersion objective(1.4 numerical aperture; Nikon). Images were acquired using aCoolSNAP-HQ cooled charge-coupled device camera (Roper Sci-entific Inc., Tucson, AZ, USA) driven by V++ imaging software(Digital Optics Ltd., Auckland, New Zealand). All raw data/imageshad their pixel intensities within the camera’s dynamic range(0–4095). The �F/F0 of the control and treatment groups wereranked and normalized to control to accommodate for variationsin culture conditions, and to allow comparisons between experi-mental batches, as we previously described [15].

2.6. Mechanical stimulation

To stimulate a solitary astrocyte of interest, we employedmechanical contact using a glass pipette filled with external solu-tion as we described elsewhere [15,17]. This approach allowsspatio-temporal control of the stimulus application without affect-ing plasma membrane integrity. The establishment of the patchpipette contact with the plasma membrane was determined byan increase in pipette resistance monitored using a patch-clampamplifier (PC-ONE; Dagan, Minneapolis, MN, USA) that delivered

prior to establishing a pipette–astrocyte contact, and �R repre-sents the increase in the resistance (0.05–0.28 M�) during thecontact, had comparable intensities under all conditions tested(Mann–Whitney U-test, P = 0.253–0.351).

122 R.C. Reyes et al. / Cell Calcium 54 (2013) 120– 125

Fig. 1. Cytosolic Ca2+ accumulation in astrocytes induced by mechanical stimulation is reduced by an antibody that binds to the pore forming region of TRPC1. (A) Arepresentative image of a solitary rat cortical astrocyte at rest and loaded with fluo-3. Scale bar = 20 �m. (B) Average kinetics of fluo-3 fluorescence from mechanicallystimulated astrocytes. (C and D) Bar graphs showing normalized peak and cumulative cytosolic Ca2+ responses to mechanical stimulation. Both responses were significantlyr in). Pd rred.

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educed when astrocytes were treated with an anti-TRPC1 antibody (30 �g/ml, 30 mirections for clarity. Arrow indicates the time when the mechanical stimulus occusterisks denote a significant change in measurement when compared to control g

.7. Statistical analysis

The comparison of the increase in pipette resistanced inifferent conditions and effects of an anti-TRPC1 antibody onechanically induced intracellular Ca2+ and Na+ loads were tested

sing Mann–Whitney U-test. Data are expressed, as means ± SEMs.

. Results

.1. Cytosolic Ca2+ responses to mechanical stimulation in ratortical astrocytes is reduced by an anti-TRPC1 antibody

Mechanical stimulation of cortical astrocytes generates a rapidncrease in cytosolic Ca2+ that slowly decays to the basal level17,20]. Previous studies conducted by us and others [8,10] pro-ided conclusive evidence that this increase, in part, results froma2+ entry through TRPC1 containing channels, and that applicationf an anti-TRPC1 antibody directed at amino acid residues 557–57121,22] inhibited this Ca2+ entry [10,12]. In this study, mechanicaltimulation of solitary astrocytes with glass pipettes induced tran-ient increase in [Ca2+]i (Fig. 1). Incubation of astrocytes with annti-TRPC1 antibody significantly reduced the peak of the [Ca2+]iransient; �F/F0 fell from 408 ± 42% in control to 320 ± 38% (n = 6,ig. 1). These changes in fluo-3 fluorescence correspond to cytosolica2+ concentrations of ∼1.7 �M and ∼860 nM, respectively; res-ing [Ca2+]i levels were ∼70 nM. Both the peak of [Ca2+]i transientsFig. 1C) and cumulative (area under the curve) [Ca2+]i responsesFig. 1D) in anti-TRPC1 treated astrocytes were significantly lowerhan those of control cells (n = 6, Mann–Whitney U-test, P < 0.01).

.2. The peak cytosolic Na+ response to mechanical stimulation inat cortical astrocytes is increased by an anti-TRPC1 antibody

Mechanical stimulation of astrocytes triggered a large transientncrease in [Na+]i (Fig. 2). Changes in peak fluorescence measured inontrol conditions averaged 58 ± 5% (�F/F0; n = 18) that correspond

oints and bars represent means ± SEM of measurements. SEMs are shown in singleNumbers in parentheses indicate the number of astrocytes studied in each group.i.e. untreated astrocytes (Mann–Whitney U-test; **p < 0.01).

to a [Na+]i increase to ∼35.5 mM from the resting level of ∼17 mM.Incubation of cultured astrocytes with an anti-TRPC1 antibodyincreased mechanically induced [Na+]i transients. An increase inthe �F/F0 peak averaged 72 ± 7% (Fig. 2; n = 18, Mann–WhitneyU-test, P < 0.01) that corresponded to the peak [Na+]i transient of∼42.6 mM. At the same time the cumulative [Na+]i response mea-sured from anti-TRPC1 antibody treated cells was not significantlydifferent when compared to control cells (Fig. 2D).

4. Discussion

Mounting highly heterogeneous and precisely controlledhomeostatic responses to a continuously changing interstitial envi-ronment of the CNS is the raison d’etre of astroglia. The rangeof these responses is remarkable as astrocytes regulate fluxesof physiologically relevant ions such as K+, Na+, Ca2+, H+, Cl−,HCO3

−, maintain glutamine–glutamate and glutamine–GABA shut-tles, supply lactate to active neuronal compartments, control watermovements, secrete scavengers of reactive oxygen species andmany more [23,24]. These responses are critically important for thefunctional connectivity in the CNS because they maintain neuronalexcitability and synaptic transmission. For this purpose, astrocytescontinuously monitor neuronal activity by multiple neurotransmit-ter receptors and ion channels [25,26]. Activation of these channelsand receptors often triggers highly localized fluctuations of Ca2+

and Na+ in astroglial cytosol that constitute a substrate for glialexcitability [4,27].

Astrocytes possess a high degree of morphological plasticity,as indeed neuronal activity results in highly dynamic redistribu-tion of water and transient changes in the volume of perisynapticastroglial processes [28,29]; similarly astroglia exhibits a vol-ume response to changes in extracellular osmotic pressure [30].

These dynamic changes in cell volume activate signalling cascadesthrough mechano-sensitive channels that mediate ion fluxes inresponse to mechano-stimulation of the plasmalemma, resultingin cytosolic Ca2+ and Na+ signals [15]. Array of mechano-sensitive

R.C. Reyes et al. / Cell Calcium 54 (2013) 120– 125 123

Fig. 2. Cytosolic Na+ accumulation in astrocytes induced by mechanical stimulation is augmented by an antibody that binds to the pore forming region of TRPC1. (A) Arepresentative image of a solitary rat cortical astrocyte at rest and loaded with CoroNaTMGreen. Scale bar = 20 �m. (B) Average kinetics of CoroNaTMGreen fluorescence fromm and cs (MannF

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echanically stimulated astrocytes. (C and D) Bar graphs showing normalized peakignificantly increased when astrocytes were treated with an anti-TRPC1 antibody

ig. 1.

stroglial channels most likely consist of several channel types, ofhich TRPC1 is a sound candidate. Indeed stretching the mem-

rane of frog oocytes or CHO-K1 cells transfected with TRPC1ctivated ionic currents, whereas inhibition of TRPC1 synthesis byntisense RNA reduced these mechano-sensitive current responses31]. Here, we further corroborate this hypothesis by demonstrat-ng that the treatment of astrocytes with an antibody against TRPC1hannel inhibited [Ca2+]i transients triggered by mechanical stim-lation. Of note, however, other mechano-sensitive channels maynderlie cation fluxes. Recently, channels of Piezo1 and Piezo2ypes have been identified as mechano-sensitive channels in dorsaloot ganglion neurones [32]. The Piezo1 channel can be selectivelynhibited by peptide toxin GsMTx4, isolated from tarantula venom,nd GsMTx4 was reported to inhibit stretch-activated channels instrocyte membranes [33].

We further confirmed that mechanical stimulation of astrocytesriggers large (up to 20 mM in amplitude) increases in [Na+]i’ whichlso were found to be sensitive to anti-TRPC1 antibody. In contrasto [Ca2+]i responses, however, inhibition of the TRPC1 channels byhe anti-TRPC1 antibody resulted in a significant increase in theeak amplitude of [Na+]i response. Thus binding of the antibodyo the TRPC1 channel decreases Ca2+ flux with a parallel increasen Na+ flux. Somewhat similar observations were gathered in site-irected mutagenesis studies of the TRPC1 and TRPC3 channels.ubstitution of seven acidic residues to basic amino acids in thehannel region of TRPC1 inhibited Ca2+ movement without affect-ng Na+ fluxes [34]. A single mutation (E630Q) of the selectivelter of TRPC3 markedly inhibited Ca2+ current, whereas increasinga+ currents through the channel especially at negative membraneotentials [35]. Incidentally, the Basic Local Alignment Search ToolBLAST®) comparison of rat TRPC1 and TRPC3 proteins reveals thathe TRPC1 antibody peptide target (amino acids 557–571) and the

utated site of TRPC3 are both located in putative selective filtershat are not highly conserved [34,35]. Taken together, these studiesuggest that at least some TRPC channels are amenable to muta-ions that disrupt Ca2+ but preserve (or even increase) Na+ entry.

umulative cytosolic Na+ responses to mechanical stimulation. The peak value was–Whitney U-test; **p < 0.01). Points, bars, arrow and numbers in parentheses as in

This observation may be important to differentially modulate TRPchannels in heath and disease.

Our results suggest that the TRPC1 antibody specifically andfunctionally affects the Ca2+ selectivity filter of the TRPC1 chan-nel. However, mechanical stimulation of astrocytes treated withthe TRPC1 antibody could simply lead to TRPC1 channel dilationwithout a change to the selectivity of the pore, causing an aug-mented Na+ and Ca2+ entry. However, that was not the outcomeof our experiments as we have recorded a dissociation betweenunilaterally affected Ca2+ responses (both the peak and cumula-tive responses were reduced) and Na+ responses (only the peakresponse was enhanced). Another intriguing, albeit unlikely, possi-bility is that the bound antibody, which affects the Ca2+ selectivityfilter, could dilate the TRPC1 channel during mechanical stimula-tion. Such conditions could allow an increase of Na+ flux, whichwould be manifested as an increase in the amplitude of the [Na+]itransient as we recorded.

It should be noted that we have not addressed the ability of anti-TRPC1 antibody to modulate ion dynamics in astrocytes at rest. Itis likely that the antibody would require an opening of the channelto bind to the pore region. Such opening events in unstimulatedastrocytes at rest are considered rare as TRPC1 channels are acti-vated when the ER store is getting depleted. Nonetheless, bindingof the antibodies to TRPC1 channels in astrocytes at rest may causecell depolarization and reduce the driving force for Ca2+ and Na+

entry to the cytosol. In such a scenario, in mechanically stimulatedastrocytes treated with the anti-TRPC1 antibody, there would be,however, a more profound decrease in [Ca2+]i and a less prominentincrease of [Na+]i than those we observed.

Another fundamental function of TRPC1 containing channels inastroglia lies in their role in SOCE. This mechanism of the Ca2+

entry is universally present in neuroglial cells and is ubiquitous

in astrocytes. Stimulation of astroglia in vitro or in situ with neuro-transmitters initiates complex [Ca2+]i responses comprising of theER Ca2+ release-dependent initial peak and the SOCE dependentlong-lasting plateau [8,10,36–38]; it should be noted that SOCE can

124 R.C. Reyes et al. / Cell Calcium

Table 1Reversal potential of astroglial plasmalemmal NCX.

Cytosolic ion concentrations Condition ENCX

[Na+]i = 16.6 mM Resting −98 mV[Ca2+]i = 73 nM

[Na+]i = 35.5 mM Mechanical stimulation; −76 mV[Ca2+]i = 1.7 �M Control

[Na+]i = 42.6 mM Mechanical stimulation; −107 mV[Ca2+]i = 861 nM TRPC1 antibody treatment

Reversal potential of NCX (ENCX) at 25 ◦C was calculated using our recorded cyto-solic Na+ and Ca2+ concentrations together with concentrations of these ions inthe external solution ([Na+]o = 140 mM and [Ca2+]o = 2 mM) and presumed NCX 3:1s

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[26] A. Verkhratsky, V. Parpura, Recent advances in (patho)physiology of astroglia,Acta Pharmacologica Sinica 31 (2010) 1044–1054.

toichiometry.

ontribute to some extent even to the peak response [10]. Similarlyeceptor independent depletion of ER Ca2+ store results in promi-ent SOCE in astrocytes [39,40]. Sites of astroglial SOCE are localizedlose to the ER thus increasing the efficacy of Ca2+ store replenish-ent [8,41]. The SOCE pathway is also prominent in pathologicallyodified glial cells such as for example in glioblastoma [42].TRPC1 containing channels have been identified as a main mech-

nism for SOCE in astroglia [5,8,10]. This endows them with anotherighly important role in the co-ordination of ionic signalling instrocytes. Being store-operated, TRPC1 channels establish a func-ional link between metabotropically induced Ca2+ signalling witha+ influx thus coordinating Ca2+ and Na+ signalling in astroglial

ub-compartments. In this study, we found that TRPC1 chan-els can potentially regulate their permeability to Ca2+ and Na+,hus being capable of dynamic control over local ionic signalling.uch changes of [Ca2+]i and [Na+]i could affect Ca2+- and/or Na+-ependent processes. Increase of [Ca2+]i can trigger regulatedxocytosis of glutamate [43], while increase of [Na+]i can triggerlycolysis leading to lactate production [44]; both events are impor-ant for the modulation of synaptic transmission and plasticity45,46]. The physiological mechanism of such regulation of TRPC1ermeability remains unknown (and it may include enzymatic orost-translational modification) and requires further investigation.

The TRPC1 channels were also reported to co-localize withstroglial plasmalemmal Na+/Ca2+ exchangers (NCX), which in turnre concentrated in perisynaptic processes, also being closely asso-iated with Na+/K+ pumps, glutamate ionotropic receptors, andlutamate/GABA Na+-dependent transporters [4]. This strategicocalization allowing focal Na+ and Ca2+ fluxes could be of criti-al importance for fast neuronal–astroglial signalling at the singleynaptic level. For example TRPC1-mediated changes in [Ca2+]i andNa+]i determined in this study can substantially affect reversalotential of astroglial NCX (ENCX); this has been suggested as a keyechanism underpinning TRPC3 signalling [47]. Indeed, the res-

ing ENCX in our experimental conditions is estimated at −98 mV, ithifts to −76 mV in astrocytes mechanically stimulated in controlonditions and to −107 mV in cells stimulated in the presence ofn anti-TRPC1 antibody (see Table 1 for details). These fluctuationsn ENCX have important functional consequences. In normal condi-ions, mechanical stimulation promotes reversal mode of NCX anda2+ entry [15], whereas after TRPC1 modification cell stimulation

avours forward mode of NCX resulting in Ca2+ extrusion and addi-ional Na+ entry. As outlined above, this can govern Ca2+- and/ora+-dependent processes in astrocytes that can in turn modulate

he operation of the tripartite synapse.In conclusion the TRPC1 channels appear as a focal point for the

o-ordination of Ca2+ and Na+ signalling in astroglia. The capabilityf TRPC1 to dynamically modify Ca2+ and Na+ fluxes may be rel-

vant for this coordination by increasing plastic potential of ionicignalling in astroglia.

[

54 (2013) 120– 125

Acknowledgments

This work was supported by the National Science Founda-tion (CBET 0943343). Reno C. Reyes was additionally funded byUCSF Neuroscience and Schizophrenia T32 (MH 089920). We thankManoj K. Gottipati for comments on a previous version of thismanuscript.

References

[1] V. Parpura, A. Verkhratsky, Homeostatic function of astrocytes: Ca2+ and Na+

signalling, Translational Neuroscience 3 (2012) 334–344.[2] V. Parpura, A. Verkhratsky, Neuroglia at the crossroads of homoeostasis,

metabolism and signalling: evolution of the concept, ASN NEURO 4 (2012)201–205.

[3] A. Verkhratsky, C. Steinhauser, Ion channels in glial cells, Brain Research. BrainResearch Reviews 32 (2000) 380–412.

[4] S. Kirischuk, V. Parpura, A. Verkhratsky, Sodium dynamics: another key toastroglial excitability? Trends in Neurosciences 35 (2012) 497–506.

[5] A. Verkhratsky, J.J. Rodriguez, V. Parpura, Calcium signalling in astroglia, Molec-ular and Cellular Endocrinology 353 (2012) 45–56.

[6] I.S. Ambudkar, H.L. Ong, Organization and function of TRPC channelosomes,Pflugers Archiv: European Journal of Physiology 455 (2007) 187–200.

[7] G. Owsianik, K. Talavera, T. Voets, B. Nilius, Permeation and selectivity of TRPchannels, Annual Review of Physiology 68 (2006) 685–717.

[8] V.A. Golovina, Visualization of localized store-operated calcium entry in mouseastrocytes. Close proximity to the endoplasmic reticulum, Journal of Physiology564 (2005) 737–749.

[9] M. Grimaldi, M. Maratos, A. Verma, Transient receptor potential channel activa-tion causes a novel form of [Ca2+]i oscillations and is not involved in capacitativeCa2+ entry in glial cells, Journal of Neuroscience 23 (2003) 4737–4745.

10] E.B. Malarkey, Y. Ni, V. Parpura, Ca2+ entry through TRPC1 channels contributesto intracellular Ca2+ dynamics and consequent glutamate release from rat astro-cytes, Glia 56 (2008) 821–835.

11] P. Pizzo, A. Burgo, T. Pozzan, C. Fasolato, Role of capacitative calcium entry onglutamate-induced calcium influx in type-I rat cortical astrocytes, Journal ofNeurochemistry 79 (2001) 98–109.

12] V. Parpura, V. Grubisic, A. Verkhratsky, Ca2+ sources for the exocytotic releaseof glutamate from astrocytes, Biochimica et Biophysica Acta 1813 (2011)984–991.

13] R. Zorec, A. Araque, G. Carmignoto, P.G. Haydon, A. Verkhratsky, V. Parpura,Astroglial excitability and gliotransmission: an appraisal of Ca2+ as a signallingroute, ASN NEURO 4 (2012), http://dx.doi.org/10.1042/AN20110061.

14] C.R. Rose, C. Karus, Two sides of the same coin: sodium homeostasis andsignaling in astrocytes under physiological and pathophysiological conditions,Glia (2013), http://dx.doi.org/10.1002/glia.22492.

15] R.C. Reyes, A. Verkhratsky, V. Parpura, Plasmalemmal Na+/Ca2+ exchangermodulates Ca2+-dependent exocytotic release of glutamate from rat corticalastrocytes, ASN NEURO 4 (2012), http://dx.doi.org/10.1042/AN20110059.

16] V. Montana, Y. Ni, V. Sunjara, X. Hua, V. Parpura, Vesicular glutamatetransporter-dependent glutamate release from astrocytes, Journal of Neuro-science 24 (2004) 2633–2642.

17] X. Hua, E.B. Malarkey, V. Sunjara, S.E. Rosenwald, W.H. Li, V. Parpura, Ca2+-dependent glutamate release involves two classes of endoplasmic reticulumCa2+ stores in astrocytes, Journal of Neuroscience Research 76 (2004) 86–97.

18] V. Parpura, P.G. Haydon, Physiological astrocytic calcium levels stimulate glu-tamate release to modulate adjacent neurons, Proceedings of the NationalAcademy of Sciences of the United States of America 97 (2000) 8629–8634.

19] S.D. Meier, Y. Kovalchuk, C.R. Rose, Properties of the new fluorescent Na+ indi-cator CoroNa Green: comparison with SBFI and confocal Na+ imaging, Journalof Neuroscience Methods 155 (2006) 251–259.

20] R.C. Reyes, V. Parpura, Mitochondria modulate Ca2+-dependent glutamaterelease from rat cortical astrocytes, Journal of Neuroscience 28 (2008)9682–9691.

21] S.Z. Xu, D.J. Beech, TrpC1 is a membrane-spanning subunit of store-operatedCa2+ channels in native vascular smooth muscle cells, Circulation Research 88(2001) 84–87.

22] S. Antoniotti, D. Lovisolo, A. Fiorio Pla, L. Munaron, Expression and functionalrole of bTRPC1 channels in native endothelial cells, FEBS Letters 510 (2002)189–195.

23] H. Kettenmann, B.R. Ransom, Neuroglia, Oxford University Press, Oxford, 2013,pp. 864.

24] A. Verkhratsky, A.M. Butt, Glial Physiology and Pathophysiology, Wiley-Blackwell, Chichester, 2013.

25] U. Lalo, Y. Pankratov, V. Parpura, A. Verkhratsky, Ionotropic receptors inneuronal–astroglial signalling: what is the role of “excitable” molecules innon-excitable cells, Biochimica et Biophysica Acta 1813 (2011) 992–1002.

27] C. Agulhon, J. Petravicz, A.B. McMullen, E.J. Sweger, S.K. Minton, S.R. Taves, K.B.Casper, T.A. Fiacco, K.D. McCarthy, What is the role of astrocyte calcium inneurophysiology? Neuron 59 (2008) 932–946.

alcium

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

R.C. Reyes et al. / Cell C

28] N.N. Haj-Yasein, V. Jensen, I. Ostby, S.W. Omholt, J. Voipio, K. Kaila, O.P. Ottersen,O. Hvalby, E.A. Nagelhus, Aquaporin-4 regulates extracellular space volumedynamics during high-frequency synaptic stimulation: A gene deletion studyin mouse hippocampus, Glia 60 (2012) 867–874.

29] M. Amiry-Moghaddam, O.P. Ottersen, The molecular basis of water transportin the brain, Nature Reviews Neuroscience 4 (2003) 991–1001.

30] H.K. Kimelberg, P. Sankar, E.R. O’Connor, T. Jalonen, S.K. Goderie, Functionalconsequences of astrocytic swelling, Progress in Brain Research 94 (1992)57–68.

31] R. Maroto, A. Raso, T.G. Wood, A. Kurosky, B. Martinac, O.P. Hamill, TRPC1 formsthe stretch-activated cation channel in vertebrate cells, Nature Cell Biology 7(2005) 179–185.

32] C. Bae, F. Sachs, P.A. Gottlieb, The mechanosensitive ion channel Piezo1 isinhibited by the peptide GsMTx4, Biochemistry 50 (2011) 6295–6300.

33] T.M. Suchyna, J.H. Johnson, K. Hamer, J.F. Leykam, D.A. Gage, H.F. Clemo, C.M.Baumgarten, F. Sachs, Identification of a peptide toxin from Grammostola spat-ulata spider venom that blocks cation-selective stretch-activated channels,Journal of General Physiology 115 (2000) 583–598.

34] X. Liu, B.B. Singh, I.S. Ambudkar, TRPC1 is required for functional store-operatedCa2+ channels. Role of acidic amino acid residues in the S5–S6 region, Journalof Biological Chemistry 278 (2003) 11337–11343.

35] M. Poteser, H. Schleifer, M. Lichtenegger, M. Schernthaner, T. Stockner,C.O. Kappe, T.N. Glasnov, C. Romanin, K. Groschner, PKC-dependent cou-pling of calcium permeation through transient receptor potential canonical3 (TRPC3) to calcineurin signaling in HL-1 myocytes, Proceedings of theNational Academy of Sciences of the United States of America 108 (2011)

10556–10561.

36] S. Kirischuk, S. Tuschick, A. Verkhratsky, H. Kettenmann, Calcium signallingin mouse Bergmann glial cells mediated by �1-adrenoreceptors andH1 histamine receptors, European Journal of Neuroscience 8 (1996)1198–1208.

[

[

54 (2013) 120– 125 125

37] T. Moller, C. Nolte, R. Burger, A. Verkhratsky, H. Kettenmann, Mechanisms of C5aand C3a complement fragment-induced [Ca2+]i signaling in mouse microglia,Journal of Neuroscience 17 (1997) 615–624.

38] S. Tuschick, S. Kirischuk, F. Kirchhoff, L. Liefeldt, M. Paul, A. Verkhratsky, H.Kettenmann, Bergmann glial cells in situ express endothelinB receptors linkedto cytoplasmic calcium signals, Cell Calcium 21 (1997) 409–419.

39] K.J. Lo, H.N. Luk, T.Y. Chin, S.H. Chueh, Store depletion-induced calciuminflux in rat cerebellar astrocytes, British Journal of Pharmacology 135 (2002)1383–1392.

40] K. Singaravelu, C. Lohr, J.W. Deitmer, Regulation of store-operated calciumentry by calcium-independent phospholipase A2 in rat cerebellar astrocytes,Journal of Neuroscience 26 (2006) 9579–9592.

41] T. Pivneva, B. Haas, D. Reyes-Haro, G. Laube, R.W. Veh, C. Nolte, G. Skibo, H.Kettenmann, Store-operated Ca2+ entry in astrocytes: different spatial arrange-ment of endoplasmic reticulum explains functional diversity in vitro and in situ,Cell Calcium 43 (2008) 591–601.

42] J. Hartmann, A. Verkhratsky, Relations between intracellular Ca2+ stores andstore-operated Ca2+ entry in primary cultured human glioblastoma cells, Jour-nal of Physiology 513 (1998) 411–424.

43] V. Parpura, R. Zorec, Gliotransmission: exocytotic release from astrocytes, BrainResearch Reviews 63 (2010) 83–92.

44] P.J. Magistretti, Neuron–glia metabolic coupling and plasticity, Journal ofExperimental Biology 209 (2006) 2304–2311.

45] A. Suzuki, S.A. Stern, O. Bozdagi, G.W. Huntley, R.H. Walker, P.J. Magistretti, C.M.Alberini, Astrocyte-neuron lactate transport is required for long-term memoryformation, Cell 144 (2011) 810–823.

46] G. Perea, A. Araque, Astrocytes potentiate transmitter release at single hip-pocampal synapses, Science 317 (2007) 1083–1086.

47] P. Eder, M. Poteser, C. Romanin, K. Groschner, Na+ entry and modulation ofNa+/Ca2+ exchange as a key mechanism of TRPC signaling, Pflugers Archiv:European Journal of Physiology 451 (2005) 99–104.