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Cellular/Molecular
Stable Membrane Expression of Postsynaptic Ca
V
1.2 CalciumChannel Clusters Is Independent of Interactions withAKAP79/150 and PDZ Proteins
ValentinaDiBiase,
1
GeraldJ.Obermair,
1
ZsoltSzabo,
1
ChristopheAltier,
2
JuanSanguesa,
2
EmmanuelBourinet,
2
andBernhardE.Flucher
1
1
Department of Physiology and Medical Physics, Innsbruck Medical University, A-6020 Innsbruck, Austria, and
2
De´partement de Physiologie, Laboratoirede Ge´nomique Fonctionnelle, Centre National de la Recherche Scientifique, 34094 Montpellier, France
InneuronsL-typecalciumcurrentscontributetosynapticplasticityandtoactivity-dependentgeneregulation.Thesubcellularlocaliza-tion of Ca
V
1.2 and its association with upstream and downstream signaling proteins is important for efficient and specific signaltransduction.HerewetestedthehypothesisthatA-kinaseanchoringproteins(AKAPs)orPDZ-proteinsareresponsibleforthetargetingandanchoringofCa
V
1.2inthepostsynapticcompartmentofglutamatergicneurons.Double-immunofluorescencelabelingofhippocam-palneuronstransfectedwithexternalHAepitope-taggedCa
V
1.2demonstratedthatclustersofmembrane-incorporatedCa
V
1.2-HAwerecolocalized with AKAP79/150 but not with PSD-95 in the spines and shafts of dendrites. To disrupt the interactions with these scaffoldproteins, we mutated known binding sequences for AKAP79/150 and PDZ proteins in the C terminus of Ca
V
1.2-HA. Unexpectedly, thedistributionpattern,thedensity,andthefluorescenceintensityofclustersweresimilarforwild-typeandmutantCa
V
1.2-HA,indicatingthat interactions with AKAP and PDZ proteins are not essential for the correct targeting of Ca
V
1.2. In agreement, brief treatment withNMDA(achemicalLTDparadigm)causedthedegradationofPSD-95andtheredistributionofAKAP79/150and
-actininfromdendriticspines into the shaft, without a concurrent loss or redistribution of Ca
V
1.2-HA clusters. Thus, in the postsynaptic compartment of hippocampal neurons Ca
V
1.2 calcium channels form signaling complexes apart from those of glutamate receptors and PSD-95. TheirnumberanddistributionindendriticspinesisnotaltereduponNMDA-induceddisruptionoftheglutamatereceptorsignalingcomplex,and targeting and anchoring of Ca
V
1.2 is independent of its interactions with AKAP79/150 and PDZ proteins.
Key words:
L-type Ca
2
channels; hippocampal neurons; dendritic spines; PSD-95;
-actinin; chemical LTD; immunofluorescencemicroscopy 
Introduction
In neurons calcium influx through ligand- and voltage-gatedchannels causes long-lasting changes of synaptic strength, whichunderlie learning and memory. NMDA receptor-mediated cal-cium influx into dendritic spines leads to the rearrangement of the postsynaptic signaling complex and ultimately to the alteredsurface expression of AMPA receptors. Activation of L-type cal-cium channels is required to transmit synaptic activity to theregulation of gene expression (Bading et al., 1993; Impey et al.,1996;Graefetal.,1999;Mermelsteinetal.,2000;Dolmetschetal.,2001). Knock-out of the L-type calcium channel Ca
V
1.2 isoformin the neocortex and hippocampus impairs NMDA receptor-independent long-term potentiation (LTP) and spatial memory (Moosmang et al., 2005). Thus, postsynaptic Ca
V
1.2 calciumchannels are critical in initiating long-lasting changes in synapticstrengththataredependentonproteinsynthesisbutindependentof NMDA receptor activation.The postsynaptic compartment of excitatory synapses con-tains two prominent classes of scaffold proteins: PDZ proteinsand AKAPs. The postsynaptic density protein-95 (PSD-95) andAKAP79/150 recruit protein kinases and phosphatases to gluta-mate receptors and are important for the modulation of synapticstrength (Lu¨scher et al., 1999). L-type calcium channels alsofunctionally interact with PDZ proteins and AKAPs. TheC-terminal PDZ-binding sequence (VSNL) of Ca
V
1.2 is essentialfor L-type calcium current-dependent activation of CREB-mediated gene-expression (Weick et al., 2003). A leucine zipperin the C terminus of Ca
V
1.2 binds AKAP79/150. This interactionisimportantforthereversiblephosphorylationofCa
V
1.2byPKAand calcineurin, and for the signaling to the nucleus throughNFATc4 (Oliveria et al., 2007). Thus, in dendritic spines of hip-pocampal neurons AKAP79/150 integrates Ca
V
1.2 in a signalingcomplex with the upstream
2
adrenergic receptor (Hooglandand Saggau, 2004) as well as with downstream signaling proteins(Davare et al., 2001).
Received July 9, 2008; revised Oct. 21, 2008; accepted Nov. 11, 2008.ThisworkwassupportedbygrantsfromtheAustrianScienceFundandtheAustrianNationalBank(P17806-B05andP20059-B05toB.E.F.andP17807-B05toG.J.O.)andfromtheTyroleanScienceFund(toG.J.O.).Thisworkispartof the thesis of V.D.B. We thank S. Baumgartner for her excellent technical assistance and P. Tuluc for functionalanalysis of calcium channel constructs.CorrespondenceshouldbeaddressedtoDr.BernhardE.Flucher,DepartmentofPhysiologyandMedicalPhysics,Inns-bruckMedicalUniversity,Fritz-Pregl-Strasse3,A-6020Innsbruck,Austria.E-mail:bernhard.e.flucher@i-med.ac.at.DOI:10.1523/JNEUROSCI.3213-08.2008Copyright©2008SocietyforNeuroscience 0270-6474/08/2813845-11$15.00/0
The Journal of Neuroscience, December 17, 2008
•
28(51):13845–13855 •
13845
 
Furthermore, PDZ proteins control the targeting of voltage-gatedcalciumchannelsintopresynapticandpostsynapticneuro-nal compartments (Maximov and Bezprozvanny, 2002; Zhang etal., 2005), and coexpression of AKAP79/150 promotes mem-brane expression of Ca
V
1.2 in
Xenopus
oocytes (Altier et al.,2002). Moreover, strong stimulation of cortical neurons has re-cently been shown to induce endocytosis of Ca
V
1.2 (Green et al.,2007). Therefore we examined whether interactions with AKAPsandPDZproteinsarenecessaryforthetargetingandstabilizationof Ca
V
1.2 calcium channels in the postsynaptic compartments of hippocampal neurons, and whether disruption of these scaffoldsin dendritic spines affects the distribution or membrane expres-sion of Ca
V
1.2.Three lines of evidence indicate that Ca
V
1.2 signaling com-plexes in dendritic spines of hippocampal neurons are structur-ally and functionally independent of the glutamate receptor sig-nalingcomplexes.First,Ca
V
1.2clusterswerenotcolocalizedwithPSD-95. Second, deletion of known interaction sequences forPDZ proteins and AKAPs did not reduce the density or size of Ca
V
1.2 clusters. Third, NMDA-induced disruption of the PSD-95/AKAPscaffoldindendriticspineswasnotaccompaniedbytheloss of Ca
V
1.2 clusters. Together, these results suggest that gluta-mate receptors and Ca
V
1.2 coexist in dendritic spines and inter-act with common scaffold proteins, but are constituents of sepa-rate signaling complexes and their membrane expression isregulated by independent mechanisms.
MaterialsandMethods
Primary culture of hippocampal neurons.
Low-density cultures of hip-pocampal neurons were prepared from 16.5-d-old embryonic BALB/cmice as described previously (Goslin et al., 1998; Obermair et al., 2003,2004). Briefly, dissected hippocampi were dissociated by trypsin treat-ment and trituration. Neurons were plated on poly-
L
-lysine-coated glasscoverslips in 60 mm culture dishes at a density of 3500 cells/cm
2
. Afterplating, cells were allowed to attach for 3–4 h before transferring thecoverslips neuron-side-down into a 60 mm culture dish with a glialfeeder layer. Neurons and glial feeder layer were cultured in serum-freeNeurobasalmedium(Invitrogen)supplementedwithGlutamaxandB27supplements (Invitrogen).
Transfection of hippocampal neurons.
Plasmids were introduced intoneurons on day 6 using Lipofectamine 2000-mediated transfection (In-vitrogen) as previously described (Obermair et al., 2004). For singletransfection (p
A-eGFP) 1
g of DNA, for cotransfection experiments(p
A-eGFP or eCFP plus HA-tagged constructs) 2
g of total DNA at amolarratioof1:2wasused.Cellswerestainedandanalyzed11–14daftertransfection.
Plasmids.
Thegenerationofp
A-eGFPandp
A-Ca
V
1.2-HA(Ca
V
1.2:GenBankaccessionno.M67515)hasbeendescribedearlier(Obermairetal.,2004).pECFP-C1(BDBiosciencesClontech)wasagiftfromD.Garc-zarczyk(InnsbruckMedicalUniversity,Innsbruck,Austria).TheCa
V
1.2C-terminal mutants were generated by standard overlapping PCR pro-tocolswithspecificoligonucleotidescarryingaprematurestopcodonfor
VSNL, or three alanine substitutions (I2046A, L2053A, I2060A) for
LZ. These PCRs were performed using pcDNA3-Ca
V
1.2 as template.C-terminalcassettes(2.2kbSbfi1-
 Not 
Ifragment)carryingthemutationswere sequenced to verify the presence of the desired mutations and thencloned into the p
A-Ca
V
1.2-HA plasmid backbone to generate p
A-Ca
V
1.2-HA(
VSNL) and p
A-Ca
V
1.2-HA(
LZ). The functional ex-pression of all Ca
V
1.2-HA constructs was confirmed using patch-clamprecordings in HEK cells and in dysgenic myotubes (data not shown).
 NMDA treatment of hippocampal neurons.
NMDA (Tocris) was ap-plied to 18- to 20-d-old neurons in neuronal medium at a final concen-tration of 25
M
for 3 min. Subsequently the cultures were washed withfresh medium and incubated in a 1:1 mix of glia-conditioned and freshmediumfor30minat37°C,beforeprocessingforimmunocytochemistry or labeling with DiIC
16
(Invitrogen). Controls were handled identically but without NMDA.
DiIC 
16 
labeling.
Living neurons were incubated for 1 min in DiIC
16
diluted
5
g/mlinconditionedmedium,washed,mountedinPBS,andimmediately observed.
Fixation and immunocytochemistry.
Neurons were either fixed in 4%paraformaldehyde, 4% sucrose in PBS (pF) at room temperature or inmethanol at
20°C for experiments involving PSD-95 (Rao and Craig,1997).Fixedneuronswereincubatedin5%normalgoatserum(NGS)inPBS containing 0.2% bovine serum albumin (BSA) and 0.2% TritonX-100(PBS/BSA/Triton)for30min.Primaryantibodieswere appliedinPBS/BSA/Triton at 4°C overnight and detected by fluorochrome-conjugated secondary antibodies (Obermair et al., 2004).
Live cell surface labeling.
For staining of surface-expressed HA-taggedCa
V
1.2 constructs living neurons were incubated with the rat anti-HAantibodyfor30minat37°C(Watschingeretal.,2008).InNMDAexper-iments this was done during the 30 min post NMDA incubation. ThenthecultureswererinsedinHBSS,pFfixedfor10min,blockedwithNGS,and incubated with the secondary antibody for 1 h (Obermair et al.,2004).
Live cell surface labeling combined with regular immunocytochemistry.
For colocalization of surface-expressed Ca
V
1.2-HA constructs and cyto-plasmic scaffold proteins, live cell-stained neurons were postfixed for 5mininpF(forAKAP79/150and
-actinin)orinmethanol(forPSD-95).ThenneuronswererinsedinPBS,permeabilized,andblockedagainwith5%NGSinPBS/BSA/Tritonandsubsequentlyincubatedwiththesecondprimary antibody overnight at 4°C. After washing, the Alexa 488-conjugatedsecondaryantibodywasappliedfor1hatroomtemperature.Coverslips were then washed and mounted in p-phenylene-diamine-glycerol to retard photobleaching (Flucher et al., 1993). Preparationswere analyzed on an Axiophot or an Axiovert 200M microscope (CarlZeiss)usinga63
,1.4NAobjective.ImageswererecordedwithacooledCCD camera (SPOT; Diagnostic Instruments) and Metavue image pro-cessing software (Universal Imaging).
 Antibodies.
Primaryantibodies:rabbitanti-AKAP79/150(4361J,poly-clonal, generously supplied by Dr. Yvonne Lai, ICOS, Bothell, WA,1:8000); mouse anti-
-actinin (monoclonal, clone EA-53, Sigma;1:10,000); rat anti-HA (monoclonal, clone 3F10, Roche Diagnostics,1:100); mouse anti-PSD-95 (monoclonal, clone 6G6–1C9, Affinity Bioreagents, 1:1000). Secondary antibodies: goat anti-mouse Alexa 488(1:2000),goatanti-rabbitAlexa488,andgoatanti-ratAlexa594(Invitro-gen, 1:4000).
Quantification of the density and fluorescent intensity of Ca
V 
1.2-HAclusters.
Fourteen-bitgray-scaleimagesofthered(HA)andgreen(eGFP)channel were acquired. Corresponding images were aligned in an imagestack and one dendritic segment of 10–15
m length was selected foranalysis. Using the eGFP image as reference, ROIs were drawn aroundthe shaft and the adjacent regions containing the spines. The shaft areawas measured and the number of spines was counted. The Ca
V
1.2-HAimage was background-flattened (MetaMorph software, Universal Im-aging) and thresholded to trace the fluorescent clusters as accurately aspossible. Using the integrated morphometric analysis option of Meta-Morph,thenumberofclusterswascountedandtheiraveragegrayvalueswere determined and corrected for background fluorescence.
Quantification of colocalization of Ca
V 
1.2-HA clusters with
-actinin, AKAP79/150,andPSD-95indendriticspines.
Fourteen-bitgray-scaleim-ages of the red (HA) and green (
-actinin, AKAP79/150, and PSD-95)channel were acquired. Corresponding images were 2D-deconvolved(MetaMorph), aligned, and background subtracted. A ROI was drawnaround the region containing the dendritic spines along a dendritic seg-ment of 20–50
m length. Colocalization was analyzed using the dis-tance based colocalization method, which is part of the JACoP plugin(Bolte and Cordelie`res, 2006) in ImageJ (W. S. Rasband, ImageJ, Na-tional Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/,1997–2007). Results are expressed as percentage of Ca
V
1.2-HA objectscolocalizing with
-actinin, AKAP79/150, or PSD-95, respectively.
Quantification of fluorescent intensity in dendritic spines and shafts.
Tocalculatethespine/shaftratio,alinewasplacedthroughthespineandtheadjacent shaft region in the eGFP image, along which background-
13846
•
J. Neurosci., December 17, 2008
•
28(51):13845–13855 Di Biase et al.
•
Stable Ca
V
1.2 Clusters in Dendritic Spines
 
subtracted intensities were recorded in theeGFP and the corresponding immunofluores-cence image (cf. Fig. 1). The threshold for de-termining average intensities of the spine andshaft was set to 68% of eGFP fluorescence be-tween the minimum intensity value of the linescan and the maximum of each peak.
Statistical analysis.
Absolute values of all themeasurements were expressed as percentage of control. Statistical significance was calculatedusingthe
t 
test(MicrosoftExcel)onnormalizeddata. Analyses were performed on 15–25 neu-rons from at least two different experimentsand at least two separate culture preparations.All data are reported as mean
95% confi-dence interval. Graphs and figures were gener-ated using Origin 7 and Adobe Photoshop 8.0software.
Results
The distribution of Ca
V
1.2 clusters inthe membrane of hippocampal neuronsoverlaps with AKAP79/150 but isdistinct from that of PSD-95 and
-actinin
AKAP79/150, PSD-95, and
-actinin areall constituents of dendritic spines.AKAP79/150 and
-actinin were reportedto interact with the Ca
V
1.2 calcium chan-nel (Hall et al., 2007; Oliveria et al., 2007)(J. W. Hell, personal communication).PSD-95isamarkerofthepostsynapticsig-naling complex of glutamatergic synapsesand recruits AKAP79/150 and the associ-ated protein kinases and phosphatases tothe postsynaptic density. If any one of these scaffold proteins participates in theCa
V
1.2 signaling complex in hippocampalneurons it is expected to be colocalizedwith this channel. We used double-immunofluorescence labeling to deter-minethedistributionpatternsofAKAP79/150, PSD-95, and
-actinin in thesomatodendritic compartment in differ-entiated cultured mouse hippocampalneurons. Figure 1
 A
shows that AKAP79/150 is widely distributed throughout theneurons, whereas PSD-95 is confined todiscretepunctaalongthedendritesandonthe soma. A magnified dendritic segmentreveals that AKAP79/150 and PSD-95 co-localize in the spine heads, where PSD-95marksthesiteoftheglutamatergicsynapse(Fig.1
B
).InthedendriticspinesAKAP79/150 reaches its highest concentrations andits distribution usually exceeds that of PSD-95 clusters in that it fills the entirespine. In addition AKAP79/150 but notPSD-95 is found in the spine neck and indendritic shafts (Fig. 1
C 
).
-Actinin alsoshows a clustered distribution pattern inmanyneurons(Fig.1
D
).However,incon-trast to AKAP79/150 and PSD-95,
-actinin clusters are typically excludedfrom the spine head but enriched in the
Figure 1.
Localization of AKAP79/150, PSD-95, and
-actinin in dendrites of cultured hippocampal neurons.
A
, A representativeglutamatergicneuron(20DIV)labeledwithanti-AKAP79/150andanti-PSD-95showsthedistributionofbothproteinsinthesomaanddendrites.
B
,AKAP79/150stainingisfoundthroughoutthedendriticshaftandisconcentratedintheheadsofdendriticspines(greeninmergedcolorimage).ClustersofPSD-95,amarkeroftheexcitatorypostsynapticcompartment,arelocalizedexclusivelyinthespineheads(red/yellow).
C 
,Alinescan(
4
m)acrossatypicalspinedemonstratesoverlappingpeaksofAKAP79/150(greenline)andPSD-95(redline)labelingintensityinthespinehead.
D 
,Ahippocampalneuron(20DIV)labeledfor
-actininshowsclustersinthesomatodendriticcompartmentandastrongaccumulationintheaxonhillock(arrowhead).
E 
,Thedendriticsegmentshowsthatthe
-actininclusters(red)areconcentratedintheproximalpartsofthespineandinthedendriticshaftwithlittleoverlapwithAKAP79/150(green/yellow).
F 
,Alinescanacrossadouble-labeledspineshowsthemaximumintensityof 
-actinin(redline)inthespineneckalternatingwithAKAP79/150peaks(greenline)intheheadsandtheshaft.Scalebars:
 A
,
D 
,10
m;
B
,
E 
,5
m.
Di Biase et al.
•
Stable Ca
V
1.2 Clusters in Dendritic Spines J. Neurosci., December 17, 2008
•
28(51):13845–13855
• 13847
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