Densin-180 Controls the Trafficking and Signaling of L-Type Voltage-Gated Ca_v1.2 Ca²⁺ Channels at Excitatory Synapses

Abstract

Voltage-gated Ca_v1.2 and Ca_v1.3 (L-type) Ca²⁺ channels regulate neuronal excitability, synaptic plasticity, and learning and memory. Densin-180 (densin) is an excitatory synaptic protein that promotes Ca²⁺-dependent facilitation of voltage-gated Ca_v1.3 Ca²⁺ channels in transfected cells. Mice lacking densin (densin KO) exhibit defects in synaptic plasticity, spatial memory, and increased anxiety-related behaviors—phenotypes that more closely match those in mice lacking Ca_v1.2 than Ca_v1.3. Therefore, we investigated the functional impact of densin on Ca_v1.2. We report that densin is an essential regulator of Ca_v1.2 in neurons, but has distinct modulatory effects compared with its regulation of Ca_v1.3. Densin binds to the N-terminal domain of Ca_v1.2, but not that of Ca_v1.3, and increases Ca_v1.2 currents in transfected cells and in neurons. In transfected cells, densin accelerates the forward trafficking of Ca_v1.2 channels without affecting their endocytosis. Consistent with a role for densin in increasing the number of postsynaptic Ca_v1.2 channels, overexpression of densin increases the clustering of Ca_v1.2 in dendrites of hippocampal neurons in culture. Compared with wild-type mice, the cell surface levels of Ca_v1.2 in the brain, as well as Ca_v1.2 current density and signaling to the nucleus, are reduced in neurons from densin KO mice. We conclude that densin is an essential regulator of neuronal Ca_v1 channels and ensures efficient Ca_v1.2 Ca²⁺ signaling at excitatory synapses.

SIGNIFICANCE STATEMENT The number and localization of voltage-gated Ca_v Ca²⁺ channels are crucial determinants of neuronal excitability and synaptic transmission. We report that the protein densin-180 is highly enriched at excitatory synapses in the brain and enhances the cell surface trafficking and postsynaptic localization of Ca_v1.2 L-type Ca²⁺ channels in neurons. This interaction promotes coupling of Ca_v1.2 channels to activity-dependent gene transcription. Our results reveal a mechanism that may contribute to the roles of Ca_v1.2 in regulating cognition and mood.

Introduction

Ca²⁺ influx through voltage-gated (Ca_v) Ca_v1.2 L-type Ca²⁺ channels regulates neuronal excitability (Lacinova et al., 2008), synaptic plasticity (Moosmang et al., 2005), and learning and memory (Moosmang et al., 2005; White et al., 2008). Like other Ca_v channels, Ca_v1.2 is composed of a pore-forming α₁ subunit (Ca_v1.2) and auxiliary β and α₂δ subunits (Simms and Zamponi, 2014). The CACNA1C gene encoding Ca_v1.2 is a major risk gene for multiple neuropsychiatric disorders, including autism spectrum disorder, attention deficit–hyperactivity disorder, schizophrenia, bipolar disorder, and major depression (Cross-Disorder Group of the Psychiatric Genomics Consortium, 2013). Therefore, an understanding of the factors regulating Ca_v1.2 is crucial for understanding the balance between normal and disordered states of cognitive and affective processing.

In addition to interactions with auxiliary subunits, neuronal Ca_v1.2 channels interact with a variety of other regulatory proteins (Calin-Jageman and Lee, 2008; Lipscombe et al., 2013). The distal C terminus of Ca_v1.2 contains a consensus site for binding to proteins containing Postsynaptic Density-95, Drosophila Discs-Large and Zona Occludens (PDZ) domains (Kurschner and Yuzaki, 1999; Weick et al., 2003). The related Ca_v1.3 channel also contains a C-terminal PDZ-binding site, which serves as a ligand for multiple proteins that regulate the function of these channels (Olson et al., 2005; Zhang et al., 2005; Calin-Jageman et al., 2007; Jenkins et al., 2010; Gregory et al., 2011; Gregory et al., 2013). Although several PDZ domain-containing proteins are known to interact with Ca_v1.2 (Kurschner et al., 1998; Kurschner and Yuzaki, 1999), the functional consequences of such interactions have not been characterized.

Densin-180 (densin) is a leucine-rich repeat and PDZ domain-containing protein that is enriched in the postsynaptic density of excitatory synapses and interacts with a variety of postsynaptic proteins including calmodulin-dependent protein kinase II (CaMKII; Apperson et al., 1996; Strack et al., 2000; Walikonis et al., 2001). We showed previously that the PDZ domain of densin interacts with Ca_v1.3 and recruits CaMKII to the channel complex, which causes Ca²⁺-dependent facilitation of Ca_v1.3 currents in transfected cells (Jenkins et al., 2010). However, mice lacking densin (densin KO) display defects in spatial memory and elevated anxiety levels (Carlisle et al., 2011), which are more similar to the behavioral phenotypes in mice lacking Ca_v1.2 (Moosmang et al., 2005; Lee et al., 2012) than in mice lacking Ca_v1.3 (Pinggera and Striessnig, 2016). Together with evidence that Ca_v1.2 is more abundant in the brain than Ca_v1.3 (Clark et al., 2003), these results suggest that densin may be a physiological relevant component of Ca_v1.2 complexes.

To test this hypothesis, we investigated whether densin functionally interacts with Ca_v1.2 in transfected cells and in neurons. We found that densin binds to, but differentially modulates, Ca_v1.2 compared with Ca_v1.3. Densin enhances the cell surface density and postsynaptic clustering of Ca_v1.2, as well as coupling of Ca_v1.2 to phosphorylation of the transcription factor cAMP response element binding protein (CREB). Our results underscore the importance of Ca_v1.2–protein interactions for neuronal Ca²⁺ signaling, which should be considered in the context of how alterations in Ca_v1.2 channel function may lead to neuropsychiatric disease.

Materials and Methods

Animals.

All procedures using animals were performed in accordance with the University of Iowa Institutional Animal Care and Use Committee. The densin KO mouse line was bred on a C57BL/6 background and has been described previously (Carlisle et al., 2011). Mice were housed three or four per cage and maintained on a 12 h reverse light/dark cycle with ad libitum access to food and water.

cDNAs.

For cell transfection, cDNAs corresponding to Ca_v1.2 subunits [rbcII (GenBank #M67515), β_2a (GenBank #NC013684) and α₂δ₁ (GenBank #M76559.1)] were expressed from the pcDNA3.1+ vector (Invitrogen). The following constructs were described previously: Ca_v1.2/pcDNA3.1+ (FLAG-tagged), Ca_v1.2ΔNT/pcDNA3.1+ (FLAG-tagged) lacking aa 1–109, GST-tagged Ca_v1.2 intracellular domains: NT (aa 1–124); loop I-II (aa 406–524); loop II-III (aa 754–902); loop III-IV (aa1170–1222); CT (aa 1942–2144) (Zhou et al., 2004; Zhou et al., 2005); GST-tagged C-terminal domain of Ca_v1.3 (aa 1962–2155) (Calin-Jageman et al., 2007); GFP-tagged densin (GenBank #NM_057142.1 (Jiao et al., 2008)); pβA-eGFP and pβA-Ca_v1.2-hemagglutinin (HA) (Altier et al., 2002; Obermair et al., 2004). GST-tagged fragments of Ca_v1.2 NT were generated by PCR amplification and subcloning into BamHI and EcoRI sites of pGEX-4T1. The pβA-Ca_v1.2-HA-ΔNT was generated by PCR amplification of the sequence encoding Ca_v1.2-HA-ΔNT using pβA-Ca_v1.2-HA as a template and subcloning the sequence encoding Ca_v1.2ΔNT into HindIII and XbaI sites of the pβA empty vector. To generate Ca_v1.2ΔB1a/pcDNA3.1+, the sequence corresponding to the B1a region (aa 65–76) was deleted from Ca_v1.2/pcDNA3.1+ using site-directed mutagenesis. The SNAP-tagged-Ca_v1.2 construct was generated by amplifying the Ca_v1.2-HA coding region in pβA-Ca_v1.2-HA and ligating into PmeI and NotI sites of the pSNAPr vector (New England Biolabs) using the NEBuilder HiFi DNA Assembly cloning system (New England BioLabs).

Cell culture and transfection.

Human embryonic kidney cells transformed with SV40 T-antigen (HEK293T; American Type Culture Collection catalog #CRL-3216, RRID: CVCL_0063) were maintained in DMEM (Thermo Fisher Scientific) with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO₂. Cells were grown to 70–80% confluence and transfected with Gene Porter reagent (Genlantis) or Fugene 6 (Promega) according to the manufacturer's protocols. For electrophysiology, cells were plated in 35 mm dishes and transfected with Ca_v1.2 subunit cDNAs: Ca_v1.2 (1.5 μg), β_2a (1 μg), and α₂δ₁ (1 μg). pEGFPN1 (0.1 μg) or GFP-densin (1 μg) was cotransfected, which facilitated identification of transfected cells by fluorescence. For pull-down assays, cells were plated in 60 mm dishes and transfected with GFP-densin (6 μg). For coimmunoprecipitation experiments, cells were plated in 60 mm dishes and transfected with Ca_v1.2 (2.4 μg), β_2a (0.8 μg), α₂δ₁ (0.8 μg), pEGFPN1 (0.5 μg), and pcDNA (3.5 μg) or GFP-densin (4 μg).

In vitro binding assays.

GST-fusion proteins containing intracellular domains of Ca_v1.2 were expressed in E. coli (BL21) and purified on glutathione-Sepharose (GE Healthcare Life Sciences) according to standard protocols. HEK293T cells transfected with GFP-densin (4 μg) were harvested and homogenized in ice-cold cell lysis buffer containing 50 mm Tris-HCl, 150 mm NaCl, 1% Triton X-100, 0.25% w/v sodium deoxycholate, 1 mm EDTA, pH 7.4, and protease inhibitors. The homogenate was rotated at 4°C for 1 h to solubilize membrane proteins and the insoluble material was separated by centrifugation at 16,100 × g for 30 min. The supernatant was incubated with immobilized GST fusion proteins (50% slurry) at 4°C overnight. The beads were washed three times with ice-cold cell lysis buffer and the bound proteins were eluted with SDS-containing sample buffer, subjected to SDS-PAGE, and transferred to nitrocellulose. Western blotting was with mouse monoclonal anti-GFP (1:3000, Santa Cruz Biotechnology catalog #sc-53882, RRID: AB_783653).

Coimmunoprecipitation assays.

Transfected HEK293T cells were harvested 48 h after transfection. Cell lysates were prepared as described for binding assays and incubated with FLAG-agarose beads (50% slurry; Sigma-Aldrich) overnight, rotating at 4°C. After three washes with cell lysis buffer, proteins were eluted and analyzed by SDS-PAGE. Coimmunoprecipitated proteins were detected by Western blotting with GFP antibodies (1:3000, Santa Cruz Biotechnology catalog #sc-53882, RRID: AB_783653). For coimmunoprecipitation from mouse brain, neural tissue was subjected to subfractionation according to previously described methods (Cox and Emili, 2006). The prefrontal cortex was dissected and homogenized in lysis buffer containing the following (in mm): 250 sucrose, 50 Tris-HCl, 5 MgCl₂, and 1 DTT, pH 7.4, plus protease inhibitors. The nuclear fraction was removed by centrifugation at 800 × g for 15 min and the supernatant was subjected to ultracentrifugation at 100,000 × g for 1 h. The membrane pellet was solubilized in solubilization buffer containing 50 mm Tris-HCl, 150 mm NaCl, pH 7.5, 1% Triton X-100, and protease inhibitors) at 4°C for 30 min and the insoluble material was removed by ultracentrifugation at 100,000 × g for 1 h. Either rabbit control IgG (Sigma-Aldrich catalog #I5006, RRID: AB_1163659) or anti-Ca_v1.2 antibodies (5 μg) were added to the solubilized membrane proteins along with Protein A Sepharose (50% slurry, Sigma-Aldrich). Reactions were continued overnight with end-over-end rotation at 4°C. The resin was collected by centrifugation and rinsed three times with solubilization buffer. Bound proteins were eluted, resolved on a SDS-polyacrylamide gel (4–12%), and Western blotted with anti-densin antibodies (1:5000, Colbran laboratory at Vanderbilt University, catalog #Ab650, RRID: AB_2619660; Jiao et al., 2008) as described above.

Preparation of neuronal cultures.

Low-density cultures of hippocampal neurons were prepared from BALB/c mice at embryonic day 18 as described previously (Obermair et al., 2004; Di Biase et al., 2011). Briefly, dissected hippocampi were dissociated by trypsin treatment and trituration. Neurons were plated on poly-l-lysine-coated glass coverslips in 60 mm culture dishes at a density of ∼3500 cells/cm². After plating, cells were allowed to attach for 3–4 h before transferring the coverslips neuron-side down into a 60 mm culture dish with a glial feeder layer. Neurons and glial feeder layer were maintained in serum-free Neurobasal medium (Invitrogen) supplemented with Glutamax and B-27 supplements (Invitrogen). Ara-C (5 μm) was added 3 d after plating and, once a week, 1/3 of the medium was removed and replaced with fresh maintenance medium.

Cortical neuron cultures were prepared from postnatal day 0–1 mouse pups. The cerebral cortex was isolated in ice-cold dissection solution (0.1 mg/ml HBSS-containing sodium pyruvate, 0.1% glucose, and 10 mm HEPES, pH 7.3). The tissue was washed and digested for 13 min in the dissection solution containing trypsin (5 mg/ml) and DNase I (0.1 mg/ml) at room temperature. The trypsin was inactivated by two washes in prewarmed plating medium containing Eagle's basal medium (Thermo Fisher Scientific), fetal bovine serum (10%), w/v glucose (0.45%), sodium pyruvate (1 mm), glutamine (2 mm), and penicillin/streptomycin (100 μg/ml). The tissue was dissociated using polished Pasteur pipettes and the cells were pelleted at 185 × g for 13 min at 4°C. The cells were gently resuspended in prewarmed plating medium and plated on 18 mm glass coverslips coated with poly-d-lysine. Neurons were maintained in maintenance medium containing Neurobasal medium (Thermo Fisher Scientific), B27 (Thermo Fisher Scientific), glutamine (2 mm), and penicillin/streptomycin (100 μg/ml) at 37°C in a humidified atmosphere with 5% CO₂ for 12–16 d before experiments.

Analysis of cell surface Ca_v1.2 and densin localization in transfected hippocampal neurons.

Expression plasmids were introduced into neurons at 6 d in vitro (DIV) using Lipofectamine 2000-mediated transfection (Invitrogen) as described previously (Obermair et al., 2004). For cotransfection of Ca_v1.2-HA + eGFP or Ca_v1.2-HA + eGFP + GFP-densin, 1.5 or 2.5 μg of total DNA was used at a molar ratio of 1:1 or 1:1:1, respectively. Cells were processed for immunostaining 12–13 d after transfection.

For surface staining of Ca_v1.2-HA, transfected neurons were incubated with anti-HA (Roche catalog #11867431001, RRID: AB_390919) for 20 min at 37°C. Coverslips were rinsed in HBSS and fixed with 4% paraformaldehyde for 10 min. After fixation, neurons were washed with PBS for 30 min, blocked with 5% goat serum for 30 min, and labeled with goat anti-rat Alexa Fluor 594 (1:4000, 1 h, Thermo Fisher Scientific catalog #A11007, RRID: AB_10561522). Coverslips were mounted in p-phenylenediamine glycerol to retard photobleaching (Flucher et al., 1993) and observed with an Axio Imager microscope (Carl Zeiss) using 63×, 1.4 numerical aperture oil-immersion objective lens. Images were recorded with cooled CCD camera (SPOT Imaging Solutions).

Fourteen-bit gray scale images of anti-HA (red channel) and eGFP (green channel) were acquired and analyzed as described previously (Di Biase et al., 2008; Di Biase et al., 2011; Stanika et al., 2016). Briefly, images were aligned and the eGFP image was used to select the regions of interest for measuring the area occupied by Ca_v1.2-HA clusters with MetaMorph software (Molecular Devices).

Electrophysiology.

HEK293T cells were subjected to whole-cell patch-clamp recordings 48 h after transfection. For each experiment, control cells were recorded on the same day in parallel with experimental cells. The external solution contained the following (in mm): 150 Tris, 1 MgCl₂, and 10 CaCl₂ or BaCl₂. The internal solution contained the following (in mm): 140 N-methyl-d-glucamine, 10 HEPES, 2 MgCl₂, 2 Mg-ATP, and 5 EGTA. The pH of both solutions was adjusted to 7.3 with methanesulfonic acid. Electrode resistances were 6–8 MΩ in the bath solution and series resistance was ∼6–8 MΩ compensated 70%. The holding voltage was −80 mV for transfected cells. Maximal gating charge was measured as the time integral of the gating current within 1.5–2 ms of a depolarizing step to the reversal potential (+70 mV) and tail current amplitudes were measured upon repolarization to −70 mV. Statistical analyses were as indicated.

For neuronal recordings, pyramidal neurons (12–16 DIV) were selected based on a pyramidal-shaped cell body and large proximal dendrites. The experimenter was blinded to mouse genotype before preparation of neuronal culture, recording, and data analysis. The external solution contained the following (in mm): 150 TEA-Cl, 5 BaCl₂, 10 HEPES, and 10 glucose, pH 7.4 with TEA-OH. The intracellular solution contained the following (in mm): 180 N-methyl-d-glucamine, 4 MgCl₂, 5 EGTA, 40 HEPES, 12 creatine-phosphate-Tris, 0.1 Leupeptin, 2 Na-ATP, and 0.4 Na-GTP, pH 7.3 with phosphoric acid. To isolate Ca_v1-mediated currents, external solution containing isradipine (5 μm; Tocris Bioscience) was applied by gravity perfusion. Currents were evoked by voltage ramps from −60 mV to +80 mV at 0.5 mV/ms from an initial holding potential of −60 mV.

Data were acquired with an EPC-9 amplifier and Patchmaster software (HEKA Elektronik) and analyzed using Igor Pro software (Wavemetrics). Statistical analysis was performed using SigmaPlot (Systat Software). Average data are presented as mean ± SEM. For current–density plots, peak current amplitudes were normalized to whole-cell capacitance. Current–voltage (I–V) relationships were fit with the following equation: I = G(V − E)/{1 + exp[(V − V_1/2)/k]} where G is conductance, V is the test potential, E is the reversal potential, V_1/2 is the voltage for the half-maximal activation, and k is the slope factor.

Analyses of cell surface Ca_v1.2 protein levels in mouse brain.

Biotinylation of cell surface proteins in brain slices were modified from methods described previously (Robertson et al., 2010). Wild-type (WT) and densin KO mice (3–6 weeks old, male) were deeply anesthetized with isoflurane (4–5%) and rapidly decapitated. The brain was removed and the prefrontal cortex was dissected and chilled in oxygenated solution containing the following (in mm): 210 sucrose, 20 NaCl, 2.5 KCl, 1 MgCl₂, and 1.2 NaH₂PO₄ · H₂O before sectioning. Coronal slices (300 μm) were collected using a vibratome (Electron Microscopy Science 5000 Oscillating Tissue Slicer) in artificial CSF (ACSF) containing the following (in mm): 125 NaCl, 2.5 KCl, 1.2 NaH₂PO₄ · H₂O, 1 MgCl₂, and 2 CaCl₂ · H₂O, oxygenated. To minimize cell damage, the highest oscillation speed and the lowest advance rate of the blade were chosen during slicing. The brain slices were immediately washed twice with ACSF before incubating with ACSF containing EZ-Link Sulfo-NHS-SS-Biotin (1 mg/ml; Thermo Fisher Scientific) for 45 min. The tissue was rinsed twice quickly and for two 10 min washes in ACSF. The reaction was quenched by washing twice for 20 min each with ACSF containing glycine before lysis in radioimmunoprecipitation assay (RIPA) buffer containing 25 mm Tris, 150 mm NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, and a mixture of protease inhibitors. Lysates were then centrifuged at 12,000 × g for 30 min at 4°C. Biotinylated proteins were isolated on streptavidin beads (Thermo Fisher Scientific) overnight at 4°C with rotation. Beads were washed 3 times with RIPA buffer (0.1% Triton X-100) and biotinylated proteins were eluted in SDS-PAGE sample loading buffer at 90°C for 10 min. Western blotting was with antibodies against Ca_v1.2, Na⁺/K⁺ ATPase (1:3000, Developmental Studies Hybridoma Bank catalog #a6F RRID:AB_2314847) and GAPDH (1:10000, Abcam catalog #ab8245 RRID:AB_2107448).

Phosphorylated CREB assays.

Cortical neurons (12–16 DIV) from WT or densin KO mice were incubated in maintenance medium containing 0.5 μm tetrodotoxin (TTX; Tocris Bioscience), 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris Bioscience), and 10 μm D-(−)-2-amino-5-phosphonopentanoic acid (APV; Tocris Bioscience) at 37°C, 5% CO₂, for 4 h. Cells were exposed to Tyrode's solution containing the following (in mm): 150 NaCl, 2 MgCl₂, 2 CaCl₂, 10 HEPES, and 10 glucose, pH 7.3, and the following (in μm): 0.5 TTX, 10 CNQX, and 10 APV, plus either 5 or 40 mm KCl for 3 min, and immediately fixed in solution containing paraformaldehyde (4%), sucrose (4%), and EGTA (20 mm) at room temperature for 10 min. Cells were permeabilized in PBS with Triton X-100 (0.1%) for 10 min and blocked with normal goat serum (10%) in PBS for 1 h. Primary antibodies were rabbit anti-phosphorylated CREB (pCREB) (1:300; Cell Signaling Technology catalog #9198S, RRID: AB_390802); mouse anti-CaMKIIα (1:2000, Thermo Fisher Scientific catalog #MA1-048, RRID: AB_325403); mouse anti-NeuN (1:100, Millipore catalog #MAB377, RRID: AB_2298772) and were diluted in PBS, added to the coverslips, and incubated overnight at 4°C. The cells were then washed 3 times with PBS and incubated in anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific catalog #A31565, RRID: AB_10373124) and goat anti-mouse Alexa Fluor 568 (1:500; Thermo Fisher Scientific catalog #A-21134, RRID: AB_2535773) for 1 h at room temperature. After additional washing, cells were incubated in Hoechst 33342 (1 μg/ml) in distilled water for 15 min before mounting on microscope slides. The cells were visualized with a 60× oil-immersion objective on a confocal microscope (Fluoview 1000; Olympus). The level of pCREB fluorescence was quantified in confocal images analyzed in ImageJ software. The nuclear marker Hoechst and anti-NeuN staining were used to identify the region of interest over each neuron. The region adjacent to the neuron was regarded as background. Mean pixel intensity for pCREB immunofluorescence was measured in the region of interest and the background level was subtracted. Genotypes of animals were revealed after the quantification.

Trafficking assays.

For endocytosis assays, transfected HEK293T cells were washed twice with Dulbecco's PBS with MgCl₂ and CaCl₂ (DPBS²⁺; Thermo Fisher Scientific) 48 h after transfection. Live cells were incubated with mouse anti-HA antibodies (1:100, Sigma-Aldrich catalog #11583816001, RRID: AB_514505) at 37°C for 15 min. After washing 3 times with DPBS²⁺ at room temperature, the cells were maintained at 37°C for varying periods to allow for channel internalization before fixing in ice-cold paraformaldehyde (4%) in DPBS²⁺ for 10 min. Cells were then blocked with normal goat serum (10%), followed by incubating with anti-Ca_v1.2 antibodies overnight and the corresponding Alexa Fluor secondary antibodies and washes as described above. Images were taken using 60× oil-immersion objectives with a constant acquisition setting on a confocal microscope (Fluoview 1000; Olympus). The cell surface HA fluorescence in Ca_v1.2-positive cells was quantified in ImageJ software using the freehand brush tool to trace the membrane region stained by anti-HA antibodies manually for measuring the cell surface fluorescence density.

For forward trafficking, cells transfected with Ca_v1.2-SNAP were incubated with BG-Block (2 μm; New England BioLabs catalog #S9106S) at 37°C for 30 min to block existing channels. The cells were then washed 3 times and incubated in complete medium for varying durations before incubating with TMR-STAR (2 μm; New England BioLabs catalog #S9105S) at 37°C for 25 min to label newly synthesized channels. After washing with complete medium, cells were fixed for 10 min at 4°C in paraformaldehyde (4%), washed in DPBS²⁺, and blocked in DPBS²⁺ containing goat serum (10%). Cells were then incubated with anti-HA antibodies for cell surface labeling and the appropriate Alexa Fluor secondary antibodies. Cell surface TMR-STAR fluorescence was quantified in ImageJ as described above. Whole-cell fluorescence densities were quantified using freehand selection, omitting the signal intensity from adjacent areas. The background fluorescence density in each channel was also taken from the same image and subtracted from the signal density. Data were acquired and analyzed without prior knowledge of protein expression profiles.

Results

Densin interacts differentially with and regulates Ca_v1.2 and Ca_v1.3

To determine whether densin is a physiological partner of Ca_v1.2 in vivo, we tested whether the two proteins form a complex in mouse brain. Antibodies that specifically recognize Ca_v1.2 (Tippens et al., 2008) were used to immunoprecipitate proteins from lysates of the mouse prefrontal cortex. Western blotting with densin antibodies revealed that densin coimmunoprecipitated with Ca_v1.2 antibodies, but not with control IgG (Fig. 1A). Because densin binds to the PDZ-binding site at the C terminus of Ca_v1.3 (Jenkins et al., 2010), we hypothesized that densin would similarly bind to the corresponding region of Ca_v1.2. In pull-down assays, GST fusion proteins containing the distal C terminus of Ca_v1.2 did not bind to GFP-densin despite interacting with the same region of Ca_v1.3 (Fig. 1B,C). GFP-densin also did not bind to the cytoplasmic loops of Ca_v1.2 (Fig. 1D). However, specific binding of GFP-densin was found for the N-terminal (NT) domain of Ca_v1.2 but not Ca_v1.3 (Fig. 1E). Moreover, deletion of the NT domain prevented coimmunoprecipitation of GFP-densin with Ca_v1.2 (Ca_v1.2ΔNT; Fig. 1F). Using GST-tagged fragments of the Ca_v1.2 NT, we found that GFP-densin bound specifically to proteins containing aa 65–91 (Fig. 1G). Because aa 77–91 are largely conserved in Ca_v1.3 (Fig. 1H), the major determinants for densin binding to the Ca_v1.2 NT appear to reside between aa 65 and 76.

Figure 1.

Densin interacts with Ca_v1.2 channels in the brain and binds to a site in the NT domain of Ca_v1.2. A, Coimmunoprecipitation of densin with Ca_v1.2 antibodies but not rabbit control IgG in lysates of mouse prefrontal cortex. Western blotting was performed using a densin antibody. B, Schematic of Ca_v1.2 α₁ subunit showing the NT domain, the C-terminal (CT) domain, and cytoplasmic loops (I–II, II–III, and III–IV). C–E, Pull-down assays using GST-tagged cytoplasmic domains of Ca_v1.2. Input represents GFP-tagged densin in lysates of transfected HEK293T cells. C, Densin binds to the CT of Ca_v1.3, but not that of Ca_v1.2. D, Densin does not bind to the cytoplasmic loops of Ca_v1.2. E, Densin binds to the NT of Ca_v1.2, but not that of Ca_v1.3. F, Coimmunoprecipitation of GFP-densin with FLAG antibodies in HEK293T cells cotransfected with FLAG-tagged Ca_v1.2 (1.2), but not with Ca_v1.2 lacking the NT (1.2ΔNT) or control cells transfected with GFP-densin alone. G, Same as in C–E except with GST-tagged fragments of the Ca_v1.2 NT. Amino acid numbers are indicated in parentheses. All blots shown are representative of at least three independent experiments. Input lanes represent 7–10% of starting material. H, Sequence alignment of the B1 regions of Ca_v1.2 and Ca_v1.3. Conserved residues are shaded in black. Numbers indicate the amino acid residues.

To elucidate the functional impact of densin on Ca_v1.2, we turned to an electrophysiological protocol we used previously to uncover the modulatory effects of densin on Ca_v1.3. When cotransfected with densin and its interacting protein, CaMKII (Strack et al., 2000; Walikonis et al., 2001), Ca_v1.3 channels undergo Ca²⁺-dependent facilitation during repetitive depolarizations (Jenkins et al., 2010). Although there was no effect of densin and CaMKII on Ca_v1.2 Ca²⁺ currents (I_Ca) with this protocol (Fig. 2A), I–V relationships revealed significantly greater current densities in cells cotransfected with GFP-densin and CaMKII than in control cells (at +10 mV, 38.0 ± 6.4 pA/pF for Ca_v1.2 + GFP-densin + CaMKII, n = 13, vs 20.7 ± 2.8 pA/pF for Ca_v1.2 + eGFP, n = 14, p = 0.02 by t test; Fig. 2B). This effect did not require CaMKII because it was reproduced in cells cotransfected with only Ca_v1.2 and GFP-densin (Fig. 2B). Densin did not augment I_Ca in cells cotransfected with channels lacking the NT aa 65–76 (Ca_v1.2ΔB1a) comprising the densin-binding site (at +10 mV, 15.4 ± 2.8 pA/pF for Ca_v1.2ΔB1a + GFP-densin, n = 14, vs 22.5 ± 5.6 pA/pF for Ca_v1.2ΔB1a + eGFP, n = 14, p = 0.29; Fig. 2C). These results indicate that densin binding to Ca_v1.2 is required for the enhanced current density in cells cotransfected with GFP-densin.

Figure 2.

Densin does not cause CDF but increases current density of Ca_v1.2 in HEK293T cells. A, I_Ca was evoked by 5 ms pulses from −80 to 10 mV at 100 Hz in cells transfected with Ca_v1.2 alone (n = 13) or cotransfected with densin and CaMKII (n = 11). Test current amplitudes were normalized to the first in the train (Fractional I) and plotted against time (right). B, I_Ca was evoked by 50 ms depolarizations from −80 mV to various voltages and normalized to cell capacitance. Representative current traces and plots of current density against voltage are shown for cells transfected with Ca_v1.2 and: eGFP (n = 13), GFP-densin (n = 15), and GFP-densin + CaMKII (n = 11). C, Same as in B except for I_Ca recorded in cells transfected with Ca_v1.2ΔB1a and eGFP (n = 14) or GFP-densin (n = 14).

GFP-densin could increase I_Ca by increasing the number of Ca_v1.2 channels and/or their activity in the cell membrane. To pinpoint the underlying mechanism, we measured the gating current upon depolarization to the reversal potential (I_gating) and the tail current (I_tail) evoked by repolarization to the holding voltage (Fig. 3A). The time integral of I_gating (Q_max) estimates the number of channels in the cell membrane and the ratio of I_tail and Q_max reflects the relative channel open probability (Wei et al., 1994; Takahashi et al., 2004). Cotransfection of GFP-densin caused a significant increase in Q_max (50.8 ± 14.1 fC for Ca_v1.2 + eGFP, n = 9 vs 94.6 ± 17.5 fC for Ca_v1.2 + GFP-densin, n = 13, p = 0.02 by t test; Fig. 3B). The slope of the I_tail–Q_max relationship was steeper (∼2-fold; Fig. 3C) in cells cotransfected with GFP-densin compared with cells transfected with Ca_v1.2 and eGFP. Therefore, densin enhances the number of functional channels as well as their open probability. To determine whether the effect of densin requires interaction with the channel, we deleted the densin-binding sequence within the NT (Ca_v1.2ΔB1a + eGFP). In cells transfected with this construct, GFP-densin did not affect Q_max (61.2.0 ± 10.4 fC for Ca_v1.2ΔB1a + eGFP, n = 14 vs 73.9 ± 5.9 fC for Ca_v1.2ΔB1a + GFP-densin, n = 14, p = 0.30 by t test; Fig. 3D,E) or the I_tail–Q_max relationship (Fig. 3F). Similar results were obtained for Ca_v1.2ΔNT (data not shown). These results demonstrate that the interaction of densin with Ca_v1.2 is required for facilitation of channel function by densin.

Figure 3.

Densin increases the number of functional Ca_v1.2 channels and their relative open probability. A, Representative traces showing I_gating and I_tail measured at the reversal potential (+70 mV). Inset shows outward gating current on expanded timescale. Shaded area indicates Q_max. B, C, Dot plot of Q_max (B) and I_tail versus Q_max (C) in cells cotransfected with Ca_v1.2 and eGFP (n = 9) or GFP-densin (n = 12). The fit by linear regression and coefficient of determination (r²) are shown. D–F, Same as in A–C except in cells transfected with Ca_v1.2ΔB1a and eGFP (n = 14) or GFP-densin (n = 14). p-values were determined by t test.

Densin promotes the forward trafficking of Ca_v1.2 channels

The effect of densin on the number of Ca_v1.2 channels could result from a larger amount of Ca_v1.2 protein in the plasma membrane either from reduced endocytosis and/or enhanced forward trafficking. To test this, we used Ca_v1.2 channels tagged with an external HA epitope (Ca_v1.2-HA; Altier et al., 2002) such that cell surface levels of channel could be measured by labeling with HA antibodies in living cells. After antibody labeling, HEK293T cells cotransfected with Ca_v1.2-HA and eGFP or GFP-densin were incubated in fresh medium for varying durations to allow for channel internalization (Fig. 4A). The level of cell surface Ca_v1.2-HA was significantly greater in cells cotransfected with GFP-densin than with eGFP at all time points (p < 0.0001 by ANOVA; Fig. 4B). However, exponential fits of the data normalized to Ca_v1.2 HA staining at the start of the experiment (time = 0 min) indicated no effect of densin on the speed of internalization of Ca_v1.2-HA (τ = 8.4 ± 0.7 min for GFP-densin, n = 62 cells, vs τ = 9.5 ± 0.8 min for eGFP, n = 60 cells, p = 0.22; Fig. 4C). These results confirm that densin increases the cell surface density of Ca_v1.2 channels, but not by inhibiting their endocytosis.

Figure 4.

Densin does not affect internalization of Ca_v1.2 in transfected HEK293T cells. A, Cells expressing Ca_v1.2-HA and eGFP (top) or GFP-densin (bottom) were incubated with HA antibodies to label cell surface channels and incubated for the indicated times (in minutes) to allow for channel internalization. The cells were then fixed and processed with Ca_v1.2 antibodies to label total channel protein. Scale bars, 10 μm. B, Cell surface HA fluorescence (arbitrary units, a.u.) was plotted against internalization time. Results are representative of three independent experiments. C, Same as B but HA intensity was normalized to that at time = 0 min. Smooth line represents fit with a single exponential equation.

To determine whether densin enhances the trafficking of Ca_v1.2 to the cell surface, we generated Ca_v1.2 constructs containing the SNAP tag, an engineered variant of the DNA repair protein O⁶-alkylguanine-DNA-alkyltransferase that uses benzylguanine as a substrate (Keppler et al., 2004). SNAP-tagged Ca_v1.2-HA (Ca_v1.2-HA-SNAP) was cotransfected with eGFP or GFP-densin in HEK293T cells that were incubated first with benzylguanine to block existing channels. Subsequent incubation of the cells at 37°C for varying durations allowed for synthesis of new, unlabeled channels that were identified with a cell-permeant fluorescent derivative of benzylguanine (TMR-STAR; Fig. 5A). The cells were then processed for live labeling with HA antibodies and colocalized TMR-STAR and HA fluorescence was used to estimate the density of cell surface Ca_v1.2 channels. As in the endocytosis experiments, cotransfection with GFP-densin increased the amount of cell surface channels at most time points (Fig. 5B). When normalized to the last time point (60 min), the maximal density of cell surface HA-Ca_v1.2 was achieved within 30 min in cells cotransfected with GFP-densin compared with 1 h for cells cotransfected with eGFP (Fig. 5C). Therefore, densin increases the cell surface density of Ca_v1.2 channels by enhancing their trafficking to, rather than decreasing their removal from, the plasma membrane.

Figure 5.

Densin accelerates the forward trafficking of Ca_v1.2 in transfected HEK293T cells. A, Cells expressing Ca_v1.2-HA-SNAP and eGFP (top) or GFP-densin (bottom) were processed for TMR-STAR labeling of newly synthesized channels (see Materials and Methods). After incubation for the indicated times to allow forward trafficking of channels, cells were subjected to labeling with HA antibodies to mark cell surface channels. Scale bars, 10 μm. B, Cell surface TMR-STAR fluorescence (arbitrary units, a.u.) was plotted against forward trafficking time. Results are representative of three independent experiments. C, Same as B but TMR-STAR signal was normalized to that at time = 60 min. Smooth line represents fit with a single exponential equation.

Densin increases the cell surface levels of Ca_v1.2 in neurons

In neurons, Ca_v1.2 and densin are primarily localized in somatodendritic regions, particularly in dendritic spines (Apperson et al., 1996; Obermair et al., 2004; Tippens et al., 2008). To determine whether densin regulates the trafficking of Ca_v1.2 in neurons, we analyzed the distribution of cell surface Ca_v1.2-HA cotransfected with eGFP or GFP-densin in dendrites of hippocampal neurons in culture under nonpermeabilized conditions. We chose this approach rather than immunolabeling endogenously expressed Ca_v1.2 because Ca_v1.2 antibodies that are currently available recognize intracellular epitopes; the detergent permeabilization required for immunofluorescent detection would not restrict analysis to cell surface channels. Because densin is primarily localized at synapses of hippocampal neurons (Carlisle et al., 2011; Stanika et al., 2016), control and experimental groups were cotransfected with eGFP because the diffuse localization of eGFP in dendrites and dendritic spines facilitates the selection of dendritic regions for the analysis of cell surface channel clusters (Obermair et al., 2004). Consistent with previous studies (Obermair et al., 2004), punctate labeling for cell surface Ca_v1.2-HA channels was evident in the soma and dendrites. This distribution of Ca_v1.2-HA was qualitatively similar in neurons transfected with GFP-densin and eGFP alone (Fig. 6A). Consistent with our results in transfected HEK293T cells (Figs. 4, 5), quantitative analyses revealed that cotransfection with GFP-densin increased the dendritic area covered by cell surface clusters of Ca_v1.2-HA channels (Fig. 6B).

Figure 6.

GFP-densin increases the cell surface levels of dendritic Ca_v1.2 channels in neurons. A, Mouse hippocampal neurons were transfected with Ca_v1.2-HA + eGFP together with or without GFP-densin and subjected to live labeling with HA antibodies to mark cell surface channels. Representative fluorescent images are shown. Scale bar, 20 μm. B, Left, Dendrites of neurons transfected as in A shown at higher magnification. Scale bar, 5 μm. Data are representative of at least two independent experiments (number of dendrites analyzed per experiment: 15). Right, Quantification of cell surface Ca_v1.2-HA in hippocampal neurons expressed as the percentage of the dendritic area (defined by eGFP fluorescence) occupied by HA fluorescence. Statistical significance was determined by Student's t test. C, D, Dendrites of neurons transfected with eGFP, GFP-densin, and Ca_v1.2-HA (C) or Ca_v1.2-HA-ΔNT (D). Neurons were subjected to live cell staining with HA antibodies (left) or HA-labeling for total channels after permeabilization (right). Scale bars, 5 μm.

To verify that densin binding to Ca_v1.2 was required for this effect, we initially analyzed neurons transfected with GFP-densin and Ca_v1.2-HA-ΔB1a, which does not interact functionally with densin in HEK293T cells (Figs. 2C, 3D–F). However, Ca_v1.2-HA-ΔB1a did not express at high levels in neurons (data not shown). Therefore, we turned to Ca_v1.2-HA-ΔNT, which lacks the entire NT domain and also does not interact with densin (Fig. 1F). Total levels of Ca_v1.2-HA-ΔNT measured by HA labeling under permeabilized conditions were not different from those of WT Ca_v1.2-HA, but we could not detect Ca_v1.2-HA-ΔNT at the cell surface when coexpressed with GFP-densin (Fig. 6C,D). Although we cannot rule out the possibility that the NT has additional roles in promoting insertion of Ca_v1.2 in the dendritic membrane, our results are consistent with the importance of the densin–Ca_v1.2 interaction in determining the postsynaptic levels of Ca_v1.2 in neurons.

Ca_v1 current density and signaling to the nucleus are weakened in neurons from densin KO mice

If densin supports the cell surface levels of Ca_v1.2 in neurons, then genetic inactivation of densin should cause a reduction in Ca_v1 current density and signaling in neurons. To test this, we used densin KO mice that were characterized previously (Carlisle et al., 2011). In whole-cell patch-clamp recordings, voltage ramps were used to evoke Ba²⁺ currents (I_Ba) in cortical neuron cultures obtained from WT and densin KO mice. Because multiple classes of Ca_v channels are expressed in these neurons (Lorenzon and Foehring, 1995), we isolated Ca_v1 currents pharmacologically by recording currents before and after bath perfusion of the Ca_v1 antagonist isradipine. Because Ca_v1.2 channels account for ∼80–90% of Ca_v1 channels in the brain (Sinnegger-Brauns et al., 2004), this strategy should largely reflect Ca_v1.2-mediated I_Ba. Using the isradipine-sensitive current as a measure of the Ca_v1 current, Ca_v1 channels accounted for ∼40% of peak I_Ba, which is similar to that reported in rat neocortical neurons (Lorenzon and Foehring, 1995). In recordings obtained from 3 separate neuronal cultures, the fractional Ca_v1 current was significantly greater (∼2-fold) in neurons from WT mice (0.42 ± 0.07; n = 8 neurons) than from densin KO mice (0.22 ± 0.05, n = 8 neurons, p < 0.02; Fig. 7A,B).

Figure 7.

Ca_v1 currents and cell surface Ca_v1.2 protein levels are reduced in cortical neurons from densin KO mice. A, I_Ba was evoked by voltage ramps from −60 to +80 mV (0.5 ms/mV) from a holding voltage of −60 mV in cortical neurons from WT (n = 8 cells from 3 mice) and densin KO (n = 8 from 3 mice) mice before (−isr) and after (+isr) bath application of isradipine (5 μm). B, Measurement of the Ca_v1-mediated current. Left, Peak currents for data collected as in A were plotted for voltage ramps evoked every minute before and after application of isradipine. Right, Fractional Ca_v1 current represents the fraction of the total I_Ba that was blocked by isradipine. Statistical significance was determined by Student's t test. C, Biotinylated cell surface proteins in prefrontal cortex of WT and densin KO mice were isolated on streptavidin agarose and subjected to Western blotting. Left, Representative Western blot probed with antibodies against Ca_v1.2, Na⁺/K⁺ ATPase, and GAPDH. Lanes represent biotinylated proteins before (input; ∼10% of total amount used for pull-down) or after streptavidin pull-down (surface). Results are representative of four independent experiments, with one WT and one densin KO littermate used per experiment. Right, Densitometric analysis of biotinylated cell surface Ca_v1.2 channels. Signals corresponding to Ca_v1.2 were normalized to that for Na⁺/K⁺ ATPase (surface) or GAPDH (input). Results are from four independent experiments. Statistical significance was determined by Student's t test.

Although these results were consistent with a role for densin in enhancing the cell surface density of Ca_v1.2 channels, upregulation of non-Ca_v1 channels may have contributed to the decrease in the isradipine-sensitive current in densin KO neurons. Moreover, at the concentration used in our experiments, isradipine (5 μm) inhibits both Ca_v1.2 and Ca_v1.3 (Koschak et al., 2001). To confirm that decreased cell surface levels of Ca_v1.2 could explain the loss of Ca_v1 currents in densin KO neurons, we isolated cell surface proteins by biotinylation in brain slices and purification on streptavidin agarose followed by Western blotting with Ca_v1.2 antibodies. Although there was no difference in total Ca_v1.2 protein levels, there was ∼50% less cell surface Ca_v1.2 in densin KO than in WT brain slices (Fig. 7C). This reduction is similar to, and thus may account for, the decrease in the Ca_v1 current in densin KO neurons (Fig. 7A,B). These results confirm that densin is required to support the cell surface density of Ca_v1.2 channels in the brain.

Activation of postsynaptic Ca_v1 channels promotes local Ca²⁺ signaling events that support activity-dependent gene transcription through phosphorylation of CREB (Wheeler et al., 2012), a transcription factor involved in synaptic plasticity and learning and memory (Deisseroth et al., 2003). If densin controls the number of postsynaptic Ca_v1.2 channels, then depolarization-induced levels of phosphorylated CREB (pCREB) should be reduced in densin KO neurons. To test this prediction, we fixed and immunolabeled neurons with antibodies recognizing pCREB after depolarization with solution containing 40 mm K⁺, which strongly activates Ca_v1-mediated CREB phosphorylation (Wheeler et al., 2012). Although pCREB levels were not different between genotypes under basal conditions (i.e., with 5 mm K⁺), the net increase in pCREB upon depolarization was significantly less in neurons from densin KO compared with WT mice (Fig. 8). We conclude that densin promotes excitation–transcription coupling in neurons, likely through enhancing the cell surface levels of postsynaptic Ca_v1.2 channels.

Figure 8.

Excitation–transcription coupling is reduced in densin KO neurons. A, Epifluorescence images of cortical neurons from WT and densin KO mice exposed to 5 or 40 mm K⁺ for 3 min and processing for double-label immunofluorescence with antibodies against pCREB and the neuronal marker, NeuN. Images are representative of three independent experiments. B, Quantification of the intensity of pCREB fluorescence (arbitrary units, a.u.) in WT and densin KO cortical neurons (n = 50–58 neurons per condition from three different sets of cultures). Statistical significance was determined by Student's t test.

Discussion

Our study provides new insights into the role of densin in regulating neuronal Ca²⁺ signaling. First, in contrast to its interaction with the C-terminal domain of Ca_v1.3, densin binds to the NT of Ca_v1.2 and increases the cell surface levels of Ca_v1.2 by promoting its forward trafficking in transfected cells. Second, densin supports the postsynaptic levels of Ca_v1.2 in neurons, which are significantly reduced in the absence of densin. Finally, densin is required for robust Ca_v1-mediated excitation-transcription coupling in neurons. These effects of densin may contribute to the role of Ca_v1.2 in synaptic plasticity and spatial memory, processes that are disrupted in densin KO mice (Carlisle et al., 2011). In addition to regulating Ca²⁺ and CaMKII-dependent facilitation Ca_v1.3 (Jenkins et al., 2010), densin also regulates the clustering of heterologously expressed Ca_v1.3 channels in dendrites and the stability of dendritic spines in hippocampal neurons in culture (Stanika et al., 2016). Together with these findings, our results reveal densin as a multifunctional regulator of neuronal Ca_v1 channels.

The inability of densin to bind to the C-terminal domain of Ca_v1.2 may seem unexpected given that this region contains a type 1 PDZ-binding sequence similar to that which binds densin in Ca_v1.3 (Jenkins et al., 2010). However, there appears to be some selectivity with which PDZ-domains associate with the Ca_v1 C termini. For example, the PDZ domains of Erbin and Shank interact with the C termini of Ca_v1.3, but not Ca_v1.2 (Zhang et al., 2005; Calin-Jageman et al., 2007). Although deletion of the Ca_v1.2 PDZ-binding site does not affect Ca_v1.2 trafficking in neurons (Di Biase et al., 2008) or cardiac myocytes (Yang et al., 2015), deletions within the NT domain increase the cell surface density and maximal open probability of Ca_v1.2 in Xenopus oocytes (Wei et al., 1996; Kanevsky and Dascal, 2006). Binding of densin to the NT domain may mimic the effect of these deletions because densin not only increases the number of functional channels, but also their relative open probability (Fig. 3). Deletion of a CaMKII-binding site in the Ca_v1.2 NT domain decreases cell surface Ca_v1.2 protein, but increases Ca_v1.2 current density in tsA-201 cells (Simms et al., 2015). Although the effect of densin in our transfected cell recordings did not require CaMKII (Fig. 2B), we cannot discount the possibility that densin modulates the interaction of CaMKII with neuronal Ca_v1.2 channels. CaMKII binds to the α₁ and β subunit of Ca_v1.2, which facilitates channel opening (Hudmon et al., 2005; Koval et al., 2010). Densin may alter such interactions because it enhances CaMKII binding to α-actinin (Walikonis et al., 2001; Robison et al., 2005), but can modulate CaMKII phosphorylation of AMPA and NMDA receptors differentially (Jiao et al., 2011).

Although Ca_v1.2 undergoes activity-dependent endocytosis in neurons (Green et al., 2007; Hall et al., 2013), the basal turnover rate of Ca_v1.2 channels in dendritic spines is slow (>1 h) and their lateral mobility is restricted (Di Biase et al., 2011). The stability of postsynaptic Ca_v1.2 channels could be due to their interactions with a variety of proteins in dendritic spines. For example, α-actinin and AKAP79/150 maintain the localization (Hall et al., 2013) and activity (Oliveria et al., 2007) of Ca_v1.2 in spines, respectively. However, activation of NMDA receptors causes a redistribution of α-actinin and AKAP79/150 into the dendritic shaft without affecting the localization of Ca_v1.2 in the spine (Di Biase et al., 2008), suggesting that these proteins are not sufficient for stabilizing dendritic Ca_v1.2 channels. In addition to CaMKII and α-actinin (Strack et al., 2000; Walikonis et al., 2001), densin interacts with Shank (Quitsch et al., 2005), δ-catenin (Izawa et al., 2002), and spinophilin (Baucum et al., 2010). Densin may help to anchor Ca_v1.2 channels within a network of interacting proteins, the dissolution of which may cause the loss of cell surface Ca_v1.2 channels in brain slices and neurons from densin KO mice (Fig. 7). This mechanism may explain how densin also promotes the dendritic clustering Ca_v1.3 (Stanika et al., 2016) despite binding to a different site than Ca_v1.2 (Fig. 1C,E).

The intracellular accumulation of Ca_v1.2-HA-ΔNT in the dendritic shaft, as well as its exclusion from spines (Fig. 6D), is similar to that of Ca_v1.2 mutants deficient in interactions with the auxiliary β subunit (Obermair et al., 2010). Considering that the forward trafficking of SNAP-tagged Ca_v1.2 channels in HEK293T cells is accelerated by densin (Fig. 5), densin may play a similar role as the β subunit in facilitating the export of Ca_v1.2 channels from their sites of synthesis and insertion into the postsynaptic membrane. This effect of densin may be particularly important to replenish Ca_v1.2 channels endocytosed during high levels of neuronal excitation (Green et al., 2007; Hall et al., 2013). The NT of Ca_v1.2 contains binding sites for multiple proteins including calmodulin (Ivanina et al., 2000), CaMKII (Simms et al., 2015), and CaBP1 (Zhou et al., 2005). Therefore, the loss of Ca_v1.2-HA-ΔNT interactions with densin and potentially other proteins could have collectively contributed to the complete absence of cell surface labeling of these mutant channels in dendrites (Fig. 6D).

As one of the major CaMKII-binding proteins in postsynaptic density (Apperson et al., 1996; Strack et al., 2000; Walikonis et al., 2001; Carlisle et al., 2011; Lowenthal et al., 2015), densin may provide a local pool of CaMKII that is necessary for coupling Ca_v1 channels to the activation of CREB in the nucleus (Wheeler et al., 2008; Wheeler et al., 2012). Ca²⁺ influx through Ca_v1 channels is sensed by calmodulin, which binds to and causes autophosphorylation of CaMKII, followed by a series of events leading to translocation of calmodulin to the nucleus and phosphorylation of CREB (Ma et al., 2014). Consistent with a role for densin in positioning CaMKII close to Ca_v1-mediated Ca²⁺ signals, the level of autophosphorylated CaMKII is profoundly reduced (∼75%) in neurons from densin KO compared with WT mice (Carlisle et al., 2011). Therefore, densin may not only ensure a sufficient density of Ca_v1.2 channels, but also of associated CaMKII enzymes available for excitation–transcription coupling. This would explain the strong reductions in Ca_v1-mediated pCREB induction in densin KO neurons (Fig. 8) and upon shRNA knockdown of densin in hippocampal neurons (Wheeler et al., 2012).

Our results suggest a potential mechanism to account for the overlap in the behavioral phenotypes of densin KO mice and those in mice with targeted deletion of Ca_v1.2. Compared with the respective control mice, anxiety-like behavior is significantly greater in densin KO mice (Carlisle et al., 2011) and mice lacking Ca_v1.2 expression in the prefrontal cortex (Lee et al., 2012). Anxiety disorders are common in patients with schizophrenia (Achim et al., 2011) and other neuropsychiatric disorders linked to polymorphisms in the CACNA1C gene encoding Ca_v1.2 (Heyes et al., 2015). It is tempting to speculate that the reduced abundance of functional Ca_v1.2 channels may contribute to the increased anxiety-like behavior and other endophenotypes of schizophrenia in densin KO mice (Carlisle et al., 2011). Indeed, molecular analyses of schizophrenia-associated polymorphisms in an intronic region of CACNA1C suggest the potential for altered levels of CACNA1C expression in patients harboring these variants (Eckart et al., 2016). Therefore, densin KO mice may serve as an appropriate mouse model with which to study how loss of function of Ca_v1.2 leads to cellular and behavioral anomalies underlying mental illness.