A Protein Phosphatase 2cα-Ca²⁺ Channel Complex for Dephosphorylation of Neuronal Ca²⁺ Channels Phosphorylated by Protein Kinase C

Yeast two-hybrid screening

Details of screening have been published previously (Chen et al., 2003; Maeno-Hikichi et al., 2003). The C terminus of α_1A (CaV2.1, amino acid residues 1819-2422; GenBank accession number X57477), α_1B (CaV2.2, 1707-2339; GenBank accession number M94172), or α_1C (CaV1.2, 1505-2171; GenBank accession number X15539) subunit was used as bait to screen a rat brain cDNA library (OriGene, Rockville, MD). We screened ∼7 × 10⁶ to 1 × 10⁸ independent clones for each bait. A total of >200 clones were sequenced out of ∼2000 positive clones.

Construction and expression of recombinant proteins

Reverse transcription-PCR was used to generate the full-length PP2cα (GenBank accession number J04503). PCR-based methods were used to generate all of the constructs used in this study. All constructs were verified by sequencing. To generate recombinant proteins, cDNA constructs were subcloned into pGEX-4T-1 (Amersham Biosciences, Arlington Heights, IL) for glutathione S-transferase (GST)-fused proteins or pQE-30 (Qiagen, Hilden, Germany) for polyhistidine-tagged (6×His) proteins. GST-fusion proteins and 6×His proteins were produced in Escherichia coli. Glutathione agarose beads (Pierce, Rockford, IL) were used to purify GST-fused proteins. Ni-NTA columns (Qiagen) were used for purification of 6×His proteins. To express the recombinant proteins in mammalian cells, cDNA constructs were subcloned into the following vectors: pCDNA3 (Invitrogen, San Diego, CA), HA-tagged pcDNA3, FLAG-tagged pcDNA3, or pEGFP-C2 (Clontech, Palo Alto, CA). The calcium phosphate method was used for transfection in human embryonic kidney 293 (HEK 293) cells or hippocampal neurons.

GST-fusion protein pulldown assays

Pulldown assays were performed as described previously (Chen et al., 2003; Maeno-Hikichi et al., 2003). Briefly, ³⁵S-labeled proteins were synthesized using an in vitro protein translation kit (Promega, Madison, WI). The assay buffer contained 100 mm NaCl, 20 mm Tris HCl, and 5% glycerol, pH 7.0, along with 1% Triton X-100 and a mixture of protease inhibitors (1 μm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.1 mg/ml benzamidine). Approximately 5 μg of GST-fused protein was incubated with 2 μl of ³⁵S-labeled protein with gentle rocking. The length of incubation varied from 2 h to overnight. All pulldown assays were performed at 4°C. The results were analyzed by SDS-PAGE and autoradiography. Nonspecific binding was insignificant.

In vitro phosphorylation and dephosphorylation assays

In vitro phosphorylation. 6×His-tagged Ca²⁺ channel I-II and II-III loops (α_1B, amino acid residues 357-463 for the I-II loop and amino acid residues 719-952 for the II-III loop) and 6×His-tagged myristoylated alanine-rich C-kinase substrate (MARCKS) were purified and quantified by the Bradford method. The purified proteins (2 μm) were phosphorylated by PKCϵ (0.01 μm; Oxford, Concord, MA) at 30°C for 10 min, in the buffer containing 50 mm Tris-HCl, 10 mm MgCl₂, and 1 mm DTT along with 0.05 mg/ml diacylglycerol, 0.5 mg/ml l-α-phosphatidylserine (Sigma, St. Louis, MO), 12.5 μm ATP, and 0.1-0.5 μCi [γ-³²P]ATP (Amersham Biosciences) for each reaction. The reaction was stopped by heat inactivation of PKCϵ (60°C for 10 min; our unpublished data).

In vitro dephosphorylation. The phosphorylated protein was aliquoted, in equal amounts, in a set of microtubes. Dephosphorylation was initiated by adding different PPs (0.02 μm), and the reaction was terminated at various time points by boiling in the SDS gel-loading buffer. For PP2b, Ca²⁺ (1 mm) and calmodulin (1 μm) were added for its activation. PP2cα was purified in the laboratory, and the others were purchased from Promega (PP2a and PP2b) and New England Biolabs (Beverly, MA) (PP1). All experiments were performed at 30°C. The results were analyzed by SDS-PAGE and quantified by a phosphoimager.

Coimmunoprecipitation and immunoblotting

Coimmunoprecipitation of PP2cα and calcium channels was performed using adult rat cortical extracts as described previously (Chen et al., 2003; Maeno-Hikichi et al., 2003). Briefly, the soluble membrane fraction of adult rat cortical extracts was incubated with anti-α_1B antibody (Alomone Laboratories, Jerusalem, Israel)-conjugated protein G-Sepharose beads overnight at 4°C. The immunoprecipitation buffer contained (in mm): 137 NaCl, 2.7 KCl, 4.3 Na₂HPO₄, 1.4 KH₂PO₄, 5 EGTA, and 5 EDTA, pH 7.5. Immune complexes were resolved by SDS-PAGE and analyzed by immunoblotting with antibodies against PP2cα (Upstate Biotechnology, Lake Placid, NY) and α_1B.

Hippocampal neuron culture

Use of animals received University Institutional Animal Care and Use Committee approval. Briefly, hippocampal neurons were cultured from embryonic day 18 (E18) to E19 embryonic rats (Sprague Dawley). The neurobasal medium was supplemented with 10% fetal bovine serum and B-27 and supplied with 5% CO₂. Typically, cultured neurons were transfected with different cDNA constructs at 10 d in vitro (DIV), and physiology experiments were performed at 12 DIV.

Immunocytochemistry

Hippocampal neurons were fixed with 4% paraformaldehyde and 4% sucrose in PBS for 10 min, permeabilized with 0.25% Triton X-100 in PBS for 5 min, and quenched with 0.1 m glycine in PBS. An anti-synapsin I antibody (1:200; Oncogene Sciences, Uniondale, NY) was used for immunostaining. An Alexa 568-conjugated secondary antibody (1:4000; Molecular Probes, Eugene, OR) was used for visualization. Fluorescent images were collected with a Zeiss (Oberkochen, Germany) Axiovert 200 inverted microscope equipped with a Hamamatsu (Hamamatsu City, Japan) CCD camera.

Electrophysiology recordings

Details for electrophysiological recordings have been published previously (Chen et al., 2003; Maeno-Hikichi et al., 2003). Briefly, whole-cell recordings were performed using an Axopatch 200B amplifier. pClamp 8.0 software (Axon Instruments, Foster City, CA) was used for data acquisition and analysis. Leak currents and capacitive transients were subtracted on-line with a P/4 protocol. The bath solution was composed of the following (in mm): 128 NaCl, 5 KCl, 10 BaCl₂, 1 MgCl₂, 10 HEPES, 10 TEA-Cl, and 10 glucose, pH 7.3. Tetrodotoxin (1 μm; Alomone Laboratories) and nifedipine (1 μm; Calbiochem, La Jolla, CA) were added in the bath solution to block Na⁺ and L-type Ca²⁺ channels. Under this condition, the primary Ca²⁺ current component should be of N- and P/Q-type with a very small percentage of R-type Ca²⁺ current (Zhang et al., 1993). The recording pipette was filled with a solution containing the following (in mm): 110 CsCl, 5 MgSO₄, 10 EGTA, 25 HEPES, 2 ATP, and 0.2 GTP, pH 7.2. Current traces were digitized at 10 kHz and low-pass filtered at 1 kHz.

Formation of a PP2cα-Ca²⁺ channel complex

To identify proteins that might interact with Ca²⁺ channels and play a role in Ca²⁺-dependent signal transduction and/or regulating the Ca²⁺ channel activity, we performed yeast two-hybrid screening of a rat brain cDNA library using as bait the full-length C termini of three different Ca²⁺ channel α₁ subunits, α_1A (CaV2.1, P/Q-type), α_1B (CaV2.2, N-type), and α_1C (CaV1.2, L-type) (Chen et al., 2003; Maeno-Hikichi et al., 2003). One clone, pulled out by both the N- and P/Q-type Ca²⁺ channel C termini (NCF and PCF), encoded a partial sequence of PP2cα, a member of PP2c family (Price and Mumby, 1999; Herzig and Neumann, 2000; Sim et al., 2003). Of the 200 plus clones we have sequenced, PP2cα is the only phosphatase identified during screening (our unpublished data). The interaction between PP2cα and the channel C termini was rigorously evaluated by several different approaches. In yeast, the interaction was established by both LacZ reporter gene assay and galactose-dependent growth in leucine-deficient medium. Binding of PP2cα to NCF was verified by GST-fusion protein pulldown assays (Fig. 1A). The interaction was further confirmed by coexpressing PP2cα and Ca²⁺ channel C termini in HEK 293 cells and coimmunoprecipitation (CoIP) assays (Fig. 1B). Reciprocal CoIP yielded the same results (Fig. 2B).

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Figure 1.

Association of PP2cα with neuronal voltage-gated Ca²⁺ channels. A, GST-fusion protein pulldown assays. Experiments were performed in the presence of 1 mm EGTA. B, PP2cα interacts with all three types of Ca²⁺ channels. GFP-tagged PP2cα and FLAG-tagged channel C termini were coexpressed in HEK 293 cells. CoIP assays were performed 2 d after transfection. C, Presence of PP2cα in the brain. Recombinant PP2cα (in pcDNA3) was expressed in HEK 293 cells. The calculated molecular mass for PP2cα is 42.2 kDa. D, Formation of the PP2cα-Ca²⁺ channel complex in vivo. Brain lysates were immunoprecipitated with an anti-α_1B antibody, and blots were detected with antibodies against PP2cα and N-type Ca²⁺ channels (α_1B). WB, Western blot.

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Figure 2.

Identification of the Ca²⁺ channel binding domain in PP2cα. A, Schematic representation of the wild-type PP2cα and its deletion mutants. The catalytic domain is from amino acid residues 22-284. D1 covers amino acid residues 1-291, and D2 covers 283-382. B, Effects of deletion mutations on the interaction between PP2cα and the N-type Ca²⁺ channel C terminus.

Multiple types of Ca²⁺ channels coexist in neurons, including L-, N-, and P/Q-types (Zhang et al., 1993; Dunlap et al., 1995; Catterall, 1998). In addition to the N-type, both L- and P/Q-type Ca²⁺ channel C termini could also bind to PP2cα (Fig. 1B). This pattern differs from what we reported for the formation of a PKC-N-type Ca²⁺ signaling complex, in which ENH binds to N-but not P/Q-type Ca²⁺ channels (Maeno-Hikichi et al., 2003). The results indicated that the PP2cα binding domain might be located at a segment with a high degree of sequence homology among all three types of Ca²⁺ channels (see below). In the rest of the paper, we refer to the interaction between PP2cα and Ca²⁺ channels, although most of the data were collected using the N-type Ca²⁺ channel (α_1B) and its mutants. Immunoblotting showed PP2cα was present in the brain (Fig. 1C), in agreement with previous reports (Price and Mumby, 1999; Herzig and Neumann, 2000). To directly demonstrate the existence of the PP2cα-Ca²⁺ channel complex, CoIP was performed using lysates prepared from the adult rat brain. Consistent with the in vitro data using expressed Ca²⁺ channels or their C termini, anti-α_1B antibody was able to immunoprecipitate PP2cα from the crude membrane fraction of the brain preparation (Fig. 1D) (also see Fig. 6A). These results demonstrate that PP2cα and Ca²⁺ channels form a complex in vivo. This conclusion is further substantiated by results of mutagenesis and functional studies (see Figs. 2, 6, 7, 8),

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Figure 6.

VGCCs are substrates for PP2cα in vivo. A, Association of PP2cα and N-type Ca²⁺ channels expressed in tsA cells. CoIP assays were performed 48 h after transfection of GFP-tagged PP2cα. B, Exemplar current traces recorded from a cell before and after application of PDBu. C, Expression of PP2cα decreases potentiation of the Ca²⁺ channel activity by PKC. V_t = 0 mV, and V_h =-80 mV. Peak Ca²⁺ currents were normalized to that recorded before application of PDBu and plotted as a function of time (n = 8). D, Summary of the effects of PDBu on the channel activity with different constructs expressed in tsA cells.

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Figure 7.

Disruption of the PP2cα-Ca²⁺ channel interaction enhances modulation of the channel activity by PDBu in hippocampal neurons. A, A simplified model of phosphorylation and dephosphorylation of Ca²⁺ channels by PKC and PP2cα. B, A dominant-negative construct, PP2cα-D2, enhances the modulatory effects of PDBu on the Ca²⁺ channel activity. Neurons were depolarized from a holding potential of -80 to 0 mV. Data are mean ± SEM. C, Exemplar current traces obtained before and after application of PDBu from a nontransfected neuron. D, I-V curves obtained before and after application of PDBu from a nontransfected control neuron. E, I-V curves obtained before and after application of PDBu from a neuron transfected with GFP-PP2cα-D2. Recordings were performed 48 h after transfection.

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Figure 8.

PP2cα is functionally associated with VGCCs in neurons. Effects of PP2cα-D2 are caused by its selective disruption of the PP2cα-Ca²⁺ channel interaction in vivo. Hippocampal neurons were transfected with different constructs as indicated, and their responses to modulation by PKC were recorded 48 h after transfection.

As an enzyme, PP2cα could, in principle, bind to its substrate, Ca²⁺ channels, through its catalytic domain. Such an interaction, common to all enzyme-mediated catalysis, would be less interesting in terms of cellular signal transduction and would not be the basis for formation of a specific signaling protein complex. We sought to locate the Ca²⁺ channel binding domain within PP2cα through deletion mutations. The phosphatase catalytic domain is located in the N-terminal two-thirds of PP2cα (Fig. 2A) (Price and Mumby, 1999; Herzig and Neumann, 2000). Mutational analysis showed that the channel binding domain was mapped to the C-terminal end of PP2cα, outside of the catalytic domain (PP2cα-D2) (Fig. 2B). The PP2cα binding domain was narrowed to a fragment of 156 aa residues right after IVS6 of the α_1B subunit (amino acid residues 1708-1864; data not shown), where all three types of Ca²⁺ channels share a high degree of homology in their primary amino acid sequences [see references listed in the study by Catterall (1998)]. Existence of a unique binding domain in PP2cα as well as in the channel C terminus provides another piece of evidence strongly supporting the specific interaction between PP2cα and the Ca²⁺ channel. In addition to understanding the molecular nature of the interaction, the deletion mutant, PP2cα-D2, which encompasses the channel binding domain (Fig. 2A), provided a useful tool for functional studies (see Figs. 7, 8).

Next, we sought to determine the subcellular localization of PP2cα in neurons using immunofluorescence microscopy. Its subcellular localization may provide a clue whether PP2cα is located in the same subcellular compartments as VGCCs. Hippocampal neurons were transfected with green fluorescent protein (GFP)-tagged PP2cα and stained with an antibody against synapsin I, a synaptic vesicle protein known to be present in presynaptic boutons (Sudhof et al., 1989). Neurons transfected with GFP-PP2cα displayed punctate green fluorescence along the neurites (Fig. 3B). This is in sharp contrast to neurons transfected with GFP alone, which showed uniform distribution of the green fluorescence throughout the neuron (Fig. 3E). These green fluorescent puncta could be simultaneously stained with the synapsin antibody (Fig. 3A,C), suggesting that these puncta are in fact presynaptic buttons and that PP2cα is enriched in presynaptic nerve terminals where VGCCs are known to be present for release of neurotransmitters (Catterall, 1998).

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Figure 3.

Presence of PP2cα in presynaptic boutons in hippocampal neurons. A, Immunostaining of a presynaptic protein, synapsin I, in GFP-PP2cα-transfected neurons. B, Green fluorescence of the same neuron, exhibiting punctate distribution of the GFP-tagged PP2cα.C, Overlay of the red (synapsin I) and green fluorescent (GFP-PP2cα) images. D, Synapsin I staining of a neuron transfected with GFP alone. E, Green fluorescence of the same neuron, exhibiting uniform distribution of GFP. The fluorescence on the cell body was saturated. F, Overlay of the red (synapsin I) and green fluorescent (GFP) images.

PP2cα is more effective in dephosphorylation of Ca²⁺ channels phosphorylated by PKCϵ

Could VGCCs, once phosphorylated by PKC, be a substrate for PP2cα? Furthermore, among different types of PPs, which one might play a major role in dephosphorylation of Ca²⁺ channels? To address these questions, we performed in vitro dephosphorylation assays using I-II and II-III loops of the Ca²⁺ channel α_1B subunit (N-type) and four different types of PPs: PP1, PP2a, PP2b, and PP2cα. Functional PKC phosphorylation sites have been identified in both I-II and II-III loops of the Ca²⁺ channel α₁ subunit (Fig. 4A) (Yokoyama et al., 1997; Zamponi et al., 1997). Phosphorylation of Ca²⁺ channels by PKC potentiates the channel activity and regulates interactions between Ca²⁺ channels and other signaling proteins (Yang and Tsien, 1993; Yokoyama et al., 1997; Zamponi et al., 1997). For instance, whereas phosphorylation of the I-II loop by PKC results in relief of the inhibitory effects by G-proteins, phosphorylation of the II-III loop promotes dissociation of N-type Ca²⁺ channels from syntaxin, a synaptic vesicle protein for release of neurotransmitters.

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Figure 4.

The Ca²⁺ channel II-III loop is a preferred substrate for PP2cα. A, Schematic representation showing the functional PKC phosphorylation sites in the Ca²⁺ channel α₁ subunit. Phosphorylation of the I-II or II-III loop by PKC regulates the interactions between Ca²⁺ channels and G-proteins or syntaxin, respectively (synprint: the syntaxin binding domain). B, Autoradiogram showing dephosphorylation of the phospho-II-III loop by these four different PPs. The II-III loop was phosphorylated by PKCϵ. Purified PP2cα was constitutively active. C, Time course of dephosphorylation of the II-III loop by four PPs. The intensity of each band was quantified by a phosphoimager and normalized to that of time 0. D, Time course of dephosphorylation of the I-II loop by four PPs. E, Time course of dephosphorylation of MARCKS by four PPs. All experiments were performed under identical conditions, and data are mean ± SEM.

Both I-II and II-III loop fragments could be phosphorylated by PKCϵ, similar to previous reports using a mixture of multiple PKCs (Yokoyama et al., 1997; Zamponi et al., 1997). Once dephosphorylation was initiated, the four PPs exhibited remarkably different capabilities in their dephosphorylation of the phosphorylated I-II and II-III loops (Fig. 4). Most notably, for the phospho-II-III loop, dephosphorylation by PP2cα developed much faster than the rest of PPs, and removal of ³²P was nearly complete after 30 min, which is significantly higher than that by other PPs (Fig. 4B,C) (p < 0.001; n = 3). Although all PPs are able to dephosphorylate the phosphorylated II-III loop, PP2cα is the most effective. The relative effectiveness for dephosphorylation of the phospho-II-III loop by these PPs is PP2cα ≫ PP2a > PP1 > PP2b. Dephosphorylation of the phospho-II-III loop by PP1, PP2a and PP2b was incomplete at their plateaus, leaving significant amount of the II-III loop remaining phosphorylated, 37.6 ± 1.3% for PP1, 26.4 ± 4.6% for PP2a, and 48.9 ± 4.7% for PP2b (compared with 5.4 ± 2.1% for PP2cα).

In contrast, for the phospho-I-II loop, none of the four PPs was very effective in removing the phospho-groups (Fig. 4C). After 60 min, a substantial amount of the I-II loop remained phosphorylated (69.0 ± 2.0% for PP1, 45.1 ± 3.1% for PP2a, 71.3 ± 3.2% for PP2b, and 56.9 ± 5.3% for PP2cα). At least for both PP2a and PP2cα, dephosphorylation had not reached the plateau after 60 min. If different PPs have distinct intrinsic activities but no preferences over different substrates, we expected to see that these four PPs should be equally effective in dephosphorylation of both I-II and II-III loops, when the assays were performed under the identical experimental conditions.

To further test whether different PPs had their preferred substrates, we turned to a different neuronal protein, MARCKS, which is a specific substrate for PKC (Aderem, 1992, 1995; Seki et al., 1995). Phospho-MARCKS is dephosphorylated by PPs, with PP1 being most effective, followed by PP2a and PP2b (Seki et al., 1995). Thus, MARCKS was used as control for the in vitro dephosphorylation assays. Polyhistidine-tagged MARCKS was purified and phosphorylated by PKCϵ, followed by dephosphorylation by PPs. The rank order for dephosphorylation of MARCKS is PP1 > PP2a > PP2b ≥ PP2cα, which is entirely different from that for the phosphorylated I-II and II-III loops (Fig. 4D). The effectiveness of PP1, PP2a, and PP2b is the same as reported previously (Seki et al., 1995). In contrast to what we saw for the phosphorylated II-III loop, PP2cα was least effective in dephosphorylation of MARCKS, with >50% of the protein remaining in the phosphorylated state 60 min after dephosphorylation was started (53.0 ± 2.3%, compared with 22.6 ± 3.2% for PP1; p < 0.002; n = 3). These results demonstrate that PPs indeed have their preferred substrates or even preferred phospho-groups. This conclusion is further supported by the observation that dephosphorylation of the I-II loop becomes more effective when all four PPs are present (Fig. 4D, filled square).

Quantitative analysis of the dephosphorylation data revealed that the apparent rate constant for dephosphorylation by PP2cα was significantly larger than that for other PPs (Fig. 5A). The preference of PP2cα for the phospho-II-III loop and the rapid rate of dephosphorylation by PP2cα should result in rapid removal of the phospho-groups from Ca²⁺ channels. For instance, >60% of ³²P was removed by PP2cα within 5 min after initiation of dephosphorylation (compared with <30% for the rest of PPs) (Fig. 5B). In contrast, for the I-II loop, fitting the data with a single exponential function was inadequate (Fig. 4D). The apparent rate constant for PP2cα is not significantly different from the others (Fig. 5C). Overall, ∼20% of the phospho-groups were removed from the phospho-I-II loop 5 min after start of dephosphorylation by these four PPs (Fig. 5D). Analysis of the dephosphorylation data from MARCKS yielded a different profile (data not shown). Collectively, these data show that among these four major PPs, PP2cα is more effective in dephosphorylation of neuronal Ca²⁺ channels after their phosphorylation by PKCϵ, at least in the in vitro settings. The results also illustrate that dephosphorylation of VGCCs by Ser/Thr protein phosphatases is more complicated than previously thought.

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Figure 5.

Quantitative analysis of dephosphorylation of the I-II and II-III loops by PP1, PP2a, PP2b, and PP2cα. A, Apparent rate constants for dephosphorylation of the II-III loop by PPs. The rate constants were derived from fit of the time course to a simple exponential function. B, Removal of ³²P from the phospho-II-III loop 5 min after initiation of the dephosphorylation reaction. C, Apparent rate constants for dephosphorylation of the I-II loop by PPs, which displayed a distinct rank order for these PPs. D, Removal of ³²P from the phospho-I-II loop 5 min after initiation of the dephosphorylation reaction.

VGCCs are a substrate for PP2cα in vivo

Are full-length Ca²⁺ channels, once phosphorylated by PKC, a substrate for PP2cα in vivo, as suggested by our in vitro assays using the channel fragments? We approached this question by expressing PP2cα in a tsA (temperature-sensitive allele of simian virus 40 large T antigen) cell line that stably expresses N-type Ca²⁺ channels (α_1B plus α₂-δ plus β₃ subunits; a generous gift from Dr. D. Lipscombe, Brown University, Providence, RI) (Lin et al., 1997). Immunoblotting showed the presence of the α_1B subunit (Fig. 6A) and PKCϵ (data not shown). Ca²⁺ currents could be recorded from this tsA cell line using Ba²⁺ as the charge carrier (Fig. 6B). Activation of PKC by PDBu (1 μm) resulted in acute potentiation of the Ca²⁺ currents (Fig. 6B,C), a response similar to that of native N-type Ca²⁺ channels in neurons (Swartz et al., 1993; Yang and Tsien, 1993).

When expressed in this tsA cell line, GFP-PP2cα was associated with the N-type Ca²⁺ channels as demonstrated by CoIP assays (Fig. 6A). Introduction of PP2cα significantly reduced the response of N-type Ca²⁺ channels to modulation by PKC (Fig. 6C). The maximal response to 1 μm PDBu was decreased from 181.3 ± 6.0 to 145.2 ± 4.7% (p < 0.001; n = 8) (Fig. 6D). Transfection of the cell line with GFP alone did not produce any significant impact on potentiation of the channel activity by PKC (177.3 ± 7.6%; n = 7) (Fig. 6D). These results suggest that the fulllength N-type Ca²⁺ channels, when phosphorylated by PKC, are a substrate for PP2cα. This conclusion is further strengthened by the results of expressing a dominant-negative construct in hippocampal neurons (Fig. 7).

PP2cα is functionally associated with VGCCs in hippocampal neurons

We next asked whether a functional PP2cα-Ca²⁺ channel complex existed in neurons, as suggested by the results of CoIP assays (Figs. 1D,6D). We sought to test whether overexpression of a dominant-negative construct could interfere with the PP2cα channel interaction and consequently the response of Ca²⁺ channels to modulation by PKC in neurons. The rationale is that overexpression of the channel binding domain from PP2cα will compete with and therefore displace endogenous PP2cα for binding to Ca²⁺ channels and effectively reduce the local PP2cα concentration near Ca²⁺ channels. As a result, more channels will become phosphorylated when PKC is activated (Fig. 7A).

Mutagenesis studies showed that the channel binding domain in PP2cα was located downstream of the catalytic domain, within a fragment of 100 aa residues (Fig. 2, PP2cα-D2). Blast of the GenBank database with this fragment did not pick up any other proteins except PP2cα itself, indicating that overexpression of PP2cα-D2 is less likely to interfere with other signaling processes in neurons. As expected, when GFP-tagged PP2cα-D2 was overexpressed in hippocampal neurons, modulation of Ca²⁺ currents by PDBu developed much faster and became more effective, compared with nontransfected control neurons (Fig. 7). The time required for modulation to develop from 10 to 90% of the maximal response (T_10-90) was reduced from 270.5 ± 44.9 s for control (n = 12) to 147.9 ± 17.1 s for PP2cα-D2-transfected neurons (n = 9; p < 0.02). Transfection of GFP-PP2cα-D2 did not affect voltage-dependent activation of the Ca²⁺ currents (Fig. 7D,E). Effects of GFP-PP2cα-D2 on potentiation of Ca²⁺ currents by PDBu were observed at all the test voltages (Fig. 7E). On average, potentiation of the Ca²⁺ currents by PDBu was increased from 163.7 ± 6.4% (n = 12) to 211.0 ± 6.6% (n = 9) as a result of overexpression of GFP-PP2cα-D2 (p < 0.001; V_t = 0 mV) (Fig. 8). Expression of GFP alone had no impact on modulation of Ca²⁺ currents by PDBu (154.0 ± 8.5%; p > 0.3) (Fig. 8).

To further test the specificity of the effects by GFP-PP2cα-D2, we transfected neurons with GFP-tagged endophilin, another Ca²⁺ channel partner protein. As we reported recently, endophilin binds to the C terminus of the Ca²⁺ channel α₁ subunit, and the channel-endophilin interaction serves to bring the endocytotic machinery into the presynaptic nerve terminal (Chen et al., 2003). On the channel C terminus, the endophilin binding site is further downstream of the PP2cα binding site, as revealed by mutagenesis studies, and endophilin does not interfere with the PP2cα channel interaction (our unpublished data). Overexpression of GFP-endophilin did not have any significant impact on the effects by PDBu (148.0 ± 8.9%; p > 0.15) (Fig. 8). Together, these data suggest that the effects of PP2cα-D2 are caused specifically by blockade of the interaction between endogenous PP2cα and VGCCs.