A protein phosphatase 2calpha-Ca2+ channel complex for dephosphorylation of neuronal Ca2+ channels phosphorylated by protein kinase C

Abstract

Phosphorylation and dephosphorylation are primary means for rapid regulation of a variety of neuronal functions, such as membrane excitability, neurotransmitter release, and gene expression. Voltage-gated Ca2+ channels are targets for phosphorylation by a variety of second messengers through activation of different types of protein kinases (PKs). Protein phosphatases (PPs), like PKs, are equally important in regulating Ca2+ channels in neurons. However, much less is understood about whether and how a particular type of PP contributes to regulating neuronal Ca2+ channel activities. This is primarily because of the lack of specific inhibitors/activators for different types of PPs, particularly the PP2c family. The functional roles of PP2c and its substrates in the brain remain virtually unknown. During our yeast two-hybrid screening, PP2calpha was pulled out by both N- and P/Q-type Ca2+ channel C termini. This raised the possibility that PP2calpha might be associated with voltage-gated Ca2+ channels for regulation of the Ca(2+) channel activity. Biochemical studies show that PP2calpha binds directly to neuronal Ca2+ channels forming a functional protein complex in vivo. PP2calpha, unlike PP1, PP2a and PP2b, is more effective in dephosphorylation of neuronal Ca2+ channels after their phosphorylation by PKC. In hippocampal neurons, disruption of the PP2calpha-Ca2+ channel interaction significantly enhances the response of Ca2+ channels to modulation by PKC. Thus, the PP2calpha-Ca2+ channel complex is responsible for rapid dephosphorylation of Ca2+ channels and may contribute to regulation of synaptic transmission in neurons.

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.

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.

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.

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.

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.

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.

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.

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.