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Department of Pharmacology and Neurobiology, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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1, 2, ß, and 
. Among these subunits, the pore-forming subunit, 1, is the largest and most important and determines the fundamental properties of the channel, which are modulated by the other subunits. It is now known that there are 10 different genes coding for 1 subunit in mammalian genome (Cav1.1–1.4, Cav2.1–2.3, and Cav3.1–3.3, also designated as 1A–1I, and 1S; for nomenclature of VDCCs, see Ertel et al. 2000). On the relationship between the functionally classified channels and structurally identified molecules, the recent consensus is as follows: Cav1 family corresponds to the L-type, Cav2.1 to P/Q-types, Cav2.2 to N-type, Cav2.3 to R-type, and Cav3 family to T-type (Ertel et al. 2000). In the nervous system, both N- and P/Q-type VDCCs are abundantly expressed and play pivotal roles in the control of neurotransmitter release (Dunlap et al. 1995). In many types of neurones, both of these channels are expressed and contribute to synaptic transmission, though the degree of the contribution seems to differ depending on the cell types (Dunlap et al. 1995). Consistent with this, immunohistochemical studies using specific antibodies to these channels revealed similar, though not completely the same, expression patterns of these channels in various types of neurones: these channels were detected in nerve terminals, soma, and dendrites (Westenbroek et al. 1992, 1995). Besides, Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels are structurally similar: both of these show similar gene organization in the chromosome, high degree of sequence homology, and similar patterns of alternative splicing (Ophoff et al. 1996; Lin et al. 1999; Soong et al. 2002; Bell et al. 2004). Thus, these two types of channels share many features in common in terms of structure and function.
Recently, an interesting mechanism for synaptic targeting of Cav2.2 channel was reported (Maximov & Bezprozvanny 2002). One variant for Cav2.2 channel, produced by an alternative splicing, carrying a longer C-terminal tail was preferentially translocated to axon and axon terminals, and the one with a shorter C-terminal tail tended to be located at cell soma and dendrites. Two motifs mediating protein–protein interactions, which are contained in the long C-terminal region, are suggested to be involved in the mechanism of synaptic targeting of this splice variant. One is a binding consensus for PDZ domain, recognized by Mint1, and the other is a proline-rich region that binds to SH3 domain of CASK (Maximov et al. 1999; Maximov & Bezprozvanny 2002). Thus, subcellular localization of Cav2.2 channel may be controlled by intrinsic cues that are regulated by alternative splicing.
As for Cav2.1 channel, though it is most similar to Cav2.2 channel in terms of primary structure and physiological function, the mechanism of subcellular localization is poorly understood. As is the case for Cav2.2, it is known that there are two splice variants with respect to the C-terminal tail. This variation results from the differential usage of the splice acceptor at intron 46/exon 47 boundary of Cav2.1 gene (Zhuchenko et al. 1997; Soong et al. 2002). Interestingly, the PDZ binding motif and SH3 binding motif, which are shown to be important for synaptic targeting of Cav2.2 channel, are also conserved in the long version of Cav2.1 (Maximov et al. 1999). Therefore, Cav2.1 channel is expected to be targeted to synapses by a similar mechanism as in the Cav2.2 channel.
Longer version of the C-terminal tail of human Cav2.1 has an additional motif specific to this channel: it contains a polyglutamine stretch, whose expansion is known to cause human neurological disease spinocerebellar ataxia type 6 (SCA6) (Zhuchenko et al. 1997). We have identified cytoplasmic aggregates containing Cav2.1 channel possessing a long C-terminal tail in Purkinje cells from SCA6 patients (Ishikawa et al. 1999, 2001). This phenotype may be related to the possible changes in the localization of Cav2.1 channel due to the SCA6 mutation.
In this study, we addressed two questions. First, is the C-terminal tail of the Cav2.1 channel involved in the determination of localization pattern of this channel in neurones, as is the case with Cav2.2 channel? Second, does the CAG repeat expansion affect the localization pattern of Cav2.1 channel in neurones? Surprisingly, the results suggest that Cav2.1 channel apparently uses different mechanism for its localization in neurones from that for Cav2.2 channel. Furthermore, expansion of polyglutamine to the disease range does not affect the localization of Cav2.1 channel.
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Results |
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As is pointed out by Maximov et al. (1999), significant sequence similarities are detected between the C-terminal tail region of the Cav2.1 channel and that of the Cav2.2 channel by the ClustalW program (Fig. 1A,B). In the C-terminal tail of these channels, two regions show high degree of sequence homology. One is proline-rich region and the other is the very C-terminus. The proline-rich region contains several PXXP motifs, which are known to bind to SH3 domain of CASK, a modular adapter protein (Hata et al. 1996; Maximov et al. 1999). The C-terminus region contains a binding consensus (DXWC-COOH) for PDZ domain of Mint1 (Okamoto & Sudhof 1997; Maximov et al. 1999).
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From the expression plasmid pCAEGFPHC13NP(–) (designated as HC hereafter), the short form of human Cav2.1 channel is expected to be produced, though the plasmid contains sequences of exon 47 and FLAG tag (see below). This predicted protein contains part of the proline-rich region and therefore contains several PXXP motifs (Fig. 1). However, it lacks both the polyglutamine stretch and the C-terminal DXWC-COOH motif.
On the other hand, both pCAEGFPHS13NP(–) and pCAEGFPHL28NP(–) (designated as HS and HL hereafter, respectively) encode the longer version, containing entire proline-rich region, polyglutamine stretch, and C-terminal DXWC-COOH motif. The number of repeat of glutamine residues in the stretch is 13 for HS and 28 for HL. The 13 repeat unit is within the range of normal person, and 28 is within the disease range for SCA6.
We also constructed pCAEGFPHS13NP(–)F and pCAEGFPHL28NP(–)F (designated as HS-F and HL-F hereafter, respectively) to examine the effects of masking the C-terminal DXWC-COOH motif. HS-F and HL-F are the same as HS and HL, respectively, except that the C-terminal sequence was modified so that the DDWC sequence was followed by the FLAG tag sequence (DYKDDDDK).
Protein expression from the expression vectors in cultured cell line
We confirmed that the full-length proteins were expressed from the EGFP-Cav2.1 expression vectors mentioned above by immunological analyses of COS-7 cells transfected with the vectors. Using an extraction buffer containing 0.6% digitonin, we extracted proteins from COS-7 cells that had been transiently transfected with one of the expression vectors for EGFP-Cav2.1 together with an expression vector for auxiliary ß3 subunit. The protein samples were then subjected to immunoprecipitation using an anti-GFP antibody. The resulting immunoprecipitates were analysed by immunoblotting using another anti-GFP antibody. In all the samples except for the negative control, specific bands appeared above the largest molecular weight marker, 250 kDa (Fig. 1D). In the case of HC, a band with a slightly higher mobility was observed, compared with the other constructs for EGFP-Cav2.1 (Fig. 1D, lane 3). This situation is consistent with the predicted molecular weights of these fusion proteins (280 kDa for HC, and 310 kDa for the other EGFP-Cav2.1 fusion proteins). Furthermore, reprobing the blot with an anti-FLAG epitope antibody revealed that the bands detected by the anti-GFP antibody in the HS-F and HL-F samples were also immunoreactive to the anti-FLAG antibody (data not shown). We therefore conclude that fusion proteins with an intact C-terminal tail were expressed from these expression vectors.
Localization patterns of EGFP-Cav2.1 fusion protein in cultured hippocampal neurones
We introduced the expression vectors for EGFP-Cav2.1 into cultured mouse hippocampal neurones by a modified calcium phosphate method, together with expression plasmids for auxiliary 2 and ß3 subunits (pC3-br2 and pC3-ß3, respectively) and observed the green fluorescence, which was thought to reflect the Cav2.1 channel expression. When HC construct was introduced, EGFP signal was observed in almost all parts of the transfected neurones (Fig. 2). To discriminate between the axon and dendrites, we used an antibody against microtubule-associated protein 2 (MAP2) as a specific marker for dendrites (Caceres et al. 1984). MAP2 immunostaining revealed that many of the EGFP-positive processes are dendrites (Fig. 2) and that neuronal process which did not show MAP2 staining was also positive for the EGFP signal. This suggests that the short version of Cav2.1 channel was actually transferred to the axon. The EGFP signals in the axon were further characterized immunocytochemically by using an antibody against synapsin I, an axonal marker (Fletcher et al. 1991). In the distal part of the axon, clusters of EGFP signals were observed and some of the clusters were co-localized with synapsin I (Fig. 3). Especially where the axon of a transfected neurone and dendrites of untransfected cells made contacts, clusters of EGFP-Cav2.1 were clearly observed at the junction. In such cases, clusters of the EGFP-Cav2.1 in the axon often ran along the dendrite (Fig. 4), possibly corresponding to the sites of synapses (Obermair et al. 2004). Clustering of EGFP-Cav2.1 also occurred even in the proximal part of the axon, where the EGFP signals were usually distributed uniformly (not shown). Such discrete punctate pattern of distribution of EGFP-Cav2.1 channel in the axon was observed in 96% of the transfected neurones (Table 1). In contrast, EGFP-Cav2.1 was distributed uniformly in both proximal and distal parts of dendrites in general, though some enriched signals of EGFP were observed. Thus, the short version of Cav2.1 channel was localized in not only dendrites and soma but also the axon including the presynaptic sites.
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Localization of endogenous Cav2.1 channel in mouse hippocampal neurones
To confirm that the localization of EGFP-Cav2.1 channel in the hippocampal neurones was not artifact, we have compared it to the localization of mouse endogenous Cav2.1 channel. Hippocampal neurones cultured in vitro for 13 days, the same duration as the transfection experiments, were subjected to immunocytochemistry using an antibody which recognized mouse Cav2.1 channel. Double-staining with the anti-MAP2 and the anti-Cav2.1 antibodies revealed that mouse endogenous Cav2.1 channels were localized to the soma, dendrites, and the axon (Fig. 5A–C). Also, double-staining using an antibody against synaptophysin, an axonal marker showing the same localization pattern as that of synapsin I (Fletcher et al. 1991), revealed that Cav2.1 channel are clustered in the synaptic boutons (Fig. 5D,E). These patterns are essentially the same as those obtained by using the EGFP-Cav2.1 fusion constructs. Although the anti-Cav2.1 antibody used was raised against a peptide corresponding to a part of the cytoplasmic loop between repeat II and III and therefore was not able to distinguish between the long and short variants of Cav2.1, essentially the same patterns of channel localization would suggest that tagging the channel with EGFP did not affect the channel localization.
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Finally, we investigated the maturity of the cultured hippocampal neurones, since maturation state of the neurone is known to affect the localization patterns of Cav2.2 channel. Cav2.2 channel with a long C-terminal tail is known to be localized to the axon of mature neurones in clustered form and uniformly distributed in the axons of immature neurones (Maximov & Bezprozvanny 2002). Therefore, we have expressed Cav2.2 with a long C-terminal tail, to assess the maturity of the cultured hippocampal neurones.
We first constructed an expression vector for EGFP-Cav2.2 with a long C-terminal tail (Fig. 1C) and introduced the vector into COS-7 cells to confirm the expression of the full-length protein with an intact C-terminal tail (Fig. 1D). Next, we introduced the vector together with pC3-ß3 and pC3-br2 into cultured hippocampal neurones in exactly the same way as in the case of Cav2.1. MAP2 immunostaining revealed that EGFP-Cav2.2 was expressed in both somatodendritic domains and the axon of neurones (Fig. 6A–C). Also, immunostaining with anti-synapsin I antibody revealed that the EGFP-Cav2.2 was localized as clusters in the axon (Fig. 6D). This punctate localization pattern was in good agreement with previous studies (Maximov & Bezprozvanny 2002; Obermair et al. 2004). This suggests that most of the neurones cultured in our system were synaptically matured.
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Discussion |
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As for Cav2.2 channel, the presence of the long C-terminal tail is necessary and sufficient for the synaptic targeting of this channel (Maximov & Bezprozvanny 2002). The synaptic targeting process of Cav2.2 channel is thought to be mediated by two types of protein–protein interactions: one is between the PXXP motif and SH3 domain of CASK, and the other is between DXWC-COOH motif and PDZ domain of Mint1. Interestingly, the PXXP motifs and DXWC-COOH motif are well conserved in Cav2.1 channel (Maximov et al. 1999). It has been shown that Mint1 can bind to the C-terminal fragment of the Cav2.1 channel containing DDWC-COOH end (Maximov et al. 1999) and that addition of foreign sequences following the C-terminus most plausibly interferes with the binding of Mint1 (Kornau et al. 1997; Songyang et al. 1997). Furthermore, previous immunohistochemical studies revealed similar patterns of distribution of Cav2.1 and Cav2.2 channels in several types of neurones (Westenbroek et al. 1992, 1995). Therefore it was quite natural to speculate that the Cav2.1 channel may also adopt a similar mechanism for its localization in neuronal cells. However, to our surprise, the C-terminal tail of Cav2.1 channel did not significantly affect the localization pattern of the channel. This suggests that C-terminal region encoded by exon 47 is dispensable for the determination of subcellular localization of Cav2.1 channel. Furthermore, addition of the FLAG tag just after the DDWC sequence at the C-terminus did not affect the localization pattern, either. From this result as well, we can speculate that the interaction between the C-terminal motif and the PDZ domain of Mint1 is not indispensable for the Cav2.1 channel localization. Overall, it is highly possible that domain(s) other than the C-terminal tail is responsible for the determination of subcellular localization of Cav2.1 channel. Results from a recent study of Catterall's group support this possibility. Mochida et al. (2003) reported that the deletion of synprint site located in the loop between repeat II and III of Cav2.1 inhibited the localization of this channel to nerve terminals, suggesting the requirement of synprint site for the synaptic targeting of Cav2.1 channel. Although the splice variant that lacks the synprint site has not yet been reported for Cav2.1 channel, such variant has been reported to be present for Cav2.2 channel (Kaneko et al. 2002) and so it is possible that the alternative splicing, which produces Cav2.1 with or without synprint site, controls the localization pattern of Cav2.1.
Another object of the present study is to examine the possible effects of the polyglutamine tract, whose expansion is responsible for SCA6, on the Cav2.1 channel localization. In cerebellar Purkinje cells from SCA6 patients, cytoplasmic aggregation containing Cav2.1 channel was detected and this phenotype was speculated to be related to the pathogenic mechanism of SCA6 (Ishikawa et al. 1999, 2001). Cav2.1 channel is a transmembrane protein and it does not form aggregate in cytoplasm in normal states. Therefore, one possible mechanism to explain the aggregates found in SCA6 is the change in the subcellular localization of Cav2.1 channel due to abnormal polyglutamine expansion. However, we did not detect the cytoplasmic aggregation of the Cav2.1 channel, after transfection of hippocampal neurones with HL constructs, nor did we notice any difference between the results of HS and HL. Thus, our conclusion is that the expanded polyglutamine stretch does not seem to cause any abnormalities in localization of the Cav2.1 channel. But at present we cannot deny the possibility that in Purkinje cells expanded polyglutamine stretch can cause changes in the localization pattern of Cav2.1 channel. Also, it remains possible that over-expression of EGFP-Cav2.1 constructs may obscure subtle differences in synaptic trafficking and localization. Thus, further studies controlling the expression level of exogenous proteins would be necessary to clarify this matter.
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Experimental procedures |
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EGFP-Cav2.1 fusion constructs
To generate an in-frame fusion construct of EGFP and human Cav2.1 channel, an SphI–XbaI 7.8 kb fragment from a Cav2.1 expression vector HL28NP(–) (Toru et al. 2000) was blunt-ended by T4 DNA polymerase (New England Biolab) and ligated to blunt-ended pEGFP-C2 (Clontech) digested with EcoRI. [NP stands for the asparagine and proline residues encoded by a short exon 31* and is known to affect the sensitivity to a Cav2.1 channel blocker (Bourinet et al. 1999; Lin et al. 1999).] Then the nucleotide sequence around the EGFP/Cav2.1 boundary of the resulting plasmid, pEGFP-HL28NP(–), was confirmed. Finally, an NheI-BamHI 8.8 kb fragment encoding the EGFP-Cav2.1 fusion protein from pEGFP-HL28NP(–) was blunt-ended and cloned into the HincII site of pCApA vector (kindly provided by Dr C. Akazawa) to make pCAEGFPHL28NP(–). Sequence encoding FLAG tag was introduced to HS13NP(–) (Toru et al. 2000) by a PCR-based method and the nucleotide sequences around the coding region for FLAG tag were confirmed. The other fusion constructs for EGFP-Cav2.1 were prepared based on pEGFP-HL28NP(–) by replacing corresponding fragments.
pCAEGFP-BIII
An expression vector for a fusion protein of EGFP and Cav2.2, pCAEGFP-BIII, was constructed on the basis of pCAEGFPHL28NP(–). First, pCAEGFPHL28NP(–) was digested with EcoRI and SalI, to remove the coding sequence for Cav2.1. The resultant plasmid was then digested with HindIII, and the 7.5 kb HindIII fragment from pKCRBIII, containing all of the coding sequence of rabbit Cav2.2 (Fujita et al. 1993), was inserted to make pCAEGFP-BIII. In-frame fusion of EGFP and Cav2.2 was confirmed by nucleotide sequencing.
pC3-br2
To construct an expression vector for rat brain 2 subunit, pC3-br2, the backbone vector of an expression plasmid pcD2 2–1 derived from pcDNAI/Neo (a generous gift from Dr H. R. Chin) was changed to pcDNA3 (Invitrogen).
pC3-ß3
To construct pC3-ß3, an expression plasmid for rabbit ß3 subunit, a 1.6 kb SalI-KpnI fragment from the plasmid SvCaB3 (a generous gift from Dr V. Flockerzi) and a HindIII-KpnI fragment from pcDNA3 was ligated using the partial fill-in method (Zabarovsky & Allikmets 1986).
Primary neuronal culture and transfection
Hippocampi were collected from ICR mouse fetuses of 16.5 day of gestation. Method for neuronal culture was essentially the same as those previously described (Goslin & Banker 1991; Maximov & Bezprozvanny 2002), except that polyethyleneimine instead of poly L-lysine was used for coating coverslips and that AraC treatment to inhibit the growth of glial cells was omitted. Plating density of neurones was 
350/mm2. Transfection was performed by the modified calcium phosphate method described by Xia et al. (1996). EGFP-Cav2.1 or –Cav2.2 fusion construct was transfected with expression plasmids for 2 and ß3 subunits in a molar ratio of 3: 1: 1. Total of 3 µg of plasmid DNA was used for one 35 mm dish. Transfection was performed 9 days after the beginning of culture, and cells were fixed for cytological analyses 4 days after transfection.
Immunocytochemistry
Cells were fixed with 4% paraformaldehyde, 4% sucrose in phosphate-buffered saline (PBS) for 10 min at room temperature. They were then washed with PBS and treated with 0.05% Triton X-100 in PBS for 5 min. After rinsing with PBS followed by blocking treatment, cells were incubated in primary antibody solution overnight at 4 °C. Primary antibodies used were mouse anti-MAP2 monoclonal antibody (Clone AP20, Neo Markers, used at 1: 400 dilution) and rabbit anti-synapsin I polyclonal antibody (Chemicon, used at 1: 500 dilution). To detect the mouse primary antibody, biotinylated donkey anti-mouse IgG (Chemicon) and Texas Red-conjugated streptavidin (Jackson ImmunoResearch Laboratories) were used. For the detection of rabbit primary antibody, Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) was used. Images were obtained and analysed using the LSM 5 Pascal confocal imaging system (Zeiss).
For the detection of endogenous mouse Cav2.1 channel, rabbit anti-Cav2.1 polyclonal antibody (Alomone lab, used at 1: 100 dilution) was used. Also, anti-synaptophysin mouse monoclonal antibody (Roche, used at 1: 100 dilution) was used for double-immunofluorescent studies.
Immunoblotting
COS-7 cells were cultured in 35 mm dishes with DMEM/F-12 (Gibco) supplemented with 10% foetal bovine serum. Transfection with one of the expression plasmids for EGFP-Cav2.1 fusion protein was performed using Lipofectamine and Plus reagent (Invitrogen) according to the manufacturer's suggestion. Two days after transfection, cells were washed with PBS, harvested with a scraper, frozen in liquid nitrogen, and stored at –80 °C. To solubilize EGFP-Cav2.1 fusion protein, cell pellet was briefly sonicated in solubilization buffer (10 mM HEPES-NaOH, pH 7.4, 0.5 M NaCl, 0.6% digitonin) supplemented with protease inhibitors (Sigma). After removing debris by centrifugation, anti-GFP antibody (Medical & Biological Laboratories Co. Ltd) was added to the solubilized protein sample, and the mixture was incubated for several hours at 4 °C. To precipitate the fusion protein bound to the antibody, protein G agarose (Sigma) was added to the sample and the mixture was incubated overnight at 4 °C with gently rotated. After centrifugation and several washes, the immunoprecipitates were resuspended in sample buffer (25 mM Tris, pH 6.8, 3% SDS, 4 M urea, 30 mM dithiothreitol) and heated for 10 min at 94 °C. These protein samples were resolved by SDS-PAGE (5.5% gel) and transferred to PVDF membrane (Immobilon P, Millipore). The blot was probed with an anti-GFP antibody (Santacruz, 1: 1000 dilution) and the bound antibody was detected by using biotinylated secondary antibody (Chemicon) and streptavidin-biotinylated horseradish peroxidase complex (Amersham-Pharmacia) in conjunction with Super Signal West Femto kit (Pierce).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: t-tanabe.mphm{at}tmd.ac.jp
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Received: 14 October 2004
Accepted: 8 November 2004
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