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Volume 17, Number 4, Issue of February 15, 1997 pp. 1339-1349
Copyright ©1997 Society for Neuroscience

Regional Expression and Cellular Localization of the ₁ and  Subunit of High Voltage-Activated Calcium Channels in Rat Brain

Andreas Ludwig, Veit Flockerzi, and Franz Hofmann

Institut für Pharmakologie und Toxikologie der Technischen Universität München, 80802 München, Germany

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

REFERENCES

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ABSTRACT

The neuronal high voltage-activated calcium channels are a family of ion channels composed from up to five different ₁ and four different  subunits. The neuronal distribution and subunit composition of calcium channels were investigated using subunit-specific antibodies and riboprobes. The  subunit-specific antibodies identified the presence of _1a in skeletal muscle; ₂ in heart; and ₂, ₃, and ₄ in brain. The ₃ protein was widely distributed in rat brain, with prominent labeling of olfactory bulb, cortex, hippocampus, and habenula. The ₄ protein was also widely expressed, most prominently in the cerebellum. ₂ protein was expressed at only low levels. In situ hybridization with  subunit-specific riboprobes confirmed the differential expression pattern of the individual subunits. Hybridization with riboprobes specific for the _{1A, 1B, 1C}, and _1D subunits showed a broad distribution of _1A and _1B transcripts, whereas the expression level of _1C and _1D mRNA was lower and more spatially restricted. The overall expression pattern and cellular localization suggested that ₄ may associate predominantly, but probably not exclusively, with the _1A subunit, and ₃ with the _1B subunit. In certain brain areas such as the habenula, the ₃ subunit may associate with other ₁ subunits too. Furthermore, the ₂ subunit may form complexes with different ₁ subunits in brain and cardiac muscle. These results demonstrate that a given  subunit may associate with different ₁ subunits in a cell type-dependent manner, contributing to the diversity of the neuronal calcium channels.

Key words: ion channel; calcium channel; brain; hippocampus; cerebellum; olfactory bulb; habenula

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INTRODUCTION

The high voltage-activated calcium channels are multimeric protein complexes containing the channel-forming ₁ subunit and the auxiliary , ₂/, and  subunits (for additional references, see Hofmann et al., 1994). The ₂/ and  subunits are encoded by single genes, whereas six and four genes have been identified for the ₁ and  subunits, respectively. The ₂/ subunit, five of the six ₁ subunits, and all four  subunits are expressed in brain, suggesting that brain calcium channels contain at least one of the five ₁ subunits, one of the four  subunits, and an ₂/ protein (Snutch and Reiner, 1992; Catterall, 1995; De Waard et al., 1996). Expression studies with the various cloned subunits have shown that each ₁ subunit reconstitutes with each  subunit to yield a functional channel. This apparent indiscriminatory channel complex formation was supported by the finding that a highly conserved region of the linker of the I-II loop of all ₁ subunits interacts with each  subunit (De Waard et al., 1994; Pragnell et al., 1994).

A careful comparison of the electrophysiological and pharmacological properties between expressed and native channels showed that the cloned channel subunits encode channels that have similar but not identical properties as the native channels (Zhang et al., 1993; Stea et al., 1994, Randall and Tsien, 1995; Reuter, 1996). An attractive explanation for the differences between expressed and native channels would be a subunit composition of the native channel that was not matched in the expression studies and the occurrence of diverse splice variants of the ₁ and  subunits. It seemed unlikely that the native calcium channels are the result of a random combination of a given ₁ subunit with any of the four  subunits. The analysis of the subunit composition of three calcium channels only partially confirmed this consideration. The purified skeletal muscle channel is a complex of the _1S/_1a/₂// subunit (for references, see Hofmann et al., 1994), whereas the neuronal _1B/₂/ subunits of the N-type channel complex were immunoprecipitated together with the ₃ and ₄ subunits (Scott et al., 1996), and the neuronal _1A/₂/ subunits of the -conotoxin MVIIC-sensitive calcium channel were associated with the _1b, ₂, ₃, and ₄ subunits (Liu et al., 1996). The latter channel was immunoprecipitated and purified from microsomes derived from whole brains. Therefore, these results do not contradict the hypothesis that an ₁ subunit associates only with one type of  subunit in each neuron at a defined subcellular localization.

We have used site-directed anti- antibodies and ₁ and  subunit-specific riboprobes to determine the regional expression and cellular colocalization of ₁ and  subunits in rat brain. This analysis did not include the _1E subunit, because this subunit is rather ubiquitiously distributed in the brain and has been described previously (Soong et al., 1993; Yokoyama et al., 1995; Day et al., 1996). Our results indicate that the calcium channel subunits do not combine in a random fashion in the brain.

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MATERIALS AND METHODS

Sprague Dawley rats and New Zealand rabbits were obtained from Charles River (Kisslegg, Germany). Keyhole limpet hemocyanin, antipain, pepstatin A, and benzamidine were obtained from Calbiochem (La Jolla, CA); m-maleimido benzoyl-N-hydroxysuccinimide and toluidine blue from Sigma (St. Louis, MO); epoxy-Sepharose 6B, CNBr-activated Sepharose 4B, and dextran from Pharmacia, Piscataway, NJ; the anti-FLAG antibody M2 from IBI; immobilon membrane from Millipore (Bedford, MA); the ECL detection kit and Hybond nitrocellulose from Amersham (Arlington Heights, IL); iodacetamide, 1,10-phenantroline, and leupeptin from Fluka, Neu-Ulm, Germany; goat anti-rabbit IgG-peroxidase conjugate from Jackson Laboratories (West Grove, PA); blotting paper from Schleicher & Schüll (Dassel, Germany); proteinase K and tRNA from Boehringer Mannheim (Mannheim, Germany); [³⁵S]UTP (1000-1500 Ci/mmol) from DuPont NEN (Wilmington, DE); T7 RNA polymerase from New England Biolabs (Beverly, MA); T3 RNA polymerase and pcDNA III from Invitrogen (San Diego, CA); and Escherichia coli BL21(DE3)pLysS from Novagen. The vector pAR(RI) was a gift from Dr. M. A. Blanar, Hormone Research Institute, University of California, San Francisco (Blanar and Rutter, 1992).

Production of  subunit-specific antibodies. Peptides B30 CDRNWQRNRPWPKDSY (aa 463-477 of ₃; Hullin et al., 1992), B35 CYNRGSPGGCSHDSRHRL (aa 504-519 of ₄; Castellano et al., 1993), B36 CDSETQESRDSAYVEPKEDY (aa 502-520 of _2a; Hullin et al., 1992), and B37 CSQRSSRHLEEDYADAYQDLY (aa 411-430 of ₃; Hullin et al., 1992) were synthesized by the solid-phase method. The N terminal C of B30, B36, and B37, as well as CY of B35, is not present in the native sequences and was added for coupling and detection purposes. The peptides were purified by reverse-phase HPLC and coupled to keyhole limpet hemocyanin with m-maleimido benzoyl-N-hydroxysuccinimide ester (Green et al., 1982). The conjugates were emulsified with Freund's complete adjuvant and injected into New Zealand white rabbits. The animals were boostered three to four times with conjugate emulsified in Freund's incomplete adjuvant. For affinity purification of antibodies, the peptides B30, B35, B36, and B37 were coupled to epoxy-Sepharose 6B. Sera were diluted in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄), pH 7.4, and were cycled over the affinity matrix for 16 hr at 4°C. Bound antibodies were eluted with 4.5 M MgCl₂ and concentrated with Centricon-30 devices (Amicon, Beverly, MA).

An antibody against a sequence common to all  subunits was generated with a fusion protein produced in E. coli. A 567 bp fragment (nt 886-1452 of ₃) was PCR amplified from a cDNA plasmid carrying _3a and cloned into pAR(RI) (Blanar and Rutter, 1992) containing the N-terminal FLAG peptide DYKDDDDKL. The 21 kDa fusion protein was expressed in BL21(DE3)pLysS, detected on immunoblots with the anti-FLAG antibody M2 (IBI) and purified from inclusion bodies by preparative SDS-PAGE with the Model 491 Prep Cell (BioRad, Hercules, CA). Rabbits were immunized with the fusion protein, as described above. Antibodies were affinity-purified by incubation of serum with fusion protein blotted onto Immobilon, and bound antibodies were eluted with 4.5 M MgCl₂. To remove contaminating cross-reactive activities, the purified antibody was adsorbed on rat liver acetone powder coupled to CNBr-activated Sepharose 4B.

The specificity of the antibodies was tested by immunoblots using the purified calcium channel from rabbit skeletal muscle (Schneider et al., 1992) and membranes from HEK 293 cells that were transfected individually with the full-length cDNA of the _2a, ₃, or ₄ subunit and the _1Ca subunit (see Fig. 2A). The four affinity-purified anti- subunit antibodies labeled no bands in immunoblots using membrane preparations from control cells transfected with the pcDNA3 vector alone (Invitrogen). The ₂ subunit-specific antibody stained specifically the _2a subunit with an apparent molecular mass of 72 kDa, which is in close agreement to the calculated M_r of 68 kDa (Hullin et al., 1992). The anti-₃ and anti-₄ subunit antibodies labeled the expressed ₃ and ₄ subunits with apparent molecular masses of 60 and 58 kDa, which are close to the predicted M_r values of 54 and 58 kDa, respectively (Hullin et al., 1992; Castellano et al., 1993). The anti-_common antibody labeled specifically the expressed full-length _2a, ₃, and ₄ subunits as well as the skeletal muscle ₁ subunit (Ruth al., 1989). Immunostaining of the  subunits was abolished by preincubation of the antibodies with the corresponding peptides respective fusion protein.

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Fig. 2. Controls for antibody specificity, histoblots, and ISH sections. A, Microsomal membranes from HEK 293 cells transfected with control vector (lane 1) or full-length ₃ cDNA expression vector (lanes 2, 3) were electrophoresed on a 7.5% SDS gel and blotted onto nitrocellulose. Lanes 1 and 2 were probed with the anti-₃ antibody preincubated in the absence () and lane 3 with the anti-₃ antibody preincubated in the presence (+) of 10 µM peptide B30. B, A sagittal histoblot section adjacent to the section of Figure 3B was probed with the anti-₂-specific antibody blocked with 10 µM peptide B36. C, A sagittal ISH section adjacent to the section of Figure 4B was labeled with the ₂ sense riboprobe.
[View Larger Version of this Image (49K GIF file)]

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Immunoblot. Microsomal membranes were prepared from rat liver, skeletal muscle, brain, uterus, and heart as well as from transfected HEK 293 cells at 4°C. Tissues were homogenized in buffer A containing (in mM): 20 MOPS, pH 7.4, 300 sucrose, 2 EDTA, 1 iodacetamide, 1 1,10-phenantroline, and 0.1 phenylmethanesulfonyl fluoride, and 1 µg/ml antipain, 1 µg/ml leupeptin, 1 µM pepstatin A, and 1 mM benzamidine. The homogenate was centrifuged for 10 min at 5000 × g. The pellet was reextracted and centrifuged for 10 min at 5000 × g. The combined supernatants were brought to 0.6 M KCl and centrifuged for 35 min at 100,000 × g. The pellet was resuspended in buffer A and stored at 70°C. Protein concentrations were determined with the BCA method (Pierce, Rockford, IL).

Membrane proteins (50-100 µg) were separated using 7.5% SDS-PAGE and blotted onto nitrocellulose membranes. The membranes were blocked with TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) containing 0.1% Tween and 5% dry milk powder and probed with the primary anti- subunit antibody and subsequently with a secondary goat anti-rabbit IgG-peroxidase-conjugated antibody. Bound antibodies were detected by the ECL chemiluminescence method(Amersham).

Histoblots. In situ detection of the  subunit proteins in rat brain slices was accomplished by a modification (Benke et al., 1995) of the original in situ blotting method (Taraboulos et al., 1992; Okabe et al., 1993). Slices of unfixed rat brain (16 µm thick) were cut on a cryostat and mounted onto nitrocellulose. The membrane was placed for 15 min on blotting paper (Schleicher and Schüll), soaked in transfer buffer (39 mM glycine, 48 mM Tris, 2% SDS, 20% methanol), and incubated subsequently in 0.1 M Tris, pH 7.0, 2% SDS, 0.1 M -mercaptoethanol for 1 hr at 45°C and for 16 hr at room temperature (RT). The membrane was blocked in TBS containing 0.1% Tween and 5% dry milk powder and labeled with the anti- subunit antibodies, followed by a secondary peroxidase-conjugated goat anti-rabbit IgG antibody. Bound antibodies were detected by ECL. Adjacent sections were processed according to standard histological techniques, stained with toluidine blue, and viewed with a stereomicroscope in comparison with overlaid histoblot film images. Neuronal structures were identified according to Paxinos and Watson (1986).

In situ hybridization (ISH). Adult Sprague Dawley rats (250-350 gm) were anesthetized with sodium pentobarbital and perfused with 50 ml ice-cold PBS, pH 7.4. The brain was removed and quickly frozen in isopentane cooled in a dry ice/ethanol bath. Sections (16 µm thick) were cut in a cryostat, thaw-mounted onto polyLlysine-coated slides, fixed with 4% paraformaldehyde in PBS, pH 7.4, and dehydrated. Before hybridization, slices were pretreated with 7 µg/ml proteinase K for 15 min at RT and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0, for 10 min. Dehydrated slices were prehybridized for 2 hr at 42°C in hybridization buffer (10 mM Tris, pH 8.0, 0.3 M NaCl, 1 mM EDTA, 1 × Denhardt's solution, 10% dextran, 50% deionized formamide, 50 mM DTT). The slices were then incubated with the radiolabeled probe (5 × 10⁶ cpm/ml hybridization buffer) for 16 hr at 55°C. After hybridization, the slides were washed two times in 2 × SSC, 1 mM DTT, 1 mM EDTA, and incubated in RNase A (20 µg/ml) for 30 min at RT and washed twice in 2 × SSC, 1 mM DTT, 1 mM EDTA. Subsequently, sections were washed at high stringency in 0.1 × SSC, 1 mM DTT, 1 mM EDTA for 2 hr at 75°C. The slices were dehydrated, dried, and exposed to Kodak BioMax MR film for 6 d. The slides were then dipped in autoradiography emulsion Kodak NTB-2, exposed for 6-8 weeks, and developed in Kodak D-19 developer. Sections were lightly counterstained with toluidine blue and examined with bright- and dark-field illumination.

For construction of vectors for in vitro transcription, 157-278 bp fragments of _1b (Pragnell et al., 1991), _2a (Perez-Reyes et al., 1992), ₃ (Castellano and Perez-Reyes, 1994), ₄ (Castellano et al., 1993), _1A (Starr et al., 1991), _1B (Dubel et al., 1992), _1C (Snutch et al., 1991), and _1D (Hui et al., 1991) were PCR-amplified from rat brain first-strand cDNA with the following primer pairs:

AL9 (5-GGTCCTTAATCCCCAGCTGTA-3) (nt 2031-2051 of _1b) and AL10 (5-GGGTCTGGGGTTTGTGGAAGA-3) (nt 2302-2282 of _1b)

AL5 (5-GGACCACTGTTTCTTGCTTGT-3) (nt 2555-2576 of _2a) and AL4 (5-CTGCTGACTTGGCATTAAGA-3) (nt 2791-2772 of _2a)

AL1 (5-CGCCACCTGGAGGAAGACTA-3) (nt 1406-1425 of ₃) and AL2 (5-CGCTGCCAGTTCCGGGTCATTG-3) (nt 1563-1544 of ₃)

AL3 (5-CAGCCATGACTCCCGACATA-3) (nt 1737-1754 of ₄) and AL4 (5-CCTCCTAGACTCAAGGGCATA-3) (nt 1952-1932 of ₄)

AL19 (5-CTCCCGAGAACAGCCTTATC-3) (nt 3257-3294 of _1A) and AL20 (5-GGGGTCTGCCTCCTCTTCCT-3) (nt 3483-3464 of _1A)

AL17 (5-GGGGATAAGGAAACTCGAAAT-3) (nt 3031-3051 of _1B) and AL18 (5-GGCCTTCCAGGTCCGTGTTA-3) (nt 3304-3285 of _1B)

AL13 (5-CTCCAGCCCAGTGAAAATGA-3) (nt 2745-2764 of _1C) and AL14 (5-GCCAGGGAGATGCTACTGAG-3) (nt 3022-3003 of _1C)

AL15 (5-GCCAACAGTGACAACAAGGT-3) (nt 2912-2931 of _1D) and AL16 (5-ACACGGATCGGGTTGGTCTT-3) (nt 3153-3134 of _1D).

A BamHI and Asp718 site was added to the 5end of sense and antisense primers, respectively. The PCR profile was as follows: initial denaturation for 1.5 min at 94°C; 40 cycles of 94°C 1 min, 52°C 1 min, 72°C 1 min, and a final extension at 72°C for 5 min. The reaction products were analyzed by restriction mapping and cloned into a pUC19-derived vector containing opposing T3 and T7 RNA polymerase promoters flanked by BamHI respective Asp718 sites. All inserts were sequenced on both strands. Antisense and sense probes were in vitro transcribed with [³⁵S]UTP and T7 or T3 RNA polymerase, respectively.

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RESULTS

Tissue-specific expression of the  subunits

In initial immunoblot experiments, the anti-_common and the ₂, ₃, and ₄ subunit-specific antibodies (for details, see Materials and Methods) were used to determine the tissue distribution of the  subunits (Fig. 1). The anti-_common antibody (Fig. 1A) recognized in skeletal muscle the ₁ subunit (55 kDa) and a 35 kDa band. The staining of the 35 kDa band varied in intensity among different membrane preparations and most likely represented a proteolytic fragment of the ₁ subunit. In brain membranes, the anti-_common antibody stained a protein of 60 kDa; at weaker intensity, a band at 58 kDa; and after longer exposure times, a faint band at 72 kDa. The antibody labeled a 72 kDa protein in heart and a major protein species of 56 kDa and a minor species of 36 kDa in aorta. Labeling of immunoblots with the  subunit-specific antibodies confirmed the  subunit expression profile detected by the _common antibody. The anti-₂ subunit antibody recognized specifically the 72 kDa protein in heart and at weaker intensity in brain (Fig. 1B). The anti-₃ subunit antibody strongly stained the 60 kDa protein in brain (Fig. 1C), whereas the anti-₄ subunit antibody recognized a 58 kDa protein in this tissue (Fig. 1D). The monoclonal antibody 7C3 that was generated against the _1a subunit from rabbit skeletal muscle (Nastainczyk et al., 1990) stained specifically the 55 kDa protein in rat skeletal muscle, but showed no signal with rat brain membranes (data not shown). None of the antibodies stained a band in rat liver membranes, providing additional evidence for the specificity of the antibodies used (Fig. 1). These immunoblots supported the following tissue distribution: the predominant subunit in skeletal muscle is _1a and that of the heart is the ₂ subunit. Brain contains at least the ₂, ₃, and ₄ subunits and, as shown previously, the _1b subunit (Pragnell et al., 1991). The anti-_common antibody recognized a 56 kDa protein species in aorta, which was not labeled by the ₃ subunit-specific antibody, suggesting that rat smooth muscle contains a  subunit that has a different C terminus than the rabbit _3a subunit (Hullin et al., 1992). To identify a C-terminal truncated splice variant of the _3a subunit, designated _3b (Murakami et al., 1994), an antibody was generated against the C terminus of the _3b subunit (aa 411-430). This sequence is identical for the _3a and _3b subunits. The new antibody labeled specifically the ₃ subunit expressed in HEK 293 cells and the ₃ subunit present in rat brain. However, this antibody was unable to immunolabel a band in membrane or cytosol preparations from rat aorta (data not shown), indicating that the  subunit of rat aorta differs considerably from that expressed in rat brain.

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Fig. 1. Expression of  subunit proteins in rat tissues. Microsomal membranes (50 µg per lane) of rat liver (Li), skeletal muscle (Sk), brain (Br), heart (He), and aorta (Ao) were separated using 7.5% SDS-PAGE and transferred to nitrocellulose. The blots were probed with the anti-_common antibody (A), the anti-₂ specific antibody (B), the anti-₃ specific antibody (C), and the anti-₄ specific antibody (D). In (B), the lane containing the separated brain proteins (Br) was from a separate immunoblot and was exposed four times longer than the other lanes. Molecular mass standards (× 10³) are indicated on the left of each blot.
[View Larger Version of this Image (76K GIF file)]

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Distribution of the  subunit proteins in rat brain

The  subunit-specific antibodies were used to map the regional expression of ₂, ₃, and ₄ subunits in cryostat sections of rat brain. No acceptable signal could be obtained using conventional immunohistochemical techniques. This failure was probably attributable to the limited accessibility of the  subunit epitopes in the native channel complex. However, the  subunit distribution was mapped in situ using the histoblot technique (Okabe et al., 1993; Benke et al., 1995). This procedure allows direct protein mapping from a single cell layer of an SDS-denatured tissue section mounted onto nitrocellulose. Histoblotting was used successfully to map among others the distribution of prion protein PrPSc (Taraboulos et al., 1992) and NMDA-receptor NR1 subunit (Benke et al., 1995) in brain. A series of horizontal, sagittal, and coronal sections spaced at 1 mm intervals was labeled with the anti- subunit antibodies (see Fig. 3; Table 1). No signal was seen in adjacent sections processed without primary antibody or with primary antibody blocked with an excess of peptide (Fig. 2B). The anti-_common antibody (Fig. 3A) labeled most intensely the olfactory bulb, cortex, hippocampus, habenula, and cerebellum. Moderate labeling was seen in the basal ganglia, amygdala, nucleus interpeduncularis, and superior and inferior colliculus (Table 1).

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Fig. 3. Histoblots showing the neuronal distribution of  subunit proteins. Sagittal sections of rat brain were transferred to nitrocellulose and labeled with the anti-_common antibody (A), the anti-₂ antibody (B), the anti-₃ antibody (C), and the anti-₄ antibody (D).
[View Larger Version of this Image (82K GIF file)]

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Table 1. Distribution of  subunit protein and mRNA and 1 subunit mRNA in the rat brain  common  2  3  4  1  2  3  4  1C  1D  1B  1A  1E Protein Protein Protein Protein mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA Olfactory bulb +++   +++ ++ ++   +++ ++ ++ ++ + ++ +++ Cortex +++   ++ ++ ++   ++ ++   + ++ ++ ++ Hippocampus Ammon's horn ++ + ++ + ++ + ++ + ++ + ++ +++ +++ Dentate gyrus +++ + ++ ++ ++ + ++ + ++ ++ ++ +++ +++ Caudate putamen ++   ++ ++ ++ + ++ +     + + ++ Amygdala ++   + + ++   ++ + + + ++ + ++ Hypothalamus +   + + ++   ++ +   + ++ ++ ++ Thalamus + + + +   + + ++ +   ++ ++ + Habenula +++   +++       +++     +     +++ Nucleus interpeduncularis ++   ++                   n.d. Superior colliculus ++   ++ + +   + +   ++ ++ + n.d. Inferior colliculus ++     ++ ++   + ++     ++ +++ n.d. Cerebellum +++ + ++ +++   + ++ +++ ++ + ++ +++ +++ Brainstem +     + +     +     ++ ++ ++ In situ hybridization signals on film images and emulsion-dipped sections and chemiluminescence signals obtained with specific antibodies on histoblots were rated as +++, strong; ++, moderate; +, low; , no signal above background; n.d., not determined. * Data from Soong et al., 1993.

The overall staining with the anti-₂ subunit antibody was much lower than that observed with the anti-₃ and anti-₄ subunit antibodies (Fig. 3B). The anti-₂ subunit antibody weakly labeled the hippocampus, thalamus, and cerebellum (Table 1). The staining observed after application of the anti-₃ subunit antibody (Figs. 3C, 5A) was strongest in the olfactory bulb and habenula. Moderate signals were detected in the cortex, hippocampus, basal ganglia, nucleus interpeduncularis, superior colliculus, and cerebellum (Table 1). Immunoreactivity in the hippocampus was found throughout the histological layers, with slightly more prominent signals in a formation that represents the stratum radiatum of Ammon's horn. In contrast to the distribution of the ₃ subunit, the expression of the ₄ subunit was highest in the cerebellum (Fig. 3D). Most prominent staining was detected over the molecular cell layer, whereas the granular cell layer was only weakly stained. Moderate ₄ subunit immunoreactivity was found in the olfactory bulb, cortex, hippocampus, basal ganglia, and inferior colliculus (Table 1). In the hippocampal formation, the ₄ subunit immunoreactivity was quite prominently localized at the molecular layer of the dentate gyrus, whereas the other hippocampal layers were stained moderately. The anti-₄ subunit antibody did not stain the habenula (see Fig. 5C).

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Fig. 5. Distribution of  subunits in rat brain. A, Histoblot of a coronal section labeled with the ₃-specific antibody. B, ISH of an adjacent coronal section with the ₃-specific riboprobe. C, Histoblot of an adjacent coronal section labeled with the ₄-specific antibody. D, ISH of a horizontal section with the riboprobe directed against ₄ mRNA.
[View Larger Version of this Image (129K GIF file)]

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Regional expression of  subunit mRNAs in rat brain

To confirm the immunocytochemical distribution of the  subunit proteins, ISH with riboprobes specific for the ₁, ₂, ₃, and ₄ subunits was performed (Fig. 4). The different riboprobes had similar lengths of 157-278 bp and contained between 60 and 70 [³⁵S]UTP molecules. Sections of one anatomical plane were processed together. These conditions were chosen to obtain information on the relative concentration of the different mRNA species. Control ISH experiments with the sense riboprobes on adjacent sections showed no signals (Fig. 2C). Hybridization with the ₁ subunit riboprobe showed moderate labeling in the olfactory bulb, cortex, hippocampus, caudate putamen, amygdala nuclei, hypothalamus, and inferior colliculus (Fig. 4A; Table 1). As already observed for the ₂ protein, the ₂ subunit mRNA was expressed at a low level (Fig. 4B) in the hippocampus, caudate putamen, thalamus, and cerebellum (Table 1).

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Fig. 4. Neuronal distribution of  subunit mRNAs. Autoradiographic film images of sagittal sections hybridized with antisense riboprobes specific for ₁ subunit mRNA (A), ₂ subunit mRNA (B), ₃ subunit mRNA (C), and ₄ subunit mRNA (D).
[View Larger Version of this Image (89K GIF file)]

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In contrast to the ₂ subunit mRNA, the ₃ subunit transcripts were highly expressed (Figs. 4C, 5B). Strong hybridization signals were detected in the olfactory bulb and habenula. Moderate signals were detected in the cortex, hippocampus, caudate putamen, amygdala nuclei, hypothalamus, and cerebellum (Table 1). The expression pattern of the ₄ subunit transcripts (Figs. 4D, 5D) showed remarkable differences to that of the other  subunits. The strongest hybridization signals were found in the cerebellum. Moderate labeling was observed in the olfactory bulb, cortex, thalamus, and inferior colliculus. In general, the intensity of the ISH signals corresponded with that of the histoblots (compare Fig. 5A with Fig. 5B and see Table 1), suggesting that the mRNA levels correlated well with the protein concentration.

Cellular localization of the  subunit mRNAs

The cellular distribution of the four transcripts was studied in the cerebellum, olfactory bulb, and frontal cortex (Fig. 6), because these regions were heavily labeled by the ₃ and ₄ subunit probes. Pronounced differences in the cellular expression profile were found. In slices of the cerebellum, the ₄ subunit riboprobe labeled intensely granule cells and the Purkinje neurons (Fig. 6D). In addition, scattered cells were labeled in the molecular layer representing basket or stellate neurons.

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Fig. 6. Cellular localization of  subunit mRNA in the cerebellum, olfactory bulb, and cortex. Dark-field microscopy views showing emulsion-dipped sagittal sections through the cerebellum labeled with antisense riboprobes specific for ₁ subunit mRNA (A), ₂ subunit mRNA (B), ₃ subunit mRNA (C), and ₄ subunit mRNA (D). M, Molecular cell layer; P, Purkinje cell layer; G, granular cell layer. E, F, Coronal sections through the olfactory bulb hybridized with the riboprobes directed against the ₃ subunit mRNA (E) and ₄ subunit mRNA (F). Gr, Granular layer; Mi, mitral cell layer; Ep, external plexiform layer; Gl, glomerular layer. G, H, Coronal section through the frontal cortex showing the expression of ₃ subunit mRNA (G) and ₄ subunit mRNA. H, I-VI, Cortical layers I-VI. I, Coronal section through the habenular complex labeled with the ₃-specific riboprobe. Magnification, 100×.
[View Larger Version of this Image (124K GIF file)]

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In comparison with the ₄ subunit signal, the cerebellar signal of the ₃ subunit riboprobe was less intense (Fig. 6C). The ₃ subunit was present in granule cells and in some cells of the molecular layer. The Purkinje cells expressed the ₃ subunit mRNA only at very low levels or not at all. Hybridization with the ₂ subunit antisense probe showed a thin line beneath the molecular layer on autoradiographic film images (Fig. 4B). On emulsion-dipped slides, this signal was identified as labeling of Purkinje neurons (Fig. 6B). Hybridization with the ₁ subunit riboprobe showed no specific labeling of cerebellar cells, suggesting that the ₁ subunit is not expressed in the cerebellum (Fig. 6A).

In the olfactory bulb, virtually no signal was observed with the ₁ and ₂ subunit-specific riboprobes (data not shown). The ₃ subunit-specific probe gave strong to moderate signals, which were confined to the granule cell layer, and weak labeling of the glomerular layer (Fig. 6E). In contrast, the ₄ subunit-specific riboprobe yielded prominent signals over mitral cells and scattered tufted cells. The granule cell layer was only weakly labeled (Fig. 6F).

No obvious difference was observed in the type of expression of ₁, ₃ (Fig. 6G), and ₄ (Fig. 6H) subunit transcripts in the cerebral cortex. In cortical layers II-VI, numerous cortical neurons were strongly labeled. All four  subunits were expressed in the neurons of the CA1, CA2, and CA3 fields of the hippocampus and in the granule cells of the dentate gyrus. Highest expression levels were observed for the ₃ transcripts followed by the ₁ mRNA. The ₄ and ₂ subunit-specific probes showed considerably weaker signals, but both subunits were clearly expressed in the neurons of the hippocampal formation. One prominent feature of the expression of the ₃ subunit that was not observed with the other  subunit riboprobes was labeling of the habenula. The medial habenulae were very strongly labeled by the ₃-specific riboprobe, both on autoradiographic film images (Fig. 5B) and on emulsion-dipped slides (Fig. 6I) and by the anti-₃ antibody on histoblots (Fig. 5A).

Expression profile of the ₁ subunit mRNAs in rat brain

In the next series of experiments, we investigated the colocalizations of the  subunits with the ₁ subunits of voltage-gated calcium channels. The expression of the _1E subunit was not investigated in detail, because previous studies showed that this subunit is rather ubiquitously expressed in the brain (Soong et al., 1993; Yokoyama et al., 1995). ISH was performed with probes specific for _1A, _1B, _1C, and _1D subunit mRNA. The probes were chosen to recognize all described splice variants of these ₁ subunits and followed the same criteria as described for the  subunit riboprobes. All four riboprobes were directed against the loop between segments IIS6 and IIIS1, which is highly variable and characteristic for each type of ₁ subunit. An abundant and broad distribution of the _1A and _1B transcripts was observed (Fig. 7C,D; Table 1) contrasting with a lower and spatially more restricted expression of the _1C and _1D mRNA (Fig. 7A,B;, Table 1). On the autoradiographic film images, moderate hybridization signals with the _1C specific riboprobe (Fig. 7A) were detected in the olfactory bulb, hippocampus, and cerebellum (Table 1). In the hippocampus, _1C transcripts were moderately expressed in the dentate gyrus and the CA2 and CA3 fields of Ammon's horn, whereas the CA1 field was only weakly labeled. The overall expression level of _1D transcripts (Fig. 7B) in rat brain was similar to that of the _1C subunit. Moderate signals were observed in the olfactory bulb, the dentate gyrus of the hippocampal formation, and the superior colliculus (Table 1). In contrast to the expression profile of both L-type ₁ calcium channel subunits, the _1B transcripts (Fig. 7C) were expressed almost ubiquitously, with moderate labeling of most brain regions (Table 1). Expression of the _1A subunit (Fig. 7D) was most prominent in the cerebellum. Strong signals were also observed in the hippocampus and inferior colliculus. Nearly all other brain regions were labeled moderately on autoradiographic film images (Table 1).

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Fig. 7. Neuronal distribution of ₁ subunit mRNAs. Autoradiographic film images of sagittal sections hybridized with antisense riboprobes specific for _1C subunit mRNA (A), _1D subunit mRNA (B), _1B subunit mRNA (C), and _1A subunit mRNA (D).
[View Larger Version of this Image (81K GIF file)]

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Cellular localization of ₁ subunit mRNAs

The cellular localization of the ₁ subunit transcripts in the cerebellum is shown in Figure 7. The _1c subunit was expressed moderately in neurons of the granular layer, whereas no labeling of Purkinje cells was observed (Fig. 8A). In contrast, the _1D transcript was moderately expressed both in granule cells and in Purkinje neurons (Fig. 8B). The _1B-specific riboprobe gave moderate signals over neurons in the granule cell layer. The Purkinje cells were not labeled significantly (Fig. 8C). Hybridization with the _1A-specific riboprobe showed heavy labeling of Purkinje neurons and granule cells as well as neurons in the molecular layer (Fig. 8D). These results are in accordance with Northern blot experiments using cerebella from mutant mice with different types of cerebellar degeneration (Mori et al., 1991).

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Fig. 8. Cellular localization of the ₁ subunit mRNAs in the cerebellum and olfactory bulb. A-D, Sagittal sections through the cerebellum hybridized with riboprobes directed against the _1C subunit mRNA (A), _1D subunit mRNA (B), _1B subunit mRNA (C) and _1A subunit mRNA (D). E, F, Sections through the olfactory bulb labeled with the riboprobes specific for _1D subunit mRNA (E) and _1A subunit mRNA (F). Abbreviations are as in the legend to Figure 6. Magnification, 100×.
[View Larger Version of this Image (174K GIF file)]

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In the olfactory bulb, the _1D (Fig. 8E) and _1C, as well as _1B (data not shown) mRNA was expressed moderately in granule cells. Mitral cells, tufted cells, and periglomerular cells were lightly labeled too. Hybridization with the _1A-specific probe (Fig. 8F) showed intense labeling of mitral cells and some scattered tufted cells. Neurons in the granular layer and periglomerular cells gave moderate signals. Expression of the _1C subunit was not detected in the cerebral cortex. The _1D probe labeled moderately and the _1A and _1B riboprobes labeled intensely most neurons throughout cortical layers II-VI (data not shown).

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DISCUSSION

This study analyzed the regional expression and localization of various ₁ and  subunits of voltage-activated calcium channels at the mRNA and protein levels. The specificity of the anti- subunit antibodies was demonstrated (see Fig. 2A) by (1) immunoblots of microsomal membranes from HEK 293 cells transfected with expression vectors coding for the various full length  subunits, (2) block of the immunostaining by preincubation of the antibodies with the specific peptides, (3) no signals with liver microsomes, and (4) the use of antibodies directed against different regions of the  subunit (C terminal vs common region). In agreement with previous Northern blot results (Hullin et al., 1992; Perez-Reyes et al., 1992; Castellano et al., 1993), these antibodies confirmed that the _1a subunit is predominantly expressed in skeletal muscle; the ₂ subunit in heart and brain; and the _1b, ₂, ₃, and ₄ subunits in brain. Labeling of histoblots with the anti-_common antibody gave signals in all brain regions where immunoreactivity with the  subunit-specific antibodies was detected. Furthermore, the close correspondence between the results from immunohistoblot and mRNA ISH demonstrated further the specificity of the antibodies and riboprobes as well as the usability of the histoblot method to determine the distribution of brain proteins. Taken together, these results demonstrate that the immunostaining pattern observed in rat brain slices was caused specifically by the expressed subunits and not by other potentially cross-reactive proteins. The staining pattern summarized in Table 1 indicates that the expression of ₁ and  subunits varies considerably among different brain regions. This highly differentiated regional and neuronal expression of the calcium channel genes is undoubtedly important for neuronal functions.

Inspection of the ISH images indicates that the _1A subunit forms a complex with the ₄ subunit in most neurons. In the cerebellum, the unique localization of the _1A transcripts matches excellently that of the ₄ subunit mRNA. Both _1A and ₄ mRNA was strongly expressed in cerebellar Purkinje cells and granule cells. In the olfactory bulb, the _1A and ₄ mRNA was coexpressed in mitral cells. Both _1A and ₄ subunits were expressed at high levels in thalamic neurons. The cellular localization of the ₄ protein in the molecular layer of the cerebellum corresponded well with the distribution of _1a immunoreactivity (Volsen et al., 1995; Westenbroek et al., 1995), which was found most prominently along dendrites of Purkinje cells and also at cell bodies and parallel fiber dendrites of cerebellar granule cells. Taken together, these results demonstrate that the _1A and ₄ subunits are expressed together in the same cells at similar or identical locations and that therefore the _1A and ₄ subunits are part of a P/Q-type calcium channel complex in most cells. However, the _1A subunit could combine also with the ₂ and/or ₃ subunits in specific neurons, because some neuronal cell types expressed the mRNA for both ₁ and ₂ or ₃ subunits. For example, the ₃ subunit mRNA was expressed in cerebellar granule cells, suggesting that the _1A subunit could also couple with the ₃ subunit. The expression of the ₂ subunit in Purkinje cells is in agreement with electrophysiological results (Stea et al., 1994), which showed that the ₂ subunit slowed inactivation of the _1A channel current, and with the recent finding that the purified _1A subunit is associated among other subunits with the ₂ protein (Liu et al., 1996).

The very broad and uniform distribution of the _1B transcripts corresponds well with the overall distribution of the ₃ mRNA. This is also true at the cellular level, e.g., both transcripts are expressed in cerebellar granule cells, suggesting that _1B associates with the ₃ subunit.

However, the strong expression of ₄ subunit mRNAs in granule cells opens the possibility that _1B may associate also with the ₄ subunit. This expression pattern is in accordance with the subunit composition of the N-type calcium channel immunoprecipitated from brain (Scott et al., 1996), which contained the ₃ and ₄ subunits in association with the _1B subunit. The ₃ subunit associates also with other ₁ subunits, as exemplified by neurons of the habenular complex. The anti-₃ and anti-_common antibodies and the ₃ antisense riboprobes gave very intense signals in the medial habenula. The nucleus interpeduncularis, which is a projection area of habenular neurons, was also labeled by the anti-_common and anti-₃ antibodies, whereas no expression of ₃ mRNA was detected in this region. This suggests that the ₃ protein is synthesized in habenular neurons and transferred via axonal transport to the nucleus interpeduncularis. None of the other  subunit-specific antibodies or riboprobes gave any signal in the habenula. Remarkably, no expression of _1B transcripts could be detected in the habenular complex. Only the _1D riboprobe gave a faint signal. This pattern indicates that the ₃ subunit is not associated exclusively with the _1B subunit, but forms complexes with other ₁ subunits, most likely with the _1E subunit. It was shown previously that transcripts of the _1E subunit are heavily expressed in the medial habenula (Soong et al., 1993). These results strongly support the hypothesis that the ₃ subunit binds to and modulates the current through the _1B and _1E subunits.

In contrast to ₁, ₃, and ₄ subunits, the ₂ subunit was expressed at low levels in rat brain, as shown by Western blots, histoblots, and mRNA ISH. Expression of the ₂ subunit was confined to pyramidal and granular cells of the hippocampus, thalamic neurons, and cerebellar Purkinje cells. The _1C and ₂ subunits are coexpressed in rat cardiac myocytes (A. Ludwig and F. Hofmann, unpublished results). The distribution pattern determined for the two L-type calcium channel _1C and _1D subunits is in accordance with results from earlier studies (Ahlijanian et al., 1990; Chin et al., 1992). Direct comparison of the slices labeled with each ₁ riboprobe demonstrated that _1C and _1D transcripts were expressed at low levels in brain. Their expression profile showed distinct differences to that of the ₂ subunit. In the cerebellum, _1D and _1A mRNA, but not _1C mRNA, was detected in Purkinje cells. Both L-type ₁ subunit transcripts were prominently expressed in the cerebellar granular layer, which labels heavily for the ₃ and ₄ subunits but not for the ₂ subunit. Moderate expression of the _1C subunit was detected in the dentate gyrus and hippocampal CA2 and CA3 fields but was observed at only low levels in the CA1. In contrast, the ₂ subunit was expressed evenly in all three hippocampal fields. Taken together, these results suggest that the ₂ subunit is associated not only with the _1C subunit, as in cardiac muscle, but also with _1A in cerebellar Purkinje cells and probably also with other ₁ subunits. Conversely, the _1C subunit may bind not exclusively ₂ but also other  subunits. In cerebellar granule cells and olfactory neurons, where _1C transcripts were prominently found, ₃ and ₄ transcripts but not ₂ mRNA were detected. This distribution pattern suggests that the ₂ and _1C subunits associate with different channel subunits in brain and heart muscle.

This study provides evidence that a given  subunit associates with different types of ₁ subunits depending on the type of neuron. This interpretation is in accordance with the finding that  subunits bind to ₁ subunits through a conserved motif located at the intracellular loop between repeat I and II of each ₁ subunit (De Waard et al., 1994; Pragnell et al., 1994). Coexpression studies of the cloned cDNAs demonstrated that all four  subunits are able to interact with a given ₁ subunit. They modulated calcium channel activity similarly but differed in their relative effectiveness (Singer et al., 1991; Welling et al., 1993; Castellano and Perez-Reyes, 1994; Olcese et al., 1994; Stea et al., 1994; Lacinova et al., 1995). The subunit composition of the calcium channel is obviously important for the identified electrophysiological properties of the channel. Beyond this functional aspects, the combination of a given ₁ subunit with different  subunits may affect the subcellular localization of the channel and interaction with G-protein subunits (Campbell et al., 1995; Herlitze et al., 1996; Ikeda, 1996). It is evident from this work that the association of an ₁ subunit with different types of  subunits contributes to the diversity of calcium channel activity in the brain.

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FOOTNOTES

Received Sept. 13, 1996; revised Dec. 2, 1996; accepted Dec. 13, 1996.

  

This work was supported by grants from Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, and Fond der Chemie. We thank Dr. M. A. Blanar, Hormone Research Institute, San Francisco, for providing the pAR(RI) vector, Dr. M. Biel for helpful discussions, Ms. S. Stief for peptide synthesis and excellent technical assistance, and Ms. E. Roller for the photography.

Correspondence should be addressed to Dr. A. Ludwig, Institut für Pharmakologie und Toxikologie, Technische Universität München, Biedersteiner Strasse 29, 80802 München, Germany.

Prof. Flockerzi's present address: Institut für Pharmakologie, Abtl. für Molekulare Pharmakologie, Universität Heidelberg, 69120 Heidelberg, Germany.

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				I. Calin-Jageman, K. Yu, R. A. Hall, L. Mei, and A. Lee Erbin Enhances Voltage-Dependent Facilitation of Cav1.3 Ca2+ Channels through Relief of an Autoinhibitory Domain in the Cav1.3 {alpha}1 Subunit J. Neurosci., February 7, 2007; 27(6): 1374 - 1385. [Abstract] [Full Text] [PDF]

				E. W. Tringham, C. E. Payne, J. R. B. Dupere, and M. M. Usowicz Maturation of rat cerebellar Purkinje cells reveals an atypical Ca2+ channel current that is inhibited by {omega}-agatoxin IVA and the dihydropyridine (-)-(S)-Bay K8644 J. Physiol., February 1, 2007; 578(3): 693 - 714. [Abstract] [Full Text] [PDF]

				P. Chameau, Y. Qin, S. Spijker, G. Smit, and M. Joels Glucocorticoids Specifically Enhance L-Type Calcium Current Amplitude and Affect Calcium Channel Subunit Expression in the Mouse Hippocampus J Neurophysiol, January 1, 2007; 97(1): 5 - 14. [Abstract] [Full Text] [PDF]

				G. Baroudi, Y. Qu, O. Ramadan, M. Chahine, and M. Boutjdir Protein kinase C activation inhibits Cav1.3 calcium channel at NH2-terminal serine 81 phosphorylation site. Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1614 - H1622. [Abstract] [Full Text] [PDF]

				P. Weissgerber, B. Held, W. Bloch, L. Kaestner, K. R. Chien, B. K. Fleischmann, P. Lipp, V. Flockerzi, and M. Freichel Reduced Cardiac L-Type Ca2+ Current in Cav{beta}2-/- Embryos Impairs Cardiac Development and Contraction With Secondary Defects in Vascular Maturation Circ. Res., September 29, 2006; 99(7): 749 - 757. [Abstract] [Full Text] [PDF]

				T. D. Helton, W. Xu, and D. Lipscombe Neuronal L-Type Calcium Channels Open Quickly and Are Inhibited Slowly J. Neurosci., November 2, 2005; 25(44): 10247 - 10251. [Abstract] [Full Text] [PDF]

				S. Dalton, S. X Takahashi, J. Miriyala, and H. M Colecraft A single CaV{beta} can reconstitute both trafficking and macroscopic conductance of voltage-dependent calcium channels J. Physiol., September 15, 2005; 567(3): 757 - 769. [Abstract] [Full Text] [PDF]

				D. Chaudhuri, B. A. Alseikhan, S. Y. Chang, T. W. Soong, and D. T. Yue Developmental Activation of Calmodulin-Dependent Facilitation of Cerebellar P-Type Ca2+ Current J. Neurosci., September 7, 2005; 25(36): 8282 - 8294. [Abstract] [Full Text] [PDF]

				N. J. Lautermilch, A. P. Few, T. Scheuer, and W. A. Catterall Modulation of CaV2.1 Channels by the Neuronal Calcium-Binding Protein Visinin-Like Protein-2 J. Neurosci., July 27, 2005; 25(30): 7062 - 7070. [Abstract] [Full Text] [PDF]

				S. Michalakis, H. Geiger, S. Haverkamp, F. Hofmann, A. Gerstner, and M. Biel Impaired Opsin Targeting and Cone Photoreceptor Migration in the Retina of Mice Lacking the Cyclic Nucleotide-Gated Channel CNGA3 Invest. Ophthalmol. Vis. Sci., April 1, 2005; 46(4): 1516 - 1524. [Abstract] [Full Text] [PDF]

				V. A. Letts, C. L. Mahaffey, B. Beyer, and W. N. Frankel A targeted mutation in Cacng4 exacerbates spike-wave seizures in stargazer (Cacng2) mice PNAS, February 8, 2005; 102(6): 2123 - 2128. [Abstract] [Full Text] [PDF]

				C. Mullner, L. A. M. Broos, A. M. J. M. van den Maagdenberg, and J. Striessnig Familial Hemiplegic Migraine Type 1 Mutations K1336E, W1684R, and V1696I Alter Cav2.1 Ca2+ Channel Gating: EVIDENCE FOR {beta}-SUBUNIT ISOFORM-SPECIFIC EFFECTS J. Biol. Chem., December 10, 2004; 279(50): 51844 - 51850. [Abstract] [Full Text] [PDF]

				P. Liao, D. Yu, S. Lu, Z. Tang, M. C. Liang, S. Zeng, W. Lin, and T. W. Soong Smooth Muscle-selective Alternatively Spliced Exon Generates Functional Variation in Cav1.2 Calcium Channels J. Biol. Chem., November 26, 2004; 279(48): 50329 - 50335. [Abstract] [Full Text] [PDF]

				D. Lipscombe, T. D. Helton, and W. Xu L-Type Calcium Channels: The Low Down J Neurophysiol, November 1, 2004; 92(5): 2633 - 2641. [Abstract] [Full Text] [PDF]

				S. Luvisetto, T. Fellin, M. Spagnolo, B. Hivert, P. F. Brust, M. M. Harpold, K. A. Stauderman, M. E. Williams, and D. Pietrobon Modal Gating of Human CaV2.1 (P/Q-type) Calcium Channels: I. The Slow and the Fast Gating Modes and their Modulation by {beta} Subunits J. Gen. Physiol., October 25, 2004; 124(5): 445 - 461. [Abstract] [Full Text] [PDF]

				J. D. Spafford, J. van Minnen, P. Larsen, A. B. Smit, N. I. Syed, and G. W. Zamponi Uncoupling of Calcium Channel {alpha}1 and {beta} Subunits in Developing Neurons J. Biol. Chem., September 24, 2004; 279(39): 41157 - 41167. [Abstract] [Full Text] [PDF]

				A. Rajadhyaksha, I. Husson, S. S. Satpute, K. D. Kuppenbender, J. Q. Ren, R. M. Guerriero, D. G. Standaert, and B. E. Kosofsky L-Type Ca2+ Channels Mediate Adaptation of Extracellular Signal-Regulated Kinase 1/2 Phosphorylation in the Ventral Tegmental Area after Chronic Amphetamine Treatment J. Neurosci., August 25, 2004; 24(34): 7464 - 7476. [Abstract] [Full Text] [PDF]

				J. Mould, T. Yasuda, C. I. Schroeder, A. M. Beedle, C. J. Doering, G. W. Zamponi, D. J. Adams, and R. J. Lewis The {alpha}2{delta} Auxiliary Subunit Reduces Affinity of {omega}-Conotoxins for Recombinant N-type (Cav2.2) Calcium Channels J. Biol. Chem., August 13, 2004; 279(33): 34705 - 34714. [Abstract] [Full Text] [PDF]

				J. D. Foell, R. C. Balijepalli, B. P. Delisle, A. M. R. Yunker, S. L. Robia, J. W. Walker, M. W. McEnery, C. T. January, and T. J. Kamp Molecular heterogeneity of calcium channel {beta}-subunits in canine and human heart: evidence for differential subcellular localization Physiol Genomics, April 13, 2004; 17(2): 183 - 200. [Abstract] [Full Text] [PDF]

				C. E. Young and C. R. Yang Dopamine D1/D5 Receptor Modulates State-Dependent Switching of Soma-Dendritic Ca2+ Potentials via Differential Protein Kinase A and C Activation in Rat Prefrontal Cortical Neurons J. Neurosci., January 7, 2004; 24(1): 8 - 23. [Abstract] [Full Text] [PDF]

				J. Stieber, S. Herrmann, S. Feil, J. Loster, R. Feil, M. Biel, F. Hofmann, and A. Ludwig The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart PNAS, December 9, 2003; 100(25): 15235 - 15240. [Abstract] [Full Text] [PDF]

				Z. Liu, J. Ren, and T. H Murphy Decoding of synaptic voltage waveforms by specific classes of recombinant high-threshold Ca2+ channels J. Physiol., December 1, 2003; 553(2): 473 - 488. [Abstract] [Full Text] [PDF]

				J. M. Schjott, S.-C. Hsu, and M. R. Plummer The Neuronal {beta}4 Subunit Increases the Unitary Conductance of L-type Voltage-gated Calcium Channels in PC12 Cells J. Biol. Chem., September 5, 2003; 278(36): 33936 - 33942. [Abstract] [Full Text] [PDF]

				T. Kleppisch, W. Wolfsgruber, S. Feil, R. Allmann, C. T. Wotjak, S. Goebbels, K.-A. Nave, F. Hofmann, and R. Feil Hippocampal cGMP-Dependent Protein Kinase I Supports an Age- and Protein Synthesis-Dependent Component of Long-Term Potentiation But Is Not Essential for Spatial Reference and Contextual Memory J. Neurosci., July 9, 2003; 23(14): 6005 - 6012. [Abstract] [Full Text] [PDF]

				H. Hibino, R. Pironkova, O. Onwumere, M. Rousset, P. Charnet, A. J. Hudspeth, and F. Lesage Direct interaction with a nuclear protein and regulation of gene silencing by a variant of the Ca2+-channel beta 4 subunit PNAS, January 7, 2003; 100(1): 307 - 312. [Abstract] [Full Text] [PDF]

				M. Murakami, B. Fleischmann, C. De Felipe, M. Freichel, C. Trost, A. Ludwig, U. Wissenbach, H. Schwegler, F. Hofmann, J. Hescheler, et al. Pain Perception in Mice Lacking the beta 3 Subunit of Voltage-activated Calcium Channels J. Biol. Chem., October 18, 2002; 277(43): 40342 - 40351. [Abstract] [Full Text] [PDF]

				H. M Colecraft, B. Alseikhan, S. X Takahashi, D. Chaudhuri, S. Mittman, V. Yegnasubramanian, R. S Alvania, D. C Johns, E. Marban, and D. T Yue Novel functional properties of Ca2+ channel {beta} subunits revealed by their expression in adult rat heart cells J. Physiol., June 1, 2002; 541(2): 435 - 452. [Abstract] [Full Text] [PDF]

				G. Wiest, J.-R. Tian, R. W. Baloh, B. T. Crane, and J. L. Demer Otolith function in cerebellar ataxia due to mutations in the calcium channel gene CACNA1A Brain, December 1, 2001; 124(12): 2407 - 2416. [Abstract] [Full Text] [PDF]

				A. Scholze, T. D. Plant, A. C. Dolphin, and B. Nurnberg Functional Expression and Characterization of a Voltage-Gated CaV1.3 ({{alpha}}1D) Calcium Channel Subunit from an Insulin-Secreting Cell Line Mol. Endocrinol., July 1, 2001; 15(7): 1211 - 1221. [Abstract] [Full Text] [PDF]

				S. Restituito, R. M. Thompson, J. Eliet, R. S. Raike, M. Riedl, P. Charnet, and C. M. Gomez The Polyglutamine Expansion in Spinocerebellar Ataxia Type 6 Causes a beta Subunit-Specific Enhanced Activation of P/Q-Type Calcium Channels in Xenopus Oocytes J. Neurosci., September 1, 2000; 20(17): 6394 - 6403. [Abstract] [Full Text] [PDF]

				L. Forti, C. Pouzat, and I. Llano Action potential-evoked Ca2+ signals and calcium channels in axons of developing rat cerebellar interneurones J. Physiol., August 15, 2000; 527(1): 33 - 48. [Abstract] [Full Text] [PDF]

				J. Q. Pan and D. Lipscombe Alternative Splicing in the Cytoplasmic II-III Loop of the N-Type Ca Channel alpha 1B Subunit: Functional Differences Are beta Subunit-Specific J. Neurosci., July 1, 2000; 20(13): 4769 - 4775. [Abstract] [Full Text] [PDF]

				A. Gerstner, X. Zong, F. Hofmann, and M. Biel Molecular Cloning and Functional Characterization of a New Modulatory Cyclic Nucleotide-Gated Channel Subunit from Mouse Retina J. Neurosci., February 15, 2000; 20(4): 1324 - 1332. [Abstract] [Full Text] [PDF]

				K. Jun, E. S. Piedras-Renteria, S. M. Smith, D. B. Wheeler, S. B. Lee, T. G. Lee, H. Chin, M. E. Adams, R. H. Scheller, R. W. Tsien, et al. Ablation of P/Q-type Ca2+ channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the alpha 1A-subunit PNAS, December 21, 1999; 96(26): 15245 - 15250. [Abstract] [Full Text] [PDF]

				F. Lehmann-Horn and K. Jurkat-Rott Voltage-Gated Ion Channels and Hereditary Disease Physiol Rev, October 1, 1999; 79(4): 1317 - 1372. [Abstract] [Full Text] [PDF]

				R. Hullin, F. Asmus, A. Ludwig, J. Hersel, and P. Boekstegers Subunit Expression of the Cardiac L-Type Calcium Channel Is Differentially Regulated in Diastolic Heart Failure of the Cardiac Allograft Circulation, July 13, 1999; 100(2): 155 - 163. [Abstract] [Full Text] [PDF]

				M. Biel, M. Seeliger, A. Pfeifer, K. Kohler, A. Gerstner, A. Ludwig, G. Jaissle, S. Fauser, E. Zrenner, and F. Hofmann Selective loss of cone function in mice lacking the cyclic nucleotide-gated channel CNG3 PNAS, June 22, 1999; 96(13): 7553 - 7557. [Abstract] [Full Text] [PDF]

				D. Walker, D. Bichet, S. Geib, E. Mori, V. Cornet, T. P. Snutch, Y. Mori, and M. De Waard A New beta  Subtype-specific Interaction in alpha 1A Subunit Controls P/Q-type Ca2+ Channel Activation J. Biol. Chem., April 30, 1999; 274(18): 12383 - 12390. [Abstract] [Full Text] [PDF]

				N. L Brice and A. C Dolphin Differential plasma membrane targeting of voltage-dependent calcium channel subunits expressed in a polarized epithelial cell line J. Physiol., March 15, 1999; 515(3): 685 - 694. [Abstract] [Full Text] [PDF]

				N. Klugbauer, L. Lacinova, E. Marais, M. Hobom, and F. Hofmann Molecular Diversity of the Calcium Channel alpha 2delta Subunit J. Neurosci., January 15, 1999; 19(2): 684 - 691. [Abstract] [Full Text] [PDF]

				B. L Soldo and H. C Moises {micro}-Opioid receptor activation inhibits N- and P-type Ca2+ channel currents in magnocellular neurones of the rat supraoptic nucleus J. Physiol., December 15, 1998; 513(3): 787 - 804. [Abstract] [Full Text] [PDF]

				T. D. Plant, C. Schirra, E. Katz, O. D. Uchitel, and A. Konnerth Single-Cell RT-PCR and Functional Characterization of Ca2+ Channels in Motoneurons of the Rat Facial Nucleus J. Neurosci., December 1, 1998; 18(23): 9573 - 9584. [Abstract] [Full Text] [PDF]

				A. Sautter, X. Zong, F. Hofmann, and M. Biel An isoform of the rod photoreceptor cyclic nucleotide-gated channel beta  subunit expressed in olfactory neurons PNAS, April 14, 1998; 95(8): 4696 - 4701. [Abstract] [Full Text] [PDF]

				D. Walker, D. Bichet, K. P. Campbell, and M. De Waard A beta 4 Isoform-specific Interaction Site in the Carboxyl-terminal Region of the Voltage-dependent Ca2+ Channel alpha 1A Subunit J. Biol. Chem., January 23, 1998; 273(4): 2361 - 2367. [Abstract] [Full Text] [PDF]

				R. Kollmar, L. G. Montgomery, J. Fak, L. J. Henry, and A. J. Hudspeth Predominance of the alpha 1D subunit in L-type voltage-gated Ca2+ channels of hair cells in the chicken's cochlea PNAS, December 23, 1997; 94(26): 14883 - 14888. [Abstract] [Full Text] [PDF]

				R. J. Lewis, K. J. Nielsen, D. J. Craik, M. L. Loughnan, D. A. Adams, I. A. Sharpe, T. Luchian, D. J. Adams, T. Bond, L. Thomas, et al. Novel omega -Conotoxins from Conus catus Discriminate among Neuronal Calcium Channel Subtypes J. Biol. Chem., November 3, 2000; 275(45): 35335 - 35344. [Abstract] [Full Text] [PDF]

				S. Wittemann, M. D. Mark, J. Rettig, and S. Herlitze Synaptic Localization and Presynaptic Function of Calcium Channel beta 4-Subunits in Cultured Hippocampal Neurons J. Biol. Chem., November 22, 2000; 275(48): 37807 - 37814. [Abstract] [Full Text] [PDF]

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