beta Subunit Heterogeneity in Neuronal L-type Ca2+ Channels*

(Received for publication, February 6, 1997)

Michaela Pichler , Tara N. Cassidy Dagger , Daniel Reimer , Hannelore Haase §, Richard Kraus , Dominique Ostler and Jörg Striessnig par

From the Institut für Biochemische Pharmakologie, Peter-Mayrstrasse 1, A-6020 Innsbruck, Austria and the § Max-Delbrück Centrum für Molekulare Medizin, D-13125 Berlin, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Heterologous expression studies have shown that the activity of voltage-gated Ca2+ channels is regulated by their beta  subunits in a beta  subunit isoform-specific manner. In this study we therefore investigated if one or several beta  subunit isoforms associate with L-type Ca2+ channels in different regions of mammalian brain.

All four beta  subunit isoforms (beta 1b, beta 2, beta 3, and beta 4) are expressed in cerebral cortex as shown in immunoblots. Immunoprecipitation of (+)-[3H]isradipine-labeled L-type channels revealed that the majority of beta  subunit-associated L-type channels was associated with beta 3 (42 ± 8%) and beta 4 (42 ± 7%) subunits, whereas beta 1b and beta 2 were present in a smaller fraction of channel complexes. beta 3 and beta 4 were also the major L-type channel beta  subunits in hippocampus. In cerebellum beta 1b, beta 2, and beta 3 but not beta 4 subunits were expressed at lower levels than in cortex. Accordingly, beta 4 was the most prominent beta  subunit in cerebellar L-type channels. This beta  subunit composition was very similar to the one determined for 125I-omega -conotoxin-GVIA-labeled N-type and 125I-omega -conotoxin-MVIIC-labeled P/Q-type channel complexes in cerebral cortex and cerebellum.

Our data show that all four beta  subunit isoforms associate with L-type Ca2+ channels in mammalian brain. This beta  subunit heterogeneity may play an important role for the fine tuning of L-type channel function and modulation in neurons.


INTRODUCTION

Voltage-gated Ca2+ channels control the depolarization-induced influx of extracellular Ca2+ into neurons and other electrically excitable cells. They exist as hetero-oligomeric complexes of different subunits (alpha 1, alpha 2-delta , and beta ). Different types of neuronal Ca2+ channels (termed L-, N-, P-, Q-, and R-type; 1) are discriminated by biophysical and pharmacological criteria (for reviews see Refs. 2-5). N- and P/Q-type channels are blocked by peptide toxins (omega -CTx1-GVIA and omega -CTx-MVIIC or omega -agatoxin-IVA, respectively), whereas L-type channels are modulated by drugs, such as dihydropyridines (6). These channel types are differentially distributed in the brain and even within a neuron (7, 8). Thereby they serve different physiological functions. N- and P/Q-type channels are abundant in nerve terminals and control Ca2+-dependent neurotransmitter release (3). L-type channels are localized mainly on neuronal cell somata and proximal dendrites where they may control Ca2+-dependent modulatory processes and excitation-transcription coupling (9).

The above Ca2+ channel types consist of different alpha 1 subunit isoforms (class A-E) that also form their drug or toxin binding domains and therefore determine their pharmacological properties (1). In contrast, important biophysical and modulatory properties, such as voltage-dependent gating (10, 11) and channel modulation by G-proteins (12, 13) and kinases (14), are determined not only by alpha 1 but also by associated alpha 2-delta and beta -subunits. Whereas only one alpha 2-delta isoform is known, four different beta  subunit isoforms (beta 1-beta 4) are expressed in mammalian brain (15, 16). Heterologous expression studies revealed that beta  subunits can affect alpha 1 function in a beta  subunit isoform-specific manner. For example, Ca2+ currents carried by alpha 1A, alpha 1E, and alpha 1C inactivate faster with coexpressed beta 3 than with beta 2 (14, 17, 18) subunits. beta 1, beta 3, and beta 4, but not beta 2, are permissive for voltage-dependent facilitation of Ca2+ channels formed by alpha 1C (19). beta 3 and beta 1 subunits confer slightly different pharmacological properties to L-type channels (20). Therefore beta  subunit heterogeneity could participate in the fine-tuning of channel function. However, it is unclear if only one or several beta  subunit isoforms associate with these channels in mammalian brain. So far only the beta  subunit composition of L-type Ca2+ channels in skeletal muscle has been studied. In this tissue exclusively beta 1a subunits are associated with the channel complex (15, 21).

Biochemical evidence for beta  subunit heterogeneity in mammalian brain has recently been provided for N-type and P/Q-type channels (22, 23). Multiple beta  subunit isoforms were found to be associated to different extents with both channel types after extraction from whole rabbit brain.

Here we report that beta  subunit heterogeneity also exists within neuronal L-type channels. We found that regional differences in the beta  subunit expression pattern affect beta  subunit composition in different regions of mammalian brain.

A preliminary report of our findings has appeared previously (24).


EXPERIMENTAL PROCEDURES

Materials

Reagents were obtained from the following sources: 125I-omega -CTx-GVIA, 125I-omega -CTx-MVIIC (2200 Ci/mmol), and (+)-[3H]isradipine from DuPont NEN (Vienna, Austria); unlabeled omega -CTx-GVIA from Sigma (Vienna, Austria); unlabeled omega -CTx-MVIIC from Saxon Biochemicals (Hannover, Germany); prestained molecular weight markers from Bio-Rad (Vienna, Austria); glutathione-Sepharose from Pharmacia (Vienna, Austria); Protein A-Sepharose from Sigma; calpain inhibitors I and II from Boehringer Mannheim (Vienna, Austria); all other protease inhibitors from Sigma.

Sequence-directed Antibodies

For antibody production in rabbits peptides were coupled to bovine serum albumin with glutaraldehyde (25) or synthesized on a lysine branch (octavalent NovaSyn PA resin, Novabiochem) for immunization. Anti-beta 2 was generated as described (26). For immunoblotting and immunoprecipitation experiments, antibodies were purified by affinity chromatography on Sepharose-4B derivatized with the antigenic peptide (25). Antigenic epitopes comprised the following amino acids (residue number is given according to the sequences in Ref. 27): beta 1b, 516-530; beta 2, 595-604; beta 3, 470-483; beta 4, 460-474. Anti-beta com was raised against residues 61-79 in beta 1a (28).

Membrane Preparation

Membranes were prepared from guinea pig or rabbit cortex, hippocampus, cerebellum, and heart muscle as described (29). Brain regions were rapidly removed from rabbit or guinea pig brains and immediately placed in ice-cold homogenization buffer containing 0.02 M NaHCO3 and a protease inhibitor mixture (2 mM EDTA, 0.2 mM PMSF, 0.5 mM benzamidine, 2 mM iodoacetamide, 1 µM pepstatin A, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 20 µg/ml calpain inhibitor I and II, 0.1 mg/ml trypsin inhibitor). The tissues were then homogenized by 10-20 strokes in a Dounce homogenizer, and microsomes were collected by centrifugation at 45,000 × g (10 min, 4 °C). Microsomes were then washed three times with 50 mM Tris-HCl, pH 7.4 (37 °C), containing the same protease inhibitor mix. Membranes were resuspended in the same buffer at a protein concentration about 5 mg/ml and stored at -80 °C until use.

Affinity Purification of beta  Subunits and Immunoblotting

Glutathione S-transferase (GST) and a GST fusion protein with the alpha 1 subunit interaction domain of the alpha 1A subunit (AIDA) were prepared as described (21). All further steps were carried out on ice or at 4 °C. Typically 20 mg of microsomal protein isolated from rabbit or guinea pig brain regions were solubilized in 9 ml of buffer A (50 mM Tris-HCl, pH 7.4, containing the protease inhibitors used for membrane preparation) supplemented with 1% (w/v) CHAPS and 1 M NaCl according to Ref. 21. 30-µl aliquots of glutathione-Sepharose equilibrated in buffer B (buffer A containing 0.1% (w/v) CHAPS, 0.1 M NaCl) were coupled with 10 µg of GST or GST-AIDA and washed three times with the above buffer. Solubilized membranes were diluted 10-fold in buffer A, and 4 ml were mixed with the coupled glutathione-Sepharose beads for 4 h or overnight. The beads were washed three times with 1.5 ml of buffer B, mixed with SDS-polyacrylamide gel electrophoresis sample buffer (15 min, 56 °C or 3 min, 95 °C), and the eluted protein separated on 10% polyacrylamide gels.

Immunoblot experiments were carried out as described (30). Prestained molecular weight markers (Bio-Rad) were run on the same gels. The apparent molecular masses of each batch were provided by the supplier.

Solubilization and Immunoprecipitation

Membrane-bound channels were prelabeled with (+)-[3H]isradipine (1-2 nM) for 60 min at 37 °C in 50 mM Tris-HCl, 0.1 mM PMSF, 1 mM CaCl2. All subsequent steps were carried out on ice or at 4 °C. Prelabeled membranes were collected, solubilized on ice for 60 min in 50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1% (w/v) digitonin, 0.2 mM PMSF, 0.5 mM benzamidine, 2 mM iodoacetamide, 1 µM pepstatin A, and nonsoluble proteins removed by centrifugation (45,000 × g, 60 min). The digitonin extracts were either used for immunoprecipitation or were further affinity-purified by chromatography on wheat germ agglutinin (WGA)-Sepharose (2 ml of packed resin for 5-20 mg of solubilized protein). Channel-associated activity was eluted from the resin in equilibration buffer (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.1 mM PMSF) containing 6% (w/v) N-acetylglucosamine. Active fractions were directly used in immunoprecipitation experiments or quickly frozen in liquid nitrogen and stored at -80 °C until use.

For immunoprecipitation of labeled channels, affinity-purified antibodies were coupled to Protein A-Sepharose. The Protein A-Sepharose-antibody complex was washed three times with 1.5 ml of ice-cold RIA buffer (solubilization buffer containing 0.1% digitonin) and then incubated for 12-16 h at 4 °C with 0.15-0.55 ml of solubilized extracts (prelabeled N- and P/Q-type channels) or WGA-Sepharose-purified channel preparations (L-type channels). Unbound radioactivity was removed by four 1.5-ml washes with RIA buffer. Bound radioactivity was determined by liquid scintillation ((+)-[3H]isradipine) or gamma-counting (125I-omega -CTx-GVIA, 125I-omega -CTx-MVIIC).

High affinity binding of (+)-[3H]isradipine to solubilized L-type Ca2+ channels was determined using a filtration assay as described (29). This assay underestimated the total specific (+)-[3H]isradipine binding activity by about 20%. This was taken into account to calculate the binding activity employed for immunoprecipitation assays.

Statistics

Data are given as means ± S.D. for the indicated number of experiments.


RESULTS

Region-specific Expression of beta  Subunit Isoforms in Mammalian Brain

To investigate the association of all known subunit isoforms with neuronal voltage-gated L-type Ca2+ channels in mammalian brain, we raised anti-peptide antibodies against unique sequences of beta 1b, beta 2, beta 3, and beta 4 subunits. In addition, an antibody against an epitope highly conserved in all beta  subunit isoforms (anti-beta com) was generated. We used these antibodies to determine their association with neuronal Ca2+ channels solubilized from rabbit or guinea pig cerebral cortex, hippocampus, and cerebellum membranes in immunoprecipitation experiments. Their expression in these brain regions was analyzed in Western blots.

To determine their relative expression densities the four beta  subunit isoforms were extracted with CHAPS from microsomes prepared from brain (beta 1b, beta 2, beta 3, beta 4) or, for control purposes, from skeletal muscle (beta 1a). Extracts were affinity-purified on GST-AIDA-Sepharose (21) in the presence of protease inhibitors as described under "Experimental Procedures." As shown in Fig. 1 for skeletal muscle beta 1a (anti-beta com staining) and neuronal beta 3 and beta 4 subunits, the enrichment of beta  subunit immunoreactivity was specific and absent when only GST was used as the affinity matrix (Fig. 1, lanes 4-6). In beta  subunit preparations from rabbit cerebral cortex anti-beta com specifically recognized a 63 ± 3/67 ± 3-kDa doublet and a 88 ± 4-kDa band (Fig. 2) (n >=  4). A ~33-kDa band was also stained to a variable extent by beta com as well as all the other beta  antibodies and corresponded to the GST-AIDA polypeptide present at relatively high amounts (10 µg) in the beta  subunit preparations.


Fig. 1. Affinity purification of beta  subunits. The results of Western blot analysis are shown. A, skeletal muscle beta 1a subunits. 2.3 mg of partially purified rabbit skeletal muscle transverse-tubule membranes were solubilized in 0.9 ml of buffer containing 1% CHAPS. 0.1 ml of the solubilized material was subjected to affinity chromatography on 5 µg of GST-AIDA (lanes 1-3) or GST alone (lanes 4-6) coupled to glutathione-Sepharose (0.03 ml) as described under "Experimental Procedures." Identical aliquots (40 µl) of the extracted starting material (lanes 1 and 4) and the supernatant after incubation with the resins (lanes 2 and 5) and resin-bound protein (lanes and 6) were separated by SDS-polyacrylamide gel electrophoresis and analyzed for beta  immunoreactivity in immunoblots employing anti-beta com. 52-56-kDa bands were stained as expected for beta 1a staining in skeletal muscle membranes (21). The same bands were stained in purified channel preparations (>= 90% pure, not shown). B, enrichment of beta 3 (left panel) and beta 4 (right panel) immunoreactivity from guinea pig cortex membranes. 20 mg of membrane protein were solubilized in a total volume of 9 ml and diluted 10-fold as described under "Experimental Procedures." 4-ml aliquots were subjected to affinity chromatography on GST-AIDA (10 µg) coupled to glutathione-Sepharose. Immunostaining was with affinity-purified anti-beta 3 (lanes 1-3, left) or anti-beta 4 (lanes 1-3, right). Lanes 1 and 4, starting material (40-µl aliquots); lanes 2 and 5, supernatants (40 µl aliquots); lanes 3 and 6, GST-AIDA resin-bound protein. No other bands were specifically enriched. The electrophoretic mobilities of the stained bands were indistinguishable from those in Fig. 2. One of two experiments yielding similar results is shown.
[View Larger Version of this Image (68K GIF file)]


Fig. 2. Expression of beta 1b, beta 3, and beta 4 subunits in different brain regions. The results of Western blot analysis are shown. beta  subunits were purified from the indicated brain regions (A and B), skeletal muscle (A) or heart microsomes (B) by GST-AIDA affinity chromatography and separated by SDS-polyacrylamide gel electrophoresis together with prestained marker proteins and subjected to immunoblot analysis with beta  subunit isoform-selective, affinity-purified antibodies. Skeletal muscle affinity chromatography on GST-AIDA-Sepharose was as in Fig. 1. For all neuronal tissues and cardiac muscle comparable amounts of membrane protein (0.45 ml of solubilization buffer per mg of protein) were used for solubilization and subsequent analysis (4 ml of diluted extract). Samples from one brain region were always separated on the same gel. In some experiments individual lanes were cut in half longitudinally and probed with different antibodies to allow an exact comparison of the relative migration of immunostained bands. The numbers denote beta  subunit antibody selectivities. C, beta com antibody recognizing an epitope common to all four beta  subunit isoforms. The arrows indicate specifically stained beta  subunits as discussed in the text. The migration of prestained molecular weight markers (105,000; 82,000; 49,000; 33,300; 28,600) is indicated on the left. CTX, cerebral cortex; CER, cerebellum; HIP, hippocampus; SKM, skeletal muscle. One of at least three independent experiments yielding similar results is shown.
[View Larger Version of this Image (87K GIF file)]

To assign the beta com-stained bands to individual beta  subunit isoforms, samples separated on the same gel were stained with isoform-specific antibodies. The 88-kDa band was composed of anti-beta 1b (Fig. 2A) and anti-beta 2 staining (Fig. 2B). In contrast, bands stained by anti-beta 4 and anti-beta 3 accounted for the beta com immunoreactivity in the 63/67-kDa doublet. The majority of anti-beta 3 immunoreactivity was associated with the larger 67-kDa beta com band, whereas anti-beta 4 recognized both bands of the doublet to a variable extent (Figs. 1, 2, 3).


Fig. 3. Relative expression densities of beta  subunit immunoreactivities in different tissues. The results of Western blot analysis are shown. beta  subunits were extracted from equal amounts of membrane protein prepared from cerebral cortex (CTX) or cerebellum (CER) as described in Fig. 2 and separated on adjacent lanes. Immunostaining was carried out with the indicated affinity-purified antibodies. Molecular weight markers are as in Fig. 2.
[View Larger Version of this Image (115K GIF file)]

beta subunit isoform staining was specific. It was completely suppressed in the presence of 1 µM of the respective antigenic peptides (not shown). As expected, only beta com but not the isoform-selective antibodies specifically recognized beta 1a extracted from partially purified skeletal muscle T-tubule membranes (Fig. 2A). beta com staining in rabbit heart represented beta 2 immunoreactivity (Fig. 2B) suggesting that other isoforms are absent or expressed at much lower levels in this tissue.

The same bands were also present in hippocampus (Fig. 2) and cerebellum extracts (Fig. 3). The relative abundance of the 88-kDa band was lowest in cerebellum because beta 1b and beta 2 expression density was lower in this region as compared with cerebral cortex (Fig. 2). When similar amounts of solubilized membrane protein from cerebral cortex and cerebellum were subjected to beta  subunit isolation and Western blotting (Fig. 3) similar beta com staining intensity was found for the 63/67-kDa doublet. beta 3-specific immunoreactivity was less abundant in cerebellum, whereas beta 4 was expressed at similar densities as in cerebral cortex (Fig. 3).

Taken together, the beta com staining pattern in mammalian brain can be explained by the presence of all four beta  subunit isoforms which are expressed in a region-specific pattern.

Neuronal L-type Ca2+ Channels Are Associated with Different beta  Subunit Isoforms in Mammalian Brain

After having established the specificity of our antibodies, we investigated if L-type Ca2+ channels are associated with only one or several beta  subunit isoforms and if beta  subunit association varies in different brain regions. We reversibly labeled neuronal L-type Ca2+ channels complexes in cerebral cortex, hippocampus, and cerebellum membranes with the L-type Ca2+ channel-selective ligand (+)-[3H]isradipine and solubilized them in buffer containing 1% (w/v) digitonin. In cerebral cortex and hippocampus 74 ± 9% (n = 4) and 91 ± 22% (n = 4) of the solubilized (+)-[3H]isradipine labeling was immunoprecipitated with saturating concentrations of an antibody directed against alpha 1C indicating that binding was associated with L-type channel complexes. 61 ± 18% (n = 5) and 80 ± 31% (n = 4) of the labeled L-type channels were immunoprecipitated by beta com. Therefore, most of the L-type channel complexes are associated with a beta  subunit which is accessible for beta com under nondenaturating conditions.

Immunoprecipitation experiments with the isoform-selective antibodies revealed the association of more than one beta  isoform with the channel complex. Affinity-purified anti-beta 3 and anti-beta 4 antibodies each immunoprecipitated 42% of the radioactivity recognized by anti-beta com (Fig. 4A). Smaller fractions were bound by anti-beta 1b and anti-beta 2 (Fig. 4A). Together our subunit-specific antibodies accounted for all (118%) beta com immunoprecipitable radioactivity in cerebral cortex.


Fig. 4. Specific immunoprecipitation of solubilized (+)-[3H]isradipine-labeled L-type Ca2+ channels from different brain regions and skeletal muscle. Immunoprecipitation was carried out as described under "Experimental Procedures." A-C, the dpm immunoprecipitated by saturating concentrations of the respective isoform-selective antibody were normalized with respect to the dpm immunoprecipitated by saturating concentrations of anti-beta com (>1500 dpm in cerebral cortex; >2600 dpm in hippocampus; >270 dpm in cerebellum). Data are shown for n = 3 with the exception of beta 1b and beta 2 immunoprecipitation (n = 2) in cerebellum. D, skeletal muscle Ca2+ channels were partially purified by affinity chromatography on WGA-Sepharose as described (37) and labeled with 2 nM (+)-[3H]isradipine. Aliquots of the labeled channel preparation were diluted with RIA buffer to a final volume of 0.3 ml and subjected to immunoprecipitation as described for neuronal channels. One of two typical experiments is shown. Numbers denote the beta  isoform to which antibodies were generated; C, beta com.
[View Larger Version of this Image (25K GIF file)]

Immunoprecipitation by these antibodies was saturable (see Fig. 5C). The nonspecific background signal observed with the same concentrations of control rabbit immunoglobulin was less than 10% (n > 3) of the radioactivity recognized by anti-bcom. In control experiments only beta com, but none of the isoform-specific antibodies, immunoprecipitated (+)-[3H]isradipine-labeled L-type channels extracted from rabbit skeletal muscle (Fig. 4B), which are exclusively associated with beta 1a (21).


Fig. 5. Specific immunoprecipitation of solubilized 125I-omega -CTx-GVIA-labeled N-type Ca2+ channels and 125I-omega -CTx-MVIIC-labeled P/Q-type Ca2+ channels from different brain regions. A-B, immunoprecipitation was as described for L-type Ca2+ channels. Reversible labeling of channels was carried out at low picomolar concentrations of 125I-omega -CTx-GVIA (<10 pM). Data are shown for n = 3-8. Numbers denote the beta  isoform to which antibodies were generated; C, beta com. C, the concentration-dependent immunoprecipitation of 125I-omega -CTx-GVIA binding activity from solubilized rabbit cerebral cortex membranes by antibodies against beta com (black-square, upper abscissa), beta 1b (square , upper abscissa), beta 3 (open circle , lower abscissa), and beta 4 (bullet , lower abscissa) is shown. One of at least two experiments yielding similar results is shown.
[View Larger Version of this Image (19K GIF file)]

A similar beta  subunit composition was observed in hippocampus (Fig. 4C). In the cerebellum only beta 4 accounted for a large portion of L-type channel-associated beta  subunits (Fig. 4D). Immunoprecipitation by beta 3 antibodies was less pronounced than in cerebral cortex (Fig. 4D). This is in good agreement with the lower relative abundance of beta 3 in this region (Fig. 3). Immunoprecipitation by beta 1b and beta 2 was difficult to detect in cerebellum (Fig. 4D) representing less than 10% of the channels immunoprecipitated by anti-beta com.

Together the isoform-selective antibodies accounted for most but not all of the beta  subunit-associated radioactivity in hippocampus (70%) and cerebellum (66%).

Similar beta  Subunit Composition of L-, N-, and P/Q-type Ca2+ Channels

Next we tested if the beta  subunit composition of L-type Ca2+ channels resembles the subunit composition of N- and P/Q-type Ca2+ channels in these regions (22, 23). For N- and P/Q-type channels it has been investigated before in digitonin extracts of whole brain membranes, but data on individual brain regions are unavailable. We therefore also subjected 125I-omega -CTx-GVIA- and 125I-omega -CTx-MVIIC-labeled channel complexes extracted from cerebral cortex and cerebellum to immunoprecipitation with our antibodies. We have previously shown that under our experimental conditions saturable high affinity 125I-omega -CTx-GVIA and 125I-omega -CTx-MVIIC binding occurs selectively to N-type and P/Q-type Ca2+ channels, respectively, with dissociation constants in the subpicomolar range (31).

In cerebral cortex and cerebellum saturating concentrations of anti-beta com recognized 85 ± 23% (n = 5) and 84 ± 13 (n = 4) of channels associated with 125I-omega -CTx-GVIA binding activity, respectively. The immunoprecipitation profile was very similar to L-type channels (Fig. 5, A and B). beta 3 and beta 4 subunits together immunoprecipitated >80% of beta com immunoprecipitable 125I-omega -CTx-GVIA binding in a saturable manner (Fig. 5C). As with L-type channels, a smaller fraction of N-type channel binding was associated with beta 1b and beta 2. In cerebellum again only beta 4 antibodies recognized substantial portions of N-type channel activity (Fig. 5B). Similar results as described for N-type and L-type channels were also obtained for 125I-omega -CTx-MVIIC-labeled P/Q-type channels in cerebral cortex (not shown). In cerebellum only beta 4 antibodies recognized significant amounts of 125I-omega -CTx-MVIIC-labeled P/Q-type channels (43 ± 18.5%, n = 3).

Isoform-selective antibodies completely accounted for the N-type (105%, Fig. 5A) and P/Q-type (>85%, not shown) channel binding recognized by anti-beta com in cerebral cortex but only for 40-50% in cerebellum. As for L-type channels this difference cannot be attributed to differences in membrane preparation because it was also found when the respective brain regions were isolated from the same animals in the same buffer and carried through the whole solubilization and immunoprecipitation procedure in parallel. It is therefore possible that in hippocampus and cerebellum immunoprecipitation by one or several of our antibodies was underestimated. At present we do not know if this is due to the expression of a yet uncharacterized beta  subunit isoform, which is immunoprecipitated by beta com but none of the other antibodies, or due to region-specific differences in proteolysis. C-terminal proteolysis could remove the C-terminal epitopes of our isoform-specific antibodies. However, we have obtained no evidence for extensive proteolytic breakdown of beta subunits in immunoblots with our beta com antibody, which recognizes an epitope located near the N terminus of the beta  subunits.


DISCUSSION

beta Subunit Heterogeneity within L-type Ca2+ Channels

The major findings of our study are as follows. 1) All known beta  subunit isoforms participate in the formation of neuronal L-type Ca2+ channels in mammalian brain. 2) beta 3 and beta 4 subunits are most often found as part of the neuronal L-type channel complexes. 3) The fractional contribution of a particular beta  subunit isoform for channel formation varies among different brain regions. 4) The beta  subunit composition and regional differences are very similar to N- and P/Q-type channels in cerebral cortex and cerebellum.

This similarity of the beta  subunit composition between L-type channels and N- as well as P/Q-type channels is interesting because the subcellular distribution of L-type channels in neurons differs significantly from the distribution of N- and P/Q-type channels. L-type alpha 1C and alpha 1D subunits are predominantly found on the cell soma and proximal dendrites, whereas N-type alpha 1B and P/Q-type alpha 1A are also found along the length of dendrites and in presynaptic terminals (8, 32). Despite these differences in neuronal targeting, these channel types do not show major differences with respect to their beta  subunit composition. Obviously different beta subunit isoforms can be targeted to different regions of a neuron.

Both alpha 1C and alpha 1D subunits participate in the formation of L-type Ca2+ channels in mammalian brain. We have made no attempts to determine if differences exist between the two L-type channels with respect to their beta  subunit composition. The fraction of channels associated with alpha 1D is small (not more than 9-26% in hippocampus and cerebral cortex as revealed by our immunoprecipitation experiments with alpha 1C; see also Ref, 33) and therefore complicates such an analysis. We cannot exclude the possibility that beta 1b and beta 2, which are found only in a minor fraction of channels, are selectively associated only with alpha 1D. However, based on our finding that alpha 1C is associated with the majority of labeled channels in cerebral cortex and hippocampus, beta -subunit heterogeneity must exist within class C L-type channels in these regions.

Implications for Neuronal L-type Ca2+ Channel Function

beta subunits strongly affect the functional properties of the pore-forming alpha 1 subunits of L-type (and non L-type) channels. As shown by heterologous coexpression in Xenopus oocytes and mammalian cells, beta  subunits affect channel gating (10, 11), modulation by G-proteins (12, 13), and phosphorylation (14) as well as Ca2+ and drug (34, 35) interaction with L-type alpha 1 subunits. Such studies also revealed that different beta  isoforms are able to confer different channel properties. For example, beta 3 confers a more rapid inactivation to currents mediated by alpha 1C (17) than does beta 2. beta  isoform-specific effects on channel inactivation were also observed for non-L-type Ca2+ channel alpha 1 subunits (11, 14, 18). Only beta 1, beta 3, and beta 4, but not beta 2, support voltage-dependent facilitation of alpha 1C-mediated Ca2+ currents (19). Similarly, small differences in the sensitivity of the channel to the Ca2+ antagonist mibefradil and the modulation by protein kinase C were observed when different beta  subunits form part of the channel complex (14, 20). We now provide direct biochemical evidence that indeed different beta  subunits participate in the formation of neuronal L-type channels suggesting that these isoform-selective effects contribute to L-type Ca2+ channel plasticity in mammalian brain. beta  subunits could be involved in the fine tuning of L-type channel function in a region-specific manner. Based on our findings future coexpression studies should preferentially focus on the comparison of the properties of L-type channels containing beta 3 or beta 4 subunits, because these isoforms seem to be present in the majority of dihydropyridine-sensitive L-type channels in cortex, hippocampus, and cerebellum.

Taken together our data demonstrate that, like in other neuronal Ca2+ channel types, several beta  subunit isoforms contribute to the formation of neuronal L-type channels. Further studies must focus on the physiological and pathophysiological consequences of this heterogeneity and investigate if changes in beta  subunit expression could account for changes in L-type Ca2+ channel function also under pathophysiological conditions, such as neurodegeneration, cerebral ischemia, or aging (36).


FOOTNOTES

*   This work was supported by the Fonds zur Förderung der Wissenschaftlichen Forschung S6602 (to J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Recipient of a Fullbright Fellowship.
   Supported by a grant from the Austrian Academy of Sciences. Work performed in partial fulfillment of a thesis.
par    To whom correspondence and reprint requests should be addressed. Tel.: 43-512-507-3164; Fax: 43-512-588627; E-mail: joerg.striessnig{at}uibk.ac.at.
1   The abbreviations used are: CTx, conotoxin; AIDA, alpha 1 interaction domain of alpha 1A; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; WGA, wheat germ agglutinin-Sepharose; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

ACKNOWLEDGEMENTS

We thank Dr. H. Glossmann for continuous support.


REFERENCES

  1. Birnbaumer, L., Campbell, K. P., Catterall, W. A., Harpold, M. M., Hofmann, F., Horne, W. A., Mori, Y., Schwartz, A., Snutch, T. P., Tanabe, T., and Tsien, R. W. (1994) Neuron 13, 505-506 [Medline] [Order article via Infotrieve]
  2. Striessnig, J., Berger, W., and Glossmann, H. (1993) Cell. Physiol. Biochem. 3, 295-317
  3. Dunlap, K., Luebke, J. I., and Turner, T. J. (1995) Trends Neurosci. 18, 89-98 [CrossRef][Medline] [Order article via Infotrieve]
  4. Catterall, W. A. (1995) Annu. Rev. Biochem. 64, 493-531 [CrossRef][Medline] [Order article via Infotrieve]
  5. Gurnett, C. A., and Campbell, K. P. (1996) J. Biol. Chem. 271, 27975-27958 [Free Full Text]
  6. Glossmann, H., and Striessnig, J. (1990) Rev. Physiol. Biochem. Pharmacol. 114, 1-105 [Medline] [Order article via Infotrieve]
  7. Westenbroek, R. E., Hell, J. W., Warner, C., Dubel, S. J., Snutch, T. P., and Catterall, W. A. (1992) Neuron 9, 1099-1115 [Medline] [Order article via Infotrieve]
  8. Westenbroek, R. E., Sakurai, T., Elliott, E. M., Hell, J. W., Starr, T. V. B., Snutch, T. P., and Catterall, W. A. (1995) J. Neurosci. 15, 6403-6418 [Medline] [Order article via Infotrieve]
  9. Murphy, T. H., Worley, P. F., and Baraban, J. M. (1991) Neuron 7, 625-635 [Medline] [Order article via Infotrieve]
  10. Singer, D., Biel, M., Lotan, I., Flockerzi, V., Hofmann, F., and Dascal, N. (1991) Science 253, 1553-1557 [Medline] [Order article via Infotrieve]
  11. Olcese, R., Qin, N., Schneider, T., Neely, A., Wei, X. Y., Stefani, E., and Birnbaumer, L. (1994) Neuron 13, 1433-1438 [Medline] [Order article via Infotrieve]
  12. Roche, J. P., Anantharam, V., and Treistman, S. N. (1995) FEBS Lett. 371, 43-46 [CrossRef][Medline] [Order article via Infotrieve]
  13. Bourinet, E., Soong, T. W., Stea, A., and Snutch, T. P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1486-1491 [Abstract/Free Full Text]
  14. Stea, A., Tomlinson, W. J., Soong, T. W., Bourinet, E., Dubel, S. J., Vincent, S. R., and Snutch, T. P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10576-10580 [Abstract/Free Full Text]
  15. Perez-Reyes, E., and Schneider, T. (1995) Kidney Int. 48, 1111-1124 [Medline] [Order article via Infotrieve]
  16. Tanaka, O., Sakagami, H., and Kondo, H. (1995) Mol. Brain. Res. 30, 1-16 [CrossRef][Medline] [Order article via Infotrieve]
  17. Hullin, R., Singer-Lahat, D., Freichel, M., Biel, M., Dascal, N., Hofmann, F., and Flockerzi, V. (1992) EMBO J. 11, 885-890 [Abstract]
  18. Ellinor, P. T., Zhang, J.-F., Randall, A. D., Zhou, M., Schwarz, T. L., Tsien, R. W., and Horne, W. A. (1993) Nature 363, 455-458 [CrossRef][Medline] [Order article via Infotrieve]
  19. Cens, T., Mangoni, M. E., Richard, S., Nargeot, J., and Charnet, P. (1996) Pfluegers Arch. 431, 771-774 [CrossRef][Medline] [Order article via Infotrieve]
  20. Welling, A., Lacinova, L., Donatin, K., Ludwig, A., Bosse, E., Flockerzi, V., and Hofmann, F. (1995) Pfluegers Arch. 429, 400-411 [Medline] [Order article via Infotrieve]
  21. Witcher, D. R., De Waard, M., Liu, H., Pragnell, M., and Campbell, K. P. (1995) J. Biol. Chem. 270, 18088-18093 [Abstract/Free Full Text]
  22. Liu, H., De Waard, M., Scott, V. E. S., Gurnett, C. A., Lennon, V. A., and Campbell, K. P. (1996) J. Biol. Chem. 271, 13804-13810 [Abstract/Free Full Text]
  23. Scott, V. E. S., De Waard, M., Liu, H., Gurnett, C. A., Venzke, D. P., Lennon, V. A., and Campbell, K. P. (1996) J. Biol. Chem. 271, 3207-3212 [Abstract/Free Full Text]
  24. Striessnig, J., Haase, H., Safayhi, H., Roenfeldt, M., Ammon, H. P. T., Morano, I., Cassidy, T. N., and Ahlijanian, M. K. (1996) Biophys. J. 70, 362 (abstr.)
  25. Striessnig, J., Glossmann, H., and Catterall, W. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9108-9112 [Abstract]
  26. Haase, H., Karczewski, P., Beckert, R., and Krause, E. G. (1993) FEBS Lett. 335, 217-222 [CrossRef][Medline] [Order article via Infotrieve]
  27. Castellano, A., Wei, X., Birnbaumer, L., and Perez-Reyes, E. (1993) J. Biol. Chem. 268, 12359-12366 [Abstract/Free Full Text]
  28. Ruth, P., Roehrkasten, A., Biel, M., Bosse, E., Regulla, S., Meyer, H. E., Flockerzi, V., and Hofmann, F. (1989) Science 245, 1115-1118 [Medline] [Order article via Infotrieve]
  29. Glossmann, H., and Ferry, D. R. (1985) Methods Enzymol. 109, 513-550 [Medline] [Order article via Infotrieve]
  30. Moebius, F. F., Hanner, M., Knaus, H.-G., Weber, F., Striessnig, J., and Glossmann, H. (1994) J. Biol. Chem. 269, 29314-29320 [Abstract/Free Full Text]
  31. Pichler, M., Wang, Z., Grabner-Weiss, C., Reimer, D., Hering, S., Grabner, M., Glossmann, H., and Striessnig, J. (1996) Biochemistry 35, 14659-14664 [CrossRef][Medline] [Order article via Infotrieve]
  32. Westenbroek, R. E., Ahlijanian, M. K., and Catterall, W. A. (1990) Nature 347, 281-284 [CrossRef][Medline] [Order article via Infotrieve]
  33. Hell, J. W., Westenbroek, R. W., Warner, C., Ahlijanian, M. K., Prystay, W., Gilbert, M. M., Snutch, T. P., and Catterall, W. A. (1993) J. Cell Biol. 123, 949-962 [Abstract]
  34. Mitterdorfer, J., Froschmayr, M., Grabner, M., Striessnig, J., and Glossmann, H. (1994) FEBS Lett. 352, 141-145 [CrossRef][Medline] [Order article via Infotrieve]
  35. Suh-Kim, H., Wei, X., and Birnbaumer, L. (1996) Mol. Pharmacol. 50, 1330-1337 [Abstract]
  36. Thibault, O., and Landfield, P. W. (1996) Science 272, 1017-1020 [Abstract]
  37. Striessnig, J., and Glossmann, H. (1991) Methods Neurosci. 4, 210-229

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.