©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Association of Native Ca Channel Subunits with the Subunit Interaction Domain (*)

(Received for publication, February 27, 1995)

Derrick R. Witcher (§) Michel De Waard Hongyan Liu (1) Marlon Pragnell (1) Kevin P. Campbell (¶)

From the Howard Hughes Medical Institute, Department of Physiology and Biophysics Program in Neuroscience, University of Iowa College of Medicine, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Subunits of voltage-dependent Ca channels play an important role in regulating Ca channel function. The sites of - subunit interaction have been localized recently to cytoplasmic domains of both subunits. The subunit interaction domain (AID) is an 18-amino-acid conserved motif located between repeats I and II on all subunits which is essential for the binding of subunits. In order to further study the interaction of subunits with AID, we have expressed a 50-amino-acid glutathione S-transferase (GST) fusion protein from the subunit that contains the AID. Mutant GST fusion proteins that contain a single amino acid change (Y392S, Y392F, and Y392W) in the AID along with control GST were coupled to glutathione-Sepharose beads to form affinity beads. Binding assays using these affinity beads with in vitro synthesized S-labeled and subunits demonstrate that the hydroxyl group on tyrosine 392 of AID is critical for binding to subunits. The affinity bead assay was also used to identify and characterize native subunits from detergent extracts of different tissues. The AID affinity beads, but not the control or Y392S beads, specifically bind subunits from detergent extracts of skeletal muscle, cardiac muscle, and brain. Immunoblot analyses demonstrate the presence of in skeletal muscle, and in cardiac muscle, and , , and in brain. The assays also demonstrate the AID beads bind to subunits from tissue homogenates extracted with low salt and no detergent suggesting the existence of a pool of subunits which is not always associated with subunits. Also, subunits from solubilized skeletal muscle triads can be affinity-purified using AID CNBr-Sepharose. Our data demonstrate that the AID binds to native subunits from detergent and non-detergent tissue extracts illustrating that this domain on the subunit is the major anchoring site for the subunit.


INTRODUCTION

Voltage-dependent calcium channels play a critical role in the regulation of many cellular processes. A number of classes (T-, L-, N-, and P-type) of voltage-sensitive Ca channels have been identified and distinguished on the bases of their electrophysiological and pharmacological properties(1, 2, 3) . The L-type voltage-sensitive Ca channel is involved in excitation-contraction coupling in skeletal and cardiac muscle while the T-type voltage-sensitive Ca channel has been shown to be involved in pacemaker activity. The N-type (-conotoxin GVIA receptor) and P-type voltage-sensitive Ca channels are found in both central and peripheral neurons and play an essential role in controlling neurotransmitter release from these neurons(4) . In addition to the regulation of transmitter release, the N-type Ca channel also helps direct the migration of immature neurons(5) . Both the skeletal muscle L-type and the neuronal N-type Ca channels have been purified, and each of these Ca channels contain common subunits (, , and ) and one variable subunit ( and 95-kDa) (6, 7) . The and subunits have been shown to associate with the purified dihydropyridine receptor and -conotoxin GVIA receptor, respectively. Biochemical studies have also demonstrated that the subunit is tightly associated with the subunit in Ca channel complexes. Sequence analysis of subunits have illustrated a lack of transmembrane segments in these proteins which suggest that these subunits are entirely cytoplasmic. Subunits are also substrates for numerous protein kinases (8) and are thus regulated by intracellular signaling.

Voltage-dependent Ca channel subunits have now been divided into several classes based on their molecular properties. The pore-forming subunit, , has been separated into six classes, S, A, B, C, D, and E, while the subunit has been separated into four classes; , , , and (9) . Also, other alternatively spliced products from these genes have been identified(10, 11, 12) . So far, only one single gene seems to encode the subunit. Electrophysiological studies have demonstrated the importance of both the and subunits on the functional expression of subunits (13, 14, 15, 16, 17) . These subunits regulate the expression of the subunit by inducing a conformational change in the structure of the subunit and/or by targeting the Ca channel to the membrane(18, 19) .

Recently, it has been shown that the subunit of voltage-dependent Ca channels binds to a cytoplasmic sequence that is located between the first and second hydrophobic repeats of all subunits(16) . This site has been named the alpha 1 subunit interaction domain or AID()(20) . The AID site encompasses an 18-amino-acid sequence of which 9 amino acids are conserved. The corresponding interaction site on the subunit has also been identified(17) . This site, a 30-amino-acid N-terminal region of the second conserved domain of the subunit, has been named the beta subunit interaction domain or BID. The sequence of the BID is also very conserved between the different subunit isoforms. It has been demonstrated by several point mutations of conserved amino acids in both epitopes that the AID and BID are required for the binding of subunits and also the changes in activation kinetics, voltage dependence of activation and inactivation, and the increase in peak currents. Since both the AID and BID are necessary for the - subunit interaction, an affinity bead assay has been developed to probe this subunit-subunit interaction(20) . The results reported here further characterize the molecular interaction between the subunit and subunit. We also demonstrate that native subunits from various tissue extracts can be identified and purified with the use of AID affinity beads.


EXPERIMENTAL PROCEDURES

Materials

The TNT-coupled reticulocyte lysate system was purchased from Promega. Isopropyl-1-thio--D-galactopyranoside is from Life Technologies, Inc., reduced glutathione from U. S. Biochemical Corp., [S]methionine from Amersham, and glutathione-Sepharose and protein G-Sepharose from Pharmacia Biotech. Horseradish peroxidase-conjugated secondary antibodies were from Boehringer Mannheim. All other chemicals were of reagent grade.

Membrane Preparation

Triads from rabbit skeletal muscle were purified as described previously(21) .

SDS-PAGE and Immunoblot Analysis

Proteins analyzed by SDS-PAGE (3-12% gradient gels) used the buffer system of Laemmli (22) . Gels were stained with Coomassie Blue or transferred to nitrocellulose for immunoblot analysis as described previously(23) .

In Vitro Translation of Subunits

The S-labeled subunits were synthesized using the TNT system. Two cDNA clones were used: (GenBank accession number X64297) and (M88751). The concentrations of the subunits were determined by trichloroacetic acid precipitation followed by scintillation counting.

Production of Specific Polyclonal Antibodies to Different Subunits

Polyclonal antibodies against specific peptides to , , , , and were produced as described previously(24) . Specific rabbit polyclonal antibodies (Rabbit 62) were produced against a 15-amino-acid peptide (DLEEDEAELGEQSGS) of the insert region of the subunit(25) , and polyclonal antibodies (Rabbit 63) were generated against the C-terminal 15-amino-acid peptide (NKNELEGWGQGVYIR) of the subunit(26) . Specific rabbit polyclonal antibodies (Rabbits 86 and 87) were produced against the C-terminal 15-amino-acid peptide (RSEAGEWNRDVYIRQ) of the subunit(27, 28) . Rabbit polyclonal antibodies (Rabbit 95) were generated against the C-terminal 15-amino-acid peptide (DRNWQRNRPWPKDSY) of the subunit (27, 29) , and polyclonal antibodies (Rabbit 127) were produced against the C-terminal amino acid peptide (RGSPGGCSHDSRHRL) of the subunit(30) . All of the rabbit polyclonal antibodies were affinity-purified from Immobilon-P transfer strips of each 15-amino-acid peptide conjugated to bovine serum albumin as described previously(31) . Also, and polyclonal antibodies were affinity-purified from Guinea Pig 48 (GP 48) (32) using Immobilon-P transfer strips of the and subunits of the skeletal muscle dihydropyridine receptor, respectively. Biotinylated affinity-purified antibodies were used in the Western blots of cardiac tissue. The biotinylation of affinity-purified antibodies were performed according to standard protocol (Amersham).

Site-directed Mutagenesis of the Epitope as a GST Fusion Protein

Site-directed mutagenesis was performed on the GST fusion protein containing the AID using the Transformer site-directed mutagenesis system (Clontech) as described previously(16) . The following primers were used to generate the mutant GST fusion proteins: 5`-CGAGCTCAACGGGTCCATGGAGTGGATCTCAAAAGC-3` (Y392S), 5`-CGAGCTCAACGGGTTCATCGAGTGGATCTCAAAAGC-3` (Y392F), and 5`-CGAGCTCAACGGGTGGATGGAGTGGATCTCAAAAGC3` (Y392W). The mutations were verified by sequence analysis.

Purification of Glutathione S-Transferase (GST) Fusion Proteins

Large bacteria cultures (300 ml) containing the pGEX plasmids (33) encoding for control and AID fusion proteins (wild-type and mutant sequences containing the subunit motif that binds the subunits) (16) were transformed into Escherichia coli DH5 cells. Overnight cultures were grown and induced according to standard procedures(23) . The cell cultures were spun down and resuspended in PBS containing 1% Triton X-100 and sonicated twice for 15 s. The sonicated material was centrifuged at 12,000 rpm for 15 min in a Beckman JA-17 rotor. The supernatant of the control GST fusion protein was incubated with glutathione-Sepharose beads. The glutathione-Sepharose beads were washed extensively with PBS, and the fusion proteins were then eluted with 50 mM Tris (pH 8.0) containing 10 mM glutathione. The procedure was more complex for the wild-type and mutant AID GST fusion proteins since >90% of these fusion proteins were insoluble in PBS containing 1% Triton X-100. For these GST fusion proteins, the pellets from the first centrifugation were resuspended in 10 ml of PBS containing 1% Sarcosyl (Sigma). After incubation for 30 min at 4 °C, the insoluble material was removed by centrifugation as described above. Triton X-100 was added to the supernatants to a final concentration of 2% and incubated with glutathione-Sepharose beads for 30 min at 4 °C. Again, the glutathione-Sepharose beads were washed extensively with PBS, and the fusion proteins were eluted with 10 mM glutathione in 50 mM Tris, pH 8.0.

Association of the Subunit with the AIDFusion Protein-Sepharose Beads

GST, AID GST fusion protein, and mutant AID GST fusion proteins were expressed and purified using glutathione-Sepharose as described above. To prepare the tissue homogenates used in the association assay, rabbit skeletal muscle, cardiac muscle, and brain tissues were homogenized in a blender (Osterizer Cycle) in a 1:5 ratio (w/v) in the homogenization buffer containing 50 mM Tris (pH 7.5), 1 M NaCl, 1% CHAPS, and several protease inhibitors: aprotinin (76.8 nM), benzamidine (0.83 mM), leupeptin (1.1 µM), pepstatin A (0.7 µM), and phenylmethylsulfonyl fluoride (0.23 mM) three times for 30 s. The tissue homogenates were incubated for 2 h at 4 °C and then centrifuged at 35,000 rpm for 35 min in a 45 Ti rotor at 4 °C. The supernatants were removed and diluted 1 to 10 in a dilution buffer containing 50 mM Tris (pH 7.5) with protease inhibitors as described above. The diluted supernatants were then incubated on ice for 1 h and centrifuged at 35,000 rpm for 35 min in a 45 Ti rotor at 4 °C to remove any precipitated material. The solubilized homogenates were subsequently incubated with the GST fusion proteins conjugated to Sepharose beads overnight. After the incubation, the Sepharose beads were washed extensively with 50 mM Tris (pH 7.4), 100 mM NaCl, 0.1% CHAPS, and protease inhibitors. Proteins bound to the Sepharose beads were analyzed by SDS-PAGE and immunoblotting. Similar experiments were also performed by homogenizing rabbit skeletal and brain tissues with a buffer that contained 50 mM Tris (pH 7.5), 100 mM NaCl, protease inhibitors, and no detergent. These homogenates were also incubated with GST, wild-type AID GST, or mutant AID GST fusion proteins conjugated to Sepharose beads overnight, washed extensively, and analyzed by SDS-PAGE and immunoblotting.

Covalent Coupling of Purified GST Fusion Proteins to CNBr-Sepharose Beads

Purified GST, AID GST fusion protein, and mutant AID GST fusion protein were coupled to CNBr-activated Sepharose beads in 100 mM NaHCO (pH 8.3), 500 mM NaCl for 3 h at room temperature and at a protein concentration of approximately 0.5 mg/ml resin. Each resin was washed four times with PBS, and the remaining active groups on the beads were blocked with 200 mM glycine (pH 8.0) for 3 h at room temperature. The resin was stored at 4 °C in PBS and 0.001% sodium azide.

Purification of the Subunit from Skeletal Muscle Triads

50 mg of skeletal muscle triads were solubilized at a protein concentration of 4 mg/ml in a buffer containing 50 mM Tris (pH 7.5), 1 M NaCl, and 1% CHAPS for 1 h at 4 °C. The mixture was spun at 35,000 rpm for 35 min in a 45 Ti rotor at 4 °C. The supernatant was diluted 10-fold with 50 mM Tris (pH 7.5) and was circulated over a 5-ml AID-Sepharose column for 6 h at 4 °C. The column was washed extensively with 50 mM Tris (pH 7.5), 150 mM NaCl, and 0.1% CHAPS, and the skeletal muscle subunit was eluted from the AID-Sepharose column with 2-ml fractions of buffer containing 50 mM glycine (pH 2.4), 150 mM NaCl, and 0.1% CHAPS. The fractions were neutralized immediately with 1 M MOPS (pH 7.6).


RESULTS AND DISCUSSION

Effect of Mutations in Tyrosine 392 of the AID on - Subunit Interactions

To examine the specific interaction between the and subunits, we have developed an in vitro binding assay(20) . In this affinity assay, the AID was expressed as a 50-amino-acid GST fusion protein and used as a ligand for in vitro translated and native subunits. To characterize the interaction of the AID with different subunits, we examined the ability of several AID GST fusion proteins, each containing point mutations at tyrosine 392, to bind to different subunits (Fig. 1A). Tyrosine 392 is one of several residues previously shown to be important for the - subunit interaction. These three mutant GST fusion proteins, AID, AID, and AID, along with GST were coupled to glutathione-Sepharose beads to form affinity beads. Both in vitro translated S-labeled and subunits were used as the substrate for the affinity binding assay (Fig. 1B). The results of the binding assay show that, compared to the wild-type AID GST fusion protein, the binding of the three mutant AID fusion proteins to both S-labeled and were dramatically reduced. Similar to what was seen with an overlay assay with S-labeled (16) , a 250 nM concentration of the mutant AID bound less than 5% of both S-labeled and subunits. Consistent with the idea that this tyrosine is critical for the - interaction, the affinity beads containing a 250 nM concentration of the mutant AID bound only 9% and 6% of the S-labeled and subunits, respectively. Also, affinity beads containing a 250 nM concentration of the mutant AID bound 20% and 30% of the S-labeled and subunits, respectively. These results demonstrate that the mutations in tyrosine 392 had a profound effect on the ability of both S-labeled and to bind to the mutant AID affinity beads. Furthermore, the dramatic decrease in the affinity between the subunit and the mutant AID affinity beads suggests that the hydroxyl group in tyrosine 392 plays an important role in the molecular interaction between the and subunits in voltage-dependent Ca channels.


Figure 1: Structural importance of the tyrosine in the subunit interaction domain (AID). A, diagram of the mutations Y392S, Y392F, and Y392W in the subunit interaction domain. The amino acid sequence of AID GST fusion protein is illustrated with a box around the AID. The conserved amino acids in the epitope are in filled lettering. B, S-labeled and subunits bound to 250 nM AID, AID, and AID expressed as a percentage of the two in vitro translated subunits bound to 250 nM wild-type AID-Sepharose beads.



Subunits from Skeletal Muscle Extracts Can Interact with the AID-Sepharose Beads

In order to investigate the ability of subunits from solubilized skeletal muscle triads to bind the AID, this epitope was expressed as a 50-amino-acid GST fusion protein. The AID GST fusion protein was coupled to glutathione-Sepharose beads and used as the substrate for the binding of native subunits in solubilized skeletal muscle triads. A mutant GST fusion protein, AID, which contains a single amino acid change in the AID, and GST were also produced and coupled to glutathione-Sepharose beads to form affinity beads. The interaction between the mutant, AID, and the subunit was shown to be greatly reduced using an overlay assay or in expression studies with Xenopus oocytes(16) . We have previously demonstrated the specificity of the AID bead assay with in vitro translated S-labeled subunits(20) . Fig. 2illustrates that AID-Sepharose beads are capable of binding subunits from solubilized skeletal muscle triads. Immunoblot analysis with affinity-purified polyclonal antibodies against the subunit of the skeletal muscle dihydropyridine receptor from GP 48 (32) revealed that the subunit was present only in solubilized skeletal muscle triads and void of the AID bead assay but not present with the AID-Sepharose affinity beads (Fig. 2A). These results suggest that the AID-Sepharose beads bind only to subunits that are not associated with in native tissues confirming the role of AID as the primary anchoring site.


Figure 2: Association of subunits from CHAPS-solubilized skeletal muscle triads with the AID affinity beads. A, an immunoblot of CHAPS-solubilized skeletal muscle triads, 75 µl (Homo.); skeletal muscle triads after incubation with 30 µl of AID-Sepharose beads (Void); and 30 µl of GST-Sepharose beads (GST), 30 µl of AID fusion protein-Sepharose beads (AID), and 30 µl of the mutant Y392S AID fusion protein (AID) after incubation with skeletal muscle triads overnight at 4 °C. This immunoblot was stained with affinity-purified polyclonal antibodies to the subunit from guinea pig 48 (GP 48)(33) . The arrow indicates the skeletal muscle subunit. B, an immunoblot containing the same samples as A stained with affinity-purified polyclonal antibodies to the subunit from GP 48. The arrow indicates the skeletal muscle subunit. Molecular mass standards ( 10) are indicated on the left.



Immunoblot analysis with affinity-purified polyclonal antibodies against the subunit from GP 48 also showed that the AID-Sepharose beads were able to bind the subunit from the solubilized skeletal muscle preparation. The affinity-purified polyclonal antibodies identified a doublet at a molecular mass of 52 kDa and a minor band at 71 kDa. Alkaline phosphatase treatment of the subunit bound to the AID-Sepharose beads demonstrated that the upper band of the doublet was a phosphorylated form of the subunit (data not shown). The minor band at a molecular mass of 71 kDa was identified by two other -specific polyclonal antibodies, but not affinity-purified antibodies on immunoblots, suggesting that this protein is a splice variant of the gene. It is also possible that this protein is an alternatively spliced form of another known or unidentified subunit. This protein is not detected in the purified skeletal muscle dihydropyridine receptor which suggests that this subunit may associate with a different subunit present in skeletal muscle or a peripheral nerve contaminant in our skeletal muscle preparation. Both GST and the mutant AID-Sepharose beads did not bind any subunits (Fig. 2B). It has been shown that the AID site anchors the subunit to the subunit(16, 17) . Recent experimental evidence suggests the existence of other sites of interaction between the subunit and the subunit(34) . Our results show that when native subunits are associated with the AID no subunits can bind to the AID- subunit complex. This demonstrates that when the primary AID-BID sites are occupied, subunits cannot bind with high affinity to sites other than the BID on subunits. Thus, our data strongly support the idea that the interaction site identified by Pragnell et al.(16) and De Waard et al.(17) are the only major high affinity sites that anchor the subunit to the subunit and that other potential interaction sites in subunits are low affinity and more labile.

Affinity bead assays were also performed on detergent-solubilized skeletal muscle homogenates. SDS-PAGE of the proteins bound to the affinity beads illustrates that the glutathione-Sepharose beads contained equal amounts of GST, AID, and AID fusion proteins (Fig. 3A). The AID-Sepharose beads, but not the GST or mutant AID-Sepharose beads, specifically bound subunit from the skeletal muscle extract. The subunit was detected by Western blot analysis using affinity-purified polyclonal antibodies from Rabbit 62 (Fig. 3B). These polyclonal antibodies were directed against the insert region of the subunit. The arrow in Fig. 3denotes the location of the subunit on the Coomassie Blue-stained polyacrylamide gel and immunoblot. The subunit present in the crude homogenate and void was almost undetectable by Western blot analysis (Fig. 3B) demonstrating the enrichment of the subunit with the AID-Sepharose affinity beads. Again, the affinity-purified polyclonal antibodies identified a splice variant of a subunit which migrated at the molecular mass of 71 kDa. The 71-kDa subunit was only detectable with the AID-Sepharose affinity beads and not in the skeletal muscle homogenate. Polyclonal antibodies against other subunit isoforms (, , and ) did not detect the presence of these isoforms on similar immunoblots. These immunoblot results parallel the results of Northern blot analysis which have shown only the transcript present in skeletal muscle(25) .


Figure 3: Association of subunits from CHAPS-solubilized skeletal muscle homogenate with the AID affinity beads. A, Coomassie Blue (CB)-stained polyacrylamide gel of skeletal muscle homogenate, 75 µl (Homo.); skeletal muscle homogenate after incubation with 30 µl of AID-Sepharose beads (Void); and 30 µl of GST-Sepharose beads (GST), 30 µl of AID fusion protein-Sepharose beads (AID), and 30 µl of the mutant Y392S AID fusion protein (AID) after incubation with skeletal muscle homogenate overnight at 4 °C as described under ``Experimental Procedures.'' B, an immunoblot stained with affinity-purified polyclonal antibody Rabbit 62 against a 15-amino-acid peptide located in a unique insert region of subunit (). The arrow indicates the skeletal muscle subunit. Molecular mass standards ( 10) are indicated on the left.



Identification of Subunits Which Bind to the AID-Sepharose Beads in Cardiac Muscle Extracts

With the use of the affinity bead assay, the identity of subunits present in CHAPS detergent cardiac muscle extract which bound to the AID-Sepharose beads was determined. Fig. 4A illustrates that the glutathione-Sepharose beads contained equal amounts of GST, AID, and AID fusion proteins when analyzed on SDS-PAGE and stained with Coomassie Blue. Immunoblot analysis of nitrocellulose blots transferred from similar polyacrylamide gels and stained with affinity-purified polyclonal antibodies to other subunit isoforms (, , , and ) demonstrated that both and were present in cardiac tissue and bound to the AID-Sepharose beads (Fig. 4B, arrows). Immunoblot analysis did not detect or subunits using this affinity bead assay suggesting that these subunits are not expressed in heart. Our data are in accordance with Northern blot analysis that shows the expression of both and subunit mRNAs in cardiac muscle(27, 28) . These results demonstrate that both the and subunits are present at the protein level in cardiac tissue and are consistent with the idea that could be associated with the subunit in cardiac muscle. Whether the subunit is also associated with the or some other subunit isoform in cardiac muscle remains to be investigated.


Figure 4: Identification of subunits which bind to AID affinity beads from CHAPS-solubilized cardiac muscle homogenate. A, Coomassie Blue (CB)-stained polyacrylamide gel of 30 µl of GST-Sepharose beads (GST), 30 µl of AID fusion protein-Sepharose beads (AID), and 30 µl of the mutant Y392S fusion protein (AID) after incubation with cardiac muscle homogenate overnight at 4 °C. B, immunoblots stained with affinity-purified polyclonal antibodies Rabbit 86 and Rabbit 95 against a 15-amino-acid C-terminal peptide of () and (), respectively. The arrows indicate the location of and . Molecular mass standards ( 10) are indicated on the left.



Identification and Distribution of Subunits from Brain Extracts

Fig. 5illustrates the results of the AID affinity bead assay from whole brain extracts. The glutathione beads used in the assay contained approximately equivalent amounts of GST, AID GST fusion protein, and the mutant AID GST fusion protein as demonstrated by the Coomassie Blue-stained polyacrylamide gel (Fig. 5A). Fig. 5B shows that C-terminal affinity-purified polyclonal antibodies from Rabbit 95 and Rabbit 127 identified and , respectively, on immunoblot analysis of the AID-Sepharose affinity beads. Again, GST and AID-Sepharose beads did not bind detectable amounts of subunits from the extracts. Both and could not be detected by immunoblot analysis using this procedure. Although both transcripts have been shown to be present in brain(26, 27, 28) , the level of protein expression of these two subunits may have made these subunits difficult to detect with this affinity bead assay. Also, the differences seen between the Northern blot analysis and the level of protein detected by this method could be due to spatial and/or temporal differences in subunit expression.


Figure 5: Identification of subunits which bind to AID affinity beads from CHAPS-solubilized brain homogenate. A, Coomassie Blue (CB) stained polyacrylamide gel of 30 µl of GST-Sepharose beads (GST), 30 µl of AID fusion protein-Sepharose beads (AID), and 30 µl of the mutant Y392S fusion protein (AID) after incubation with brain homogenate overnight at 4 °C. B, immunoblots stained with affinity-purified polyclonal antibodies Rabbit 95 and Rabbit 127 against a 15-amino-acid C-terminal peptide of () and (), respectively. The arrows indicate the location of and . Molecular weight masses ( 10) are indicated on the left.



In order to determine the protein expression of the four subunit isoforms in different regions of the rabbit brain, AID-Sepharose bead assays were performed on tissue extracts from the cerebellum, cortex, hippocampus, basal ganglia, and brain stem. The same amount of tissue (2.5 g) from each brain region was homogenized with the extraction buffer containing detergent. With the use of C-terminal affinity-purified polyclonal antibodies from Rabbit 63, immunoblot analysis revealed that (83 kDa) was present in both the hippocampus and cortex extracts, very weakly in cerebellum, but absent or undetectable in the basal ganglia and brain stem (Fig. 6). The protein expression level was higher in the hippocampus than in the cortex for the subunit. The levels of protein expression of this subunit parallel exactly the level of the mRNA for this subunit on Northern blot analysis(26) . Immunoblot analysis of the AID-Sepharose bead from whole brain extracts did not detect the subunit (Fig. 5). This suggests that in whole brain extracts the subunit is below the detection limit for the affinity bead assay. Immunoblot analysis of the subunits bound to the AID-Sepharose affinity beads from different brain regions also demonstrated that both and subunits were expressed in all brain regions. The subunit appeared to be expressed at a higher level throughout the brain compared to both and subunits. Although C-terminal affinity-purified polyclonal antibodies against were also used on a similar immunoblot, no staining was detected in any of the different brain regions. This suggests that the subunit is either absent or at levels that are below detection using this assay throughout the rabbit brain.


Figure 6: Distribution of subunits in brain regions. , an immunoblot of 30 µl of AID fusion protein-Sepharose beads after incubation with whole brain homogenate (WB) and homogenates from different regions of the brain: cerebellum (Cereb.), cortex (Cort.), hippocampus (Hipp.), basal ganglia (Bas. G.), and brain stem (BS) stained with affinity-purified polyclonal antibody Rabbit 63 against the 15-amino-acid C-terminal peptide of . , an immunoblot stained with affinity-purified polyclonal antibody Rabbit 95. , an immunoblot stained with affinity-purified polyclonal antibody Rabbit 127. The arrows indicate the location of , , and . Molecular mass standards are indicated on the left.



Identification of Nonassociated Subunits from Skeletal and Brain Tissue

To determine the existence of subunits that were not assembled into voltage-sensitive Ca channel complexes, AID-Sepharose bead assay was performed with tissue homogenates which were extracted using a low salt buffer (100 mM NaCl) and no detergent. As we have demonstrated in the previous experiments, the AID in the subunit is capable of binding native subunits from skeletal muscle, cardiac muscle, and brain. However, the buffer used to extract the different tissues contained high salt and CHAPS. While the disassociation of in vitro translated S-labeled subunits bound to the AID GST fusion protein could be partially induced (10%) by these extraction conditions, even milder conditions, low salt and no detergent, were used in the present tissue extractions. Fig. 7A illustrates that the AID-Sepharose affinity beads bound to native nonassociated subunits that were present in this skeletal muscle extract using immunoblot analysis with affinity-purified polyclonal antibodies from Rabbit 62. The AID-Sepharose beads also bound the subunit (58 kDa) from low salt brain extracts (Fig. 7B). Again, demonstrating the specificity of the bead assay, both GST and AID-Sepharose beads did not bind the subunits in the tissue extracts. The and subunits were not detected in the homogenates and voids (approximately 100 µg of each analyzed on SDS-PAGE) of the bead assay. These results indicate that there are subunits which are not tightly complexed with the subunits. Coexpression studies with the and subunits in Xenopus oocytes have shown that the subunit modulates the properties of the subunit by enhancing the current amplitude and altering voltage dependence and kinetics of the resulting Ca channels(13, 14, 15, 16, 17) . These studies have shown that subunits which are tightly associated with subunits play an important role in modulating the properties of the pore-forming subunit. Nonassociated subunits may also play a role in Ca homeostasis. It is possible that nonassociated subunits could be shuttled to the membrane to bind with subunits upon electrical or chemical stimulation of the membrane or by the result of intracellular signaling events such as protein phosphorylation. These subunits would then associate with subunits in the plasma membrane to increase their Ca channel current amplitudes and alter their voltage dependence and kinetics.


Figure 7: Identification of subunits from non-detergent tissue homogenates. A, an immunoblot of non-detergent skeletal muscle homogenate, 20 µl (Homo.); skeletal muscle homogenate after incubation with 30 µl of AID-Sepharose beads (Void); and 30 µl of GST-Sepharose beads (GST), 30 µl of AID fusion protein-Sepharose beads (AID), and 30 µl of the mutant Y392S AID fusion protein (AID) after incubation with skeletal muscle homogenate overnight at 4 °C as described under ``Experimental Procedures.'' This immunoblot was stained with affinity-purified polyclonal antibodies to the subunit from Rabbit 62 (Skeletal). B, an immunoblot of non-detergent brain homogenate, 20 µl (Homo.); brain homogenate after incubation with 30 µl of AID-Sepharose beads (Void); and 30 µl of GST-Sepharose beads (GST), 30 µl of AID fusion protein-Sepharose beads (AID), and 30 µl of the mutant Y392S AID fusion protein (AID) after incubation with brain homogenate overnight as described previously. The arrow indicates the skeletal muscle subunit. Molecular mass standards ( 10) are indicated on the left.



Affinity Purification of Subunits from Skeletal Muscle

AID CNBr-Sepharose beads were used to purify subunits from solubilized skeletal muscle triads. In Fig. 8A, SDS-PAGE demonstrated that a protein of a molecular mass of 52 kDa eluted from the AID CNBr-Sepharose column at low pH in fractions 9 and 10. Immunoblot analysis with affinity-purified polyclonal antibodies from Rabbit 62 confirmed the identification of the 52-kDa protein as the subunit (Fig. 8B). With the use of the AID affinity assay, the purified subunit retains its ability to bind to the AID-Sepharose affinity beads but not the mutant AID-Sepharose beads (data not shown).


Figure 8: Purification of subunits from skeletal muscle using AID CNBr-Sepharose chromatography. A, a Coomassie Blue-stained polyacrylamide gel of 100-µl aliquots of the column void (Void) and 2-ml fractions of washes (fractions 1-6) and elutions (fractions 7-11) of a 5-ml AID CNBr-Sepharose column with 20 mM glycine and 150 mM NaCl (pH 2.5) as described under ``Experimental Procedures'' (CB). B, an immunoblot stained with affinity-purified rabbit polyclonal antibody against the internal 15-amino-acid peptide of the skeletal muscle subunit (Rab 62). The arrow indicates the skeletal muscle subunit. Molecular mass standards ( 10) are indicated on the left.



In conclusion, our results demonstrate that both nonconservative and conservative mutations in the tyrosine residue of the AID have a profound effect on the ability of the AID to bind to subunits. Deletion of the aromatic ring containing the hydroxyl group disrupted the subunit-subunit interaction. Furthermore, a deletion of only the hydroxyl group on the tyrosine had a dramatic effect on subunits binding to the AID, suggesting that this hydroxyl group may play an important role in the molecular interactions between and subunits. Our results also illustrate that the AID binds to native subunits in both detergent and non-detergent tissue extracts. This not only provides a unique method of characterizing different subunits from various tissues but also suggests that subunits may not always be tightly associated with subunits.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Fellowship T32-AI07343 from the National Institutes of Health.

Investigator of the Howard Hughes Medical Institute. To whom correspondence and reprint requests should be addressed: Howard Hughes Medical Institute, University of Iowa College of Medicine, 400 Eckstein Medical Research Bldg., Iowa City, IA 52242. Tel.: 319-335-7867; Fax: 319-335-6957; kevin-campbell{at}uiowa.edu

The abbreviations used are: AID, subunit interaction domain; BID, subunit interaction domain; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.


ACKNOWLEDGEMENTS

We thank C. Gurnett, D. Jung, and V. Scott for their helpful comments.


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