(Received for publication, February 27, 1995)
From the
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 Voltage-dependent Ca Recently, it has been shown
that the
Figure 1:
Structural importance of the tyrosine
in the
Figure 2:
Association of
Immunoblot
analysis with affinity-purified polyclonal antibodies against the
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
Figure 3:
Association of
Figure 4:
Identification of
Figure 5:
Identification of
In order to determine the
protein expression of the four
Figure 6:
Distribution of
Figure 7:
Identification of
Figure 8:
Purification of
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
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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.
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) .
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.
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
The Subunits
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
Polyclonal antibodies against specific peptides
to Subunits
,
,
,
, 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
Site-directed
mutagenesis was performed on the GST fusion protein containing the
AIDEpitope as a GST Fusion Protein
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
GST, AID Subunit with the AID
Fusion Protein-Sepharose Beads
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
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 Subunit from Skeletal Muscle
Triads
-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).
Effect of Mutations in Tyrosine 392 of the AID on
To examine
the specific interaction between the -
Subunit Interactions
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.
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.
In order to
investigate the ability of Subunits from Skeletal Muscle Extracts Can Interact
with the AID
-Sepharose Beads
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.
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.
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.
, 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) .
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
With the use of the affinity bead assay, the
identity of Subunits Which Bind to the
AID
-Sepharose Beads in Cardiac Muscle
Extracts
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.
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
Fig. 5illustrates the results of the
AID Subunits from
Brain Extracts
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.
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.
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.
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
To determine the existence of Subunits from
Skeletal and Brain Tissue
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.
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
AID Subunits from Skeletal
Muscle
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).
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.
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.
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.
We thank C. Gurnett, D. Jung, and V. Scott for their
helpful comments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.