(Received for publication, April 17, 1997, and in revised form, May 29, 1997)
From the Department of Molecular Pharmacology and
Biological Chemistry, Northwestern University Medical School, Chicago,
Illinois 60611 and the
Department of
Pharmacology, University of Illinois at Chicago,
Chicago, Illinois 60612
The properties of cardiac L-type channels have
been well characterized electrophysiologically, and many such studies
have demonstrated that the channels are regulated by a
cAMP-dependent pathway. However, the subunit composition of
native cardiac L-type calcium channels has not been completely defined.
Furthermore, a very important question exists regarding the status of
the C-terminal domain of the pore-forming 1
subunit, as this domain has the potential to be the target of protein
kinases but may be truncated as a result of post-translational
processing. In the present studies, the
1C and
2 subunits were identified by subunit-specific
antibodies after partial purification from heart membranes, or
immunoprecipitation from cardiac myocytes. Both the
2
and the full-length
1C subunits were found to be
expressed and co-localized in intact cardiac myocytes along T-tubule
membranes. Using a quantitative antibody binding analysis, we
demonstrated that the majority of the
1C subunits in
intact cardiac myocytes appear to be full-length. In addition, we
observed that adenylyl cyclase is localized in a pattern similar to the
channel subunits in cardiac myocytes. Taken together, our results
provide new insights into the structural basis for understanding the
regulation of L-type calcium channels by a cAMP-mediated signaling
pathway.
The regulation of ion channels by protein phosphorylation and
dephosphorylation is a common theme in neurobiology. One of the most
extensively studied examples involves the cAMP-mediated regulation of
the voltage-activated L-type calcium channel in heart. The activation
of -adrenergic receptors by norepinephrine facilitates the opening
of cardiac L-type channels and regulates cardiac contractility through
a protein kinase A (PKA)1 -mediated phosphorylation of the
channels or related proteins (1, 2).
Although many electrophysiological studies have centered on this
important regulatory pathway, very little is known about the
biochemical properties of the rare membrane proteins that comprise the
channels and the molecular events that underlie their regulation.
Important issues need to be resolved to fully understand the regulatory
processes that occur in this prototypical system. An essential first
step is to understand the subunit composition of cardiac calcium
channels. Most voltage-activated calcium channels are multisubunit
complexes composed minimally of pore-forming 1 subunits
along with accessory
and
2
subunits. Earlier
studies have demonstrated that purified L-type calcium channels contain an
1 subunit and the universal
2
subunit (3, 4). Subsequently, cDNA cloning predicted the
1C isoform to be part of the cardiac calcium channels
(5). While the
1C cDNA predicts a protein with a
molecular mass of 242 kDa (5), L-type channel proteins purified from
avian and mammalian heart contained
1 subunits of
190-200 kDa (3, 6). Recent studies have confirmed the suspicion that
the purified proteins were
1C subunits that were truncated at the C terminus (7). Similar C-terminal truncations have
been identified in
1 subunits from skeletal and neuronal L-type channels (8-10) and the N-type calcium channel (11). It is
important to ascertain if these truncations arise as a result of
post-translational modifications or as artifacts of isolation and
purification of the channel, since the truncations could have serious
consequences for channel function and regulation. For example, the
truncated cardiac
1C subunit is not a substrate of PKA
in vitro (3, 12), while the full-length protein is a
substrate (7, 12). In addition, a truncated
1C subunit has been shown to conduct much larger currents than the full-length protein (13). Thus, an important question is: are the
1C
subunits full-length or truncated proteins in the cardiac myocyte?
Another important unanswered question is which
-subunit(s) complex
with the
1C to form cardiac L-type channels? This
question also directly relates to the mechanism of regulation of the
channels by PKA, as several
-isoforms are predicted to be potential
substrates of PKA, while others are not. Northern analysis and
polymerase chain reaction cloning have indicated that mRNAs for
1b,
2,
3, and
4 subunits are present in cardiac muscle (14). However, no studies have been performed to demonstrate the expression and the
distribution of the
subunits at the protein level in myocytes. In
addition, the regulation of the L-type channel by PKA appears to be
self-limiting as the cAMP-generating adenylyl cyclase that is present
in cardiac myocytes appears to be negatively regulated by
Ca2+ influx through L-type channels (15). However, it is
not known if the channel subunits and adenylyl cyclase are spatially
localized in cardiac myocytes in such a way that the increased
Ca2+ resulting from calcium channel activation and/or
Ca2+-induced Ca2+ release could cause direct
inhibition of the adenylyl cyclase. To address these issues, we used
multiple channel-specific antibodies to determine whether the
1C subunit is a full-length or truncated protein in the
intact myocyte and to identify its partner
subunit. In addition, we
demonstrated the expression and the localization of the calcium channel
subunits and adenylyl cyclase in cardiac myocytes.
Adult male rabbits were purchased from commercial
suppliers. The human embryonic kidney (HEK) 293 cells were a gift from
Dr. Ron Kopito (Stanford University). A stable HEK293 cell line
expressing type V AC (HEK/ACV) was a gift from Dr. Jack
Krupinski, Weis Center for Research, Danville, PA. FITC- and
TRITC-coupled secondary antibodies were obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Monoclonal anti--actinin
(sarcomeric) (EA-53) antibodies were obtained from Sigma. All other
reagents were from standard sources.
Calcium channel
subunit-specific antibodies including Card C, Card I, 2,
and
gen were described previously (16, 17). The Card C
antibodies were purified by protein G chromatography following standard
procedures. The Card I antibody was affinity purified against
1C proteins heterologously expressed in Sf9 insect
cells.2 Crude Sf9 cell
membranes containing the
1C proteins were separated by
SDS-PAGE and transferred to nitrocellulose. The region on the blots
containing the
1C proteins were cut out and incubated
with Card I overnight at 4 °C. After extensive washing, the bound
antibody was eluted with 0.2 M glycine, pH 2.5, and rapidly
neutralized with 1 M K2HPO4. The
specificity of purified Card I was tested using expressed proteins on
immunoblots (data not shown).
Partial cDNA clones for the type V and VI adenylyl cyclase (AC)
were obtained and sequenced after screening a chicken heart library
(Stratagene). A generic AC antibody, ACcom, was generated against a chick type VI AC sequence in the C-terminal loop
corresponding to amino acids 993-1112 in the dog type VI AC (18),
which was identified by sequence alignment using LaserGene to be highly conserved in all ACs. An antibody targeted to type V and VI isozymes, ACV/VI, was generated against a sequence from the 6-7 loop
region of the chick type V AC corresponding to amino acids 547-661 in the dog type V AC (19). This sequence is highly conserved between type
V and VI isozymes but has low homology with other AC isozymes (identified by LaserGene alignment). To produce fusion proteins for
antibody production, the cDNAs encoding the chick type V and type
VI sequences noted above were generated by polymerase chain reaction of
the partial chick heart clones and subcloned into pGEX-4T3 (Pharmacia
Biotech Inc.), resulting in an in-frame fusion of the AC residues to
glutathione S-transferase. While both fusion proteins were
insoluble, they were purified by SDS-PAGE and electroelution. Briefly,
isopropyl-1-thio--D-galactopyranoside-induced bacteria were resuspended and sonicated in PBS containing 1% Triton X-100. The
insoluble protein pellet was washed three times in the same buffer to
remove soluble proteins, and the final protein pellet was solubilized
in SDS sample buffer and subjected to SDS-PAGE directly. The insoluble
fusion protein was the major protein band on the gel revealed by
Coomassie staining (data not shown). The fusion proteins were purified
by excision from the gel followed by electroelution into SDS
electrophoresis buffer (20). Antibodies to the purified fusion proteins
were prepared in goats and rabbits at Bethyl Laboratories (Montgomery,
TX). Four antibodies were obtained: rabbit ACcom, goat
ACcom, rabbit ACV/VI, and goat
ACV/VI (see "Results").
To avoid the background problem
caused by secondary antibodies in studies of low abundance proteins
when the same antibody is used for immunoblotting after
immunoprecipitation, Card I, gen, and
2
antibodies were biotinylated using an ImmunoPure NHS-LC-Biotinylation
kit (Pierce) following the standard procedures suggested by the
manufacturers.
Adult rabbit cardiac myocytes were isolated using previously described procedures (21). After isolation, myocytes were transferred to M199 media (Life Technologies, Inc.) and kept at 37 °C until used.
Membrane Preparation and Partial Purification of Calcium Channels from Rabbit HeartsCrude membranes from rabbit heart were
prepared as described previously (22) except for the inclusion of
protease inhibitors used in Ref. 16. For immunoprecipitation, membranes
were solubilized in solubilization buffer (50 mM Tris, pH
7.4, 5 mM EDTA, 5 mM EGTA, 0.1%SDS, 1% Triton
X-100 and protease inhibitors (16)) and incubated with the
gen antibody coupled to protein G. Immunoprecipitates were subjected to SDS-PAGE and channel subunits were identified by
immunoblotting. For channel purification, membranes were prelabeled with (+)-[3H]PN 200-110 (Amersham) after which channel
proteins were solubilized and partially purified with wheat germ
agglutinin (WGA)-Sepharose chromatography (Sigma) as described
previously (3). Fractions containing the peak of dihydropyridine
binding were concentrated, and the protein components of the channel
were analyzed by SDS-PAGE followed by immunoblotting.
Freshly isolated
rabbit myocytes were homogenized in buffer A (250 mM
sucrose, 0.25 M KCl, 10 mM imidazole, pH 7.4, 5 mM MgCl2, 10 mM EDTA, and protease
inhibitors (16)) using a Tri-R Dounce homogenizer. The homogenates were
centrifuged at 5,000 × g for 10 min. Pellets were
rapidly washed three times with buffer A containing 0.6 M
KCl to extract myosin. The pellets were then washed once with buffer B
(50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, and protease inhibitors), and resuspended in
buffer B containing 1% SDS. Solubilized proteins were diluted 5-fold in buffer C (buffer B containing 0.8% digitonin, 0.25% cholate, 0.2 M NaCl) and immunoprecipitated using Card I and/or the
gen antibodies coupled to immobilized protein G
(Pierce). Pellets were washed 5 times in 20 volumes of buffer C
containing 0.5 M NaCl. Immunoprecipitates were analyzed by
SDS-PAGE followed by immunoblotting. Channel subunits were detected
with enhanced chemiluminescence (Pierce) and horseradish peroxidase as
described (16). When biotinylated antibodies were used for
immunoblotting, proteins were visualized using neutroavidin and
biotin-conjugated horseradish peroxidase (Pierce).
To test the specificity of the AC antibodies, HEK/ACV cells were sonicated in homogenization buffer (20 mM HEPES, 1 mM EDTA, pH 7.4, and protease inhibitors (16)) and crude membrane fractions were obtained by centrifugation. For immunoprecipitation, membranes were solubilized in immunoprecipitation buffer (40 mM sodium phosphate, pH 7.4, 50 mM sodium fluoride, 5 mM EDTA, 5 mM EGTA, 0.5 M NaCl, 1% Nonidet P-40, 0.5% deoxylcholate, 0.1% SDS, and protease inhibitors) and solubilized proteins were incubated with the indicated AC antibodies immobilized to protein G (Pierce). Immunoprecipitates were washed with immunoprecipitation buffer, subjected to SDS-PAGE and immunoblotting.
Immunofluorescence StainingFreshly isolated rabbit
myocytes were seeded onto poly-L-lysine-coated coverslips
and incubated at 37 °C for 1-2 h in M199 media (Life Technologies,
Inc.). Prior to staining, myocytes were washed twice with relaxation
buffer (0.1 M KCl, 5 mM EGTA, 5 mM MgCl2, 0.25 mM dithiothreitol in
phosphate-buffered saline (PBS), pH 6.8), followed by fixing in
pre-cooled (20 °C) methanol/acetone (1:1) for 5-10 min at
4 °C. Fixed myocytes were incubated overnight in labeling buffer
(1% bovine serum albumin, 2% normal goat serum in PBS) to block
nonspecific binding. Different primary antibodies were diluted in
labeling buffer and incubated with myocytes overnight at 4 °C. In
the cases of double labeling, two primary antibodies were used
simultaneously. Different mixtures of secondary antibodies were used
subsequently, including: FITC-conjugated rabbit anti-goat IgG,
FITC-conjugated goat anti-rabbit IgG, and TRITC-conjugated goat
anti-mouse IgG. Coverslips were mounted onto slides and viewed on a
scanning confocal microscope (Zeiss). For all the channel and AC
antibodies, negative control experiments were performed using preimmune
antibody, secondary antibody alone, and/or antigen-preabsorbed antibody; in each case we observed no specific staining (data not
shown).
Transient expression of different calcium channel subunits in HEK293 cells has been described previously (16, 17).
Quantifying Binding of Antibodies to Proteins in Intact CellsAs will be described below, it was of interest to determine
the relative binding efficiencies of different antibodies to recognize the same proteins. Specifically, we designed experiments to allow comparison of recognition of the 1C subunit by Card I
versus Card C. To do this we used heterologously expressed
channel subunits as the standards. HEK293 cells were transiently
transfected with plasmids encoding the
1C,
2a, and the
2
subunits (16). Cells were split into poly-L-lysine-coated 24-well plates and
were fixed ~48 h post-transfection using the method described above
for immunofluorescence studies. Cells were then incubated with 2%
bovine serum albumin in PBS for 1-2 h at room temperature, followed by
overnight incubation with the indicated primary antibodies at 4 °C.
After washing out unbound primary antibodies, 125I-labeled
protein G (NEN Life Sciences Products) was incubated with the cells for
2-4 h at 4 °C. After extensive washing with PBS, bound
radioactivity was eluted from the plates using 2% SDS and quantified
using a
-counter. Nonspecific binding from primary antibodies was
determined after preabsorbing antibodies with their immunizing
antigens.
To identify
which components comprise the cardiac calcium channels, we partially
purified the L-type calcium channels from rabbit heart using
WGA-Sepharose chromatography. Cardiac membranes were prelabeled with
the dihydropyridine ligand (+)-[3H]PN 200-110 (3) and
purification was followed by the specific binding (Fig.
1A). Fractions enriched in
binding were analyzed with the 1C subunit specific
antibody Card I (Fig. 1B). The major component recognized by
Card I, which is directed against an internal domain of the
1C subunit (16), was a diffuse protein migrating at
~200 kDa (Fig. 1B). This is likely to be the truncated
form of the
1C subunit of the L-type calcium channel (3,
7). The fractions enriched in PN 200-110 binding were pooled and
analyzed for reactivity with Card C, which is directed against the
C-terminal region of the
1C subunit (16). Card C will
only detect full-length
1C and not react with a
C-terminal truncated protein. The Card C antibody recognized a protein
migrating at 240 kDa (Fig. 1C, lane 2), that migrated
similarly to Sf9 cell expressed
1C subunit (Fig.
1C, lane 1) which was included as a positive control, since previous studies showed that this heterologously expressed
1C subunit is a full-length protein with intact N and C
termini (16).2 Card I, which will recognize the full-length
and C-terminal truncated protein, recognized essentially only the
smaller 190-200-kDa protein (Fig. 1C, lane 3). The lack of
significant staining at the 240-kDa position with this antibody
suggested that most of the
1C subunit in the partially
purified preparation is truncated at its C terminus. The stronger
staining of the full-length
1C subunit by Card C versus Card I reflected the greater sensitivity of the Card
C antibody to detect denatured
1C proteins on Western
blots (this was also observed using the Sf9 full-length
1C as a standard).
To detect which subunit co-purified with the
1C
subunit, we stained the pooled WGA-Sepharose eluates with
subunit-specific antibodies. In the same fractions that contained
1C, a diffuse protein of 68-70 kDa was specifically
immunostained by the
2-specific antibody (Fig. 1C,
lane 6), and exhibited mobility similar to the heterologously
expressed
2a subunit from Sf9 cells that was run as a
control (Fig. 1C, lane 1). Taken together, the results from
the WGA-Sepharose purification suggest that
1C and
2 subunits are likely components of cardiac L-type
calcium channels.
To begin to address whether the truncated 1C subunit is
an artifact of isolation or a physiological component of the channels, we sought to minimize possible proteolysis during the isolation procedure by using cardiac myocytes. In addition, since heart is a
heterogeneous tissue, studies with the cardiac myocytes were performed
to ensure that the
1C and
2 subunits were
indeed expressed in cardiac myocytes versus other cell types
(e.g. smooth muscle) that are present in whole heart
preparations. Crude membranes were quickly prepared from freshly
isolated myocytes, solubilized in 1% SDS, and directly subjected to
immunoprecipitation with a mixture of the Card I and
gen
(a generic
subunit antibody (17)) antibodies. The
immunoprecipitates were subjected to SDS-PAGE and analyzed for
1C content by immunoblotting with either Card C or
biotinylated-Card I (Bio-I). A 240-kDa protein was detected by Card C
in the immunoprecipitates, which corresponded to the full-length
1C subunit with an intact C terminus (Fig.
2A, lane 1). On the other
hand, a major component of ~200 kDa and a trace of the 240-kDa
component corresponding to the truncated and the full-length forms of
the
1C subunit, respectively, were identified by Bio-I
in the immunoprecipitates. Despite the fact that multiple protease
inhibitors were used, the majority of the
1C subunit was
truncated at the C terminus. The immunostained proteins migrated over a
broad range, suggesting the presence of multiple proteolytic products
that might arise as a result of proteolytic breakdown that occurs
during isolation of the protein.
The immunoprecipitates from myocytes were also analyzed for the
2 subunit. The same 68-70-kDa proteins discussed
earlier were immunoprecipitated by the
gen antibody from
myocytes and were identified by subsequent immunoblotting with the
2 antibody (Fig. 2A, lane 1). In addition,
blotting of the immunoprecipitates with biotinylated-
gen
(Bio-
gen) resulted in staining of the same proteins
(Fig. 2A, lane 4). Thus the
2 subunit is
present in cardiac myocytes.
We asked whether any other subunits could be identified from heart
by the
gen antibody (17). As a positive control, we ran
heterologously expressed
1b,
2a,
3, and
4 subunits, which were readily
detected by Bio-
gen (Fig. 2B, lanes 3-6).
However, the only immunoreactive protein detected in the
immunoprecipitates from rabbit heart membranes by
Bio-
gen was the same as that detected by the
2 antibody (Fig. 2B, lanes 1 and
2). These results suggested that
2 is the
major cardiac isoform of the
subunit as detected by our
antibodies.
L-type
calcium channels have been localized to the T-tubular system in
skeletal muscle (23, 24), but only very limited information is
available concerning their distribution in mammalian cardiac muscle. We
investigated the subcellular localization of the 2 and
1C subunits in cardiac myocytes using immunofluorescence staining. Previous studies have demonstrated that the T-tubule network
is closely apposed to the Z-lines in ventricular myocytes (25, 26). An
anti-
-actinin antibody was used to label the Z-lines in intact
myocytes (27), to obtain an indication of T-tubule localization. To
reveal the distribution of the
2 subunit, freshly
isolated rabbit ventricular myocytes were co-stained with the
2 and the anti-
-actinin antibodies, and visualized
using FITC- and TRITC-conjugated secondary antibodies, respectively (Fig. 3, A-C). The staining
pattern for both the
2 subunits and
-actinin were
observed as regularly-spaced and evenly-distributed transverse bands
(Fig. 3, A and B). To further determine whether the
2 subunit is expressed on T-tubules, two confocal
images obtained from the same section of the myocyte (Fig. 3,
A and B) were overlaid using Adobe software (Fig.
3C). The predominant yellow color in the merged
image indicated that the
2 subunit and
-actinin are
closely associated. The most reasonable interpretation of these data is
that the
2 subunit is localized along T-tubule membranes
in cardiac myocytes. To corroborate these findings, we used the
gen and the anti-
-actinin antibodies to co-label the
myocytes; similar striated staining associated with the T-tubules was
also observed (data not shown). These results provided the first
demonstration that the
2 subunits are expressed in
cardiac myocytes and localized on the T-tubules.
We next investigated the presence and the subcellular localization of
the 1C subunits using immunofluorescence staining in intact cardiac myocytes. We were particularly interested in the possibility of detecting the full-length
1C subunit in
the intact cells where the possibility of proteolysis should be
minimal. Either the purified Card I (pCard I) or the purified Card C
(pCard C) antibodies were applied separately or co-applied with the
anti-
-actinin antibody to stain the fixed myocytes. Images obtained
from pCard I (Fig. 3D) showed the same distribution as
obtained with the
2 antibody. The images of pCardI and
the anti-
-actinin antibody (data not shown) were merged, and the
resulting overlaid image is shown in Fig. 3E. The staining
pattern again formed regularly-spaced transverse bands in yellow
color indicating that the
1C subunit is also
associated with T-tubule membranes as is the
2 subunit. Similar results were obtained with pCard C staining (Fig. 3,
F and G). Importantly, the images obtained with
pCard C (Fig. 3F) resulted in a staining pattern that was
indistinguishable from that of pCard I (compare Fig. 3D with
3F, and 3E with 3G), and that of the
2 subunit (Fig. 3C). These results strongly
suggested that the full-length
1C subunits are present
in intact myocytes and likely associated with the
2
subunit (as well as the
2
subunits (26)) along the
T-tubules. Experiments to co-stain the myocytes with combinations of
1C subunit specific and
subunit-specific antibodies
(e.g. Card I with anti-
2 or Card C with
gen) resulted in no specific staining (despite the fact
that strong signals were observed when used separately). These results
were likely due to steric hindrance between the two antibodies trying
to access two closely associated antigens (the
1C and
the
2 subunits).
Cardiac L-type calcium channels can be
modulated by cAMP-dependent phosphorylation. The
1C subunit is a potential target for PKA-mediated
phosphorylation, however, an intact C terminus of the
1C
subunit is required (7, 12), since the truncated
1C
subunit is not a PKA substrate (3). The data discussed so far have
demonstrated that the full-length
1C subunits were present and localized on T-tubules in intact myocytes (Fig.
3F), however, the majority of the immunoprecipitated or
purified
1C proteins were truncated (Figs. 1 and 2). It
was therefore of particular interest to attempt to assess the amount of
the full-length
1C subunits in the intact myocytes. To
do so we developed a quantitative assay using Card C and Card I to
analyze the ratio of full-length versus truncated
1C proteins in situ. Methanol fixation of
intact cells was used to avoid possible proteolysis during cell lysis, and 125I-labeled protein G was used to determine the amount
of primary antibody bound to the antigens. Fluorescence-conjugated
secondary antibodies were not used because it is very difficult to
quantify and normalize the fluorescent signals produced by different
secondary antibodies.
Since the method relies on quantifying the signal from Card I
versus Card C, it was very important to determine the
relative sensitivities of Card I and Card C toward the
1C subunits in native cells, as we previously recognized
differences in the ability of the antibodies to detect the proteins
after SDS-PAGE and immunoblotting. However, we did not use the
differences detected by Western blotting to quantify antibody
recognition of
1C, since it is well recognized that
antibodies differ in their ability to detect antigens in various types
of immunodetection assays. To quantify and compare Card I and Card C
reactivity, we used heterologously expressed full-length
1C protein as a standard. In previous studies we demonstrated expression of functional calcium channels in HEK293 cells,
in which all the
1C subunits were expressed as the
full-length form (16). HEK cells were transfected, fixed, and incubated with Card I and Card C followed by 125I-protein G as
described under "Experimental Procedures." As shown in Table
I, the total binding from both antibodies
was first determined (value T). The Card I and Card C antibodies were
preabsorbed with the antigens, and nonspecific bindings were obtained
(Table I, value NS). The relative sensitivity (value
S) of Card I or Card C antibodies to detect a similar amount
of expressed
1C subunit was determined by subtracting
the nonspecific counts from the total bound 125I-labeled
protein G counts (Table I, S = T
NS). By averaging the results from four independent
experiments, the relative binding sensitivity of the Card I
versus the Card C antibody
(SI/SC) was assessed to be
2.95 ± 0.4 (n = 4). This ratio implied that the
signals produced by the specific binding of the Card I antibody to an
equivalent amount of the
1C subunit under these
conditions was ~3 times stronger compared with that of the Card C
antibody.
|
We next asked how much full-length 1C subunit was
present in intact cardiac myocytes. The rationale was that the ratio of Card I/Card C binding would reflect the proportion of full-length versus truncated
1C subunits in the intact
myocytes. If all the
1C was full-length, the ratio
should be as in the transfected cells. However, if a significant
portion of the
1C was truncated in the intact myocyte,
then the binding of Card C (which only binds the full-length protein)
would decrease while the binding of Card I would be unchanged (as Card
I recognizes both the truncated and full-length forms of
1C). Accordingly, the Card I/Card C binding ratio would
become larger. To compare the results with those obtained from
transfected HEK cells, the myocytes were stained with Card I and Card C
followed by 125I-protein G incubation under identical
conditions as described above. We quantified the binding of these
antibodies in the myocytes in at least four separate experiments, and
the results indicated that the ratio of radioactivity due to the
specific binding of the two antibodies to the
1C
subunits in the myocytes was 2.8 ± 0.3 (n = 4)
(Table I). This ratio is similar to that obtained in the transfected
cells (Table I), indicating that the total amount of the
1C subunits detected by Card C was similar to that detected by Card I in the intact myocytes. These results provided the
first experimental evidence that all the
1C subunits
expressed in myocytes contain an intact C terminus, and the truncation
observed in the isolated protein likely occurs artificially during
channel isolation.
Previous mRNA analysis predicted
that heart preparations contain the type V and VI isoforms of AC (28),
however, no data are available to demonstrate the presence of those
isozymes at the protein level. Expressed type V and VI AC have been
demonstrated to be inhibited by micromolar Ca2+ (28), and
indeed, adenylyl cyclase in cardiac myocytes is known to be inhibited
by Ca2+ (15). This Ca2+-dependent
inhibition can be relieved by blockers of L-type calcium channels (15).
To test the hypothesis that the inhibition of AC by Ca2+
influx through the L-type channel was achieved by a close spatial proximity of the channel and AC protein, we developed two new antibodies (described under "Experimental Procedures") that should recognize the cardiac AC isoforms. The specificity of the antibodies was tested using an HEK293 cell line stably expressing rat type V AC
isozyme (HEK/ACV). Fig. 4,
panel A, shows a Western blot in which a crude membrane
preparation of HEK/ACV cells was solubilized, immunoprecipitated with polyclonal goat sera (G-ACV/VI or
G-ACcom), and probed with rabbit ACV/VI
(R-ACV/VI) or preimmune sera. A major protein band of
~200 kDa was detected by the R-ACV/VI antibodies from the
crude solubilized membrane preparation by immunoblotting (Fig.
4A, lane 1) and in preparations that were immunoprecipitated by either the G-ACcom or G-ACV/VI antibodies
and subsequently immunobloted with the R-ACV/VI antibody
(Fig. 4A, lanes 2 and 4). The size of this
protein is the same as that expected for glycosylated rat type V
adenylyl cyclase.3 This
protein band was not detected in the immunoblots by the preimmune
rabbit serum (Fig. 4A, lanes 3 and 5), when
preimmune goat sera were used in the immunoprecipitation step (data not shown), or when untransfected HEK293 cells were probed with
R-ACV/VI antibody (data not shown). It is thus clear that
the R-ACV/VI antibody specifically recognizes the type V AC
protein in immunoblotting experiments and that both the
G-ACV/VI and G-ACcom antibodies specifically
immunoprecipitate the expressed type V AC protein.
Rabbit cardiac myocytes were analyzed in immunofluorescence studies
with G-ACV/VI to assess the subcellular distribution of the
cardiac enzyme. Images obtained from immunofluorescence staining indicated that the antibody recognized the cardiac AC and that it had a
distribution pattern identical to the channel subunits in cardiac
myocytes (Fig. 4B). Co-staining of the cells with
G-ACV/VI and anti--actinin antibodies demonstrated close
localization of these two proteins, and, as was the case for the
channel subunits, strongly suggested a T-tubule localization of the AC
protein (Fig. 4C). Identical results were obtained with
G-ACcom (data not shown). Experiments that attempted to
co-stain the myocytes with the AC antibodies and channel-specific
antibodies together revealed no specific staining, as was the case in
similar studies that attempted to use two channel antibodies together.
These results provide the first direct visualization at the protein
level of cardiac AC. Since the enzyme could be detected with an
antibody targeted to type V or VI AC, the results strongly suggested
that cardiac myocytes express a type V or VI AC. In addition, they are
the first to demonstrate the subcellular localization of the AC and its
close proximity to the L-type calcium channels.
The results of the present study have demonstrated the expression
and localization of L-type calcium channel subunits and adenylyl
cyclase in cardiac myocytes using biochemical and immunocytochemical approaches. The novel findings were: (i) cardiac myocytes express the
2 subunit of L-type calcium channels and it is
co-localized on T-tubule membranes with the
1C subunit;
(ii) the
1C subunit is a full-length protein in cardiac
myocytes, and the C-terminal truncated version of the protein likely
arises during isolation procedures; (iii) cardiac myocytes express a
type V and/or VI isoform of AC and it co-localizes with the L-type
calcium channels on the T-tubule membranes. Further discussion of these
points follows.
Recent studies have revealed that the subunits of calcium channels
play multiple roles in modulating channel formation and function in
heterologous systems. The direct interaction between the
subunits
and the
1 subunits has been shown in vitro
(29). However, there are only a few reports demonstrating the
expression of
subunits and their association with the
1 subunits in native tissues (30). In the present study,
the association of the
2 subunit with the
1C subunit in cardiac myocytes has been clearly established. First, we have shown that the
2 subunit
co-purified through WGA-Sepharose chromatography with the
1C subunits from cardiac muscle. Second, the
2 subunit co-localized with the
1C subunit to the T-tubule network in cardiac myocytes.
Although the results of Northern analyses have suggested that there
could be more than one isoform present in heart (14, 31), we did
not detect the expression of other
subunits besides the
2 isoform using the
gen antibody. It
could be argued that this might be due to the lack of sensitivity of
the
gen antibody to other
isoforms, however, this
antibody readily detects the other expressed
subunits (17). Thus, a
more likely explanation of the present results is that, if there are
other
subunits expressed in heart, the level of expression compared
with that of the
2 subunit is low and beyond the
detection of the
gen antibody. The results here strongly
suggest that the
2 subunit is the major cardiac
isoform.
An important finding of the present study is that all the
1C subunit appears to be full-length in cardiac
myocytes. The development of a quantitative assay to assess the amount
of full-length
1C subunits provided the first
demonstration that the C terminus of the
1C subunit is
intact in myocytes. Since the C terminus of the
1C
subunit may play critical roles in mediating phosphorylation and
regulation of the calcium channels (2), this finding provides an
important piece of information concerning the receptor-mediated regulation of the channels. If the C terminus was truncated in the
intact cells, as it is after isolation of the channels by purification
or immunoprecipitation, it would be difficult to assign a role to the
1 subunit in channel regulation by PKA, because the
truncated
1C subunit is not a PKA substrate while the
full-length protein is a substrate (3, 7, 12). The finding that the C
terminus is intact in myocytes allows serious consideration of the
1C subunit as a major PKA target in vivo. Several possible scenarios may explain why the majority of the
1C subunit appears to be truncated when examined in
purified preparations. The truncation may result from proteolysis that occurs by a protease whose activity is not inhibited by the protease inhibitors used by ourselves and others. Alternatively, the proteolysis may occur in vivo as a result of post-translational
modification, but the C terminus may remain associated with the rest of
the channel and is "lost" only upon cell disruption. Other
scenarios are also possible and further testing will be required to
resolve this issue.
Many electrophysiological studies have shown that calcium channel
function is regulated by -adrenergic receptor agonists through
adenylyl cyclase and the cAMP-dependent signaling pathway (1). However, this pathway appears to have an internal turn-off mechanism, as cardiac AC activity is counter-regulated by
Ca2+ influx through L-type calcium channels (15). The
Ca2+ that enters cardiac myocytes through L-type calcium
channels is known to induce further increases in Ca2+ from
ryanodine-sensitive calcium-release channels. One mechanism to explain
calcium-mediated inhibition of cardiac AC would be close association of
cyclase with the calcium channels. In the present study, we
demonstrated that either the type V or type VI
Ca2+-sensitive AC is localized in rabbit myocytes and is
present on the T-tubule membranes in a spatial distribution
indistinguishable from the calcium channel complex. Other studies have
suggested a close localization of the L-type calcium channels with the
calcium-release channels (26). Taken together, these findings suggest
that the regulation of the calcium channel by cAMP signaling and the
regulation of the AC by Ca2+ are likely facilitated by a
close spatial association between the channel and the AC proteins.
In summary, we have shown that the 2 subunit of the
L-type calcium channel is expressed and localized on T-tubule membranes in association with the full-length
1C subunit. Thus the
minimal proven subunit composition of cardiac L-type channels is
1C,
2, and
2
. The C
terminus of the
1C subunit is intact in cardiac myocytes
and loss of the C terminus of the
1C subunit likely occurs during cell lysis. Moreover, we demonstrated a similar subcellular localization of the Ca2+-sensitive type V
and/or VI adenylyl cyclase with the calcium channel in cardiac
myocytes.
We thank Dr. Robert S. Decker of Northwestern University and Dr. Annelise O. Jorgensen of University of Toronto for their valuable advice and innovative suggestions for immunocytochemical studies, and Dr. Jack Krupinski of Weis Center for Research for providing the stable HEK/ACV cell line.