(Received for publication, August 23, 1995; and in revised form, October 6, 1995)
From the
We report several unexpected findings that provide novel
insights into the properties and interactions of the and
subunits of dihydropyridine-sensitive L-type channels.
First, the
subunit was expressed as multiple species
of 68-72 kDa; the 70-72-kDa species arose from
post-translational modification. Second, cell fractionation and
immunocytochemical studies indicated that the hydrophilic
subunit, when expressed alone, was membrane-localized. Third, the
subunit increased the membrane localization of the
subunit and the number of cells expressing L-type
Ca
currents, without affecting the total amount of
the expressed
subunit. Expression of maximal
currents in
/
co-transfected cells
paralleled the time course of expression of the
subunit. Taken
together, these results suggest that the
subunit plays multiple
roles in the formation, stabilization, targeting, and modulation of
L-type channels.
Calcium channels are multisubunit proteins that minimally
consist of ,
, and
subunits(1, 2) . The
subunit is the
pore-forming channel subunit and is the ``signature'' subunit
that determines many of the basic characteristics of the different
types of Ca
channels. The roles of the
``accessory'' subunits are less certain, and much remains to
be revealed about the roles of the accessory subunits in Ca
channel formation, processing, regulation, and function.
Co-expression of the
and/or
subunits
clearly results in larger Ca
currents from expressed
subunits(3, 4, 5, 6, 7, 8, 9) ,
and several reports have demonstrated that the
subunits can
modulate the activation and inactivation kinetics of the
channels(3, 4, 5, 7, 8, 10, 11) .
While channel-specific ``
'' subunits appear to play
roles in certain Na
and K
channels,
none of these channels are as complex as the Ca
channels. The
subunits are particularly intriguing since they are
very hydrophilic and are not predicted to possess membrane-spanning
domains. To begin to address how these proteins participate in channel
function, we have analyzed the properties and interactions of the
subunit and
subunits of
Ca
channels in transiently transfected human
embryonic kidney (HEK) (
)cells. The particular
and
subunits chosen for analysis are expressed
in the heart, brain and other tissues. Based on mRNA studies, they
appear to be critical components of what has been traditionally
referred to as the ``cardiac'' L-type channel, and variants
of these subunits are likely to also comprise ``brain''
L-type channels. Our results reveal previously unknown characteristics
of the proteins and their interactions, and suggest that the
subunit plays multiple roles in the formation, stabilization,
targeting, and modulation of the channel complex. In addition, these
are the first results to characterize the properties of these rare
membrane proteins at the biochemical level and provide new insights
into the poorly understood properties of these proteins.
Cell lines were maintained in DMEM (Life Technologies, Inc.)
containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37
°C in 5% CO. Transfections were done using the
HEPES-buffered calcium phosphate method (13) in 100-mm
polystyrene tissue culture plates. Cells were refed with fresh media
1-3 h prior to addition of DNA. Unless otherwise indicated in the
text, transfections were performed with 30 µg of each expression
plasmid, with all transfections normalized to 60 µg of total DNA
using either pRBG4 vector or sheared salmon sperm DNA (Life
Technologies, Inc.). Cells were incubated with DNA for 6-8 h,
after which a 30% DMEM-Me
SO solution was added to a final
concentration of 10% Me
SO, followed by a 6-min incubation.
Subsequently, cells were refed with fresh media and incubated at 37
°C until further evaluation. Post-transfection times reflect hours
following Me
SO shock, which is considered as 0 h. In
co-transfection experiments, similar results were seen when DNA amounts
were normalized with either salmon sperm DNA or pRBG4 vector or when
DNA amounts were not normalized (data not shown). Experiments using
cycloheximide contained cycloheximide at a concentration of 100
µg/ml.
Figure 1:
Characterization of expressed and
calcium channel subunits in transiently
transfected HEK cells using subunit-specific antisera. A, a
linear representation of the
protein delineating the
epitopes used to generate the Card C and Card I antisera, as described
under ``Experimental Procedures.'' B, detection of
the expressed
protein using immune and preimmune
antisera for Card C and Card I. The figure shows immunoreactivity
against membranes from non-transfected HEK cells (lanes 1, 3, 6, and 8) and membranes from transiently
transfected
cells (lanes 2, 4, 5, and 7). Immune Card C and Card I antisera both
recognized a protein in transfected cells with a relative mobility of
240 kDa. C, linear representation of the
protein, delineating the location of the two regions conserved
among known
isoforms, as well as the region in the C terminus
used to generate the
antisera as described under
``Experimental Procedures.'' D, detection of the
expressed
subunit by the
antisera
in membranes isolated from transiently transfected
cells (lane 1), non-transfected cells (lane 2),
and transverse tubule membranes purified from skeletal muscle (lane
3). Samples were electrophoresed on a 8% polyacrylamide
gel.
To visualize expressed proteins in preparations from transfected cells by immunoblotting, either whole cells or crude membranes were used. Cells were homogenized in 50 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA and protease inhibitors (0.2 mM phenylmethysulfonyl fluoride, 100 nM aprotinin, 1 µM leupeptin, 1 µM pepstatin A, 1 mM soybean trypsin inhibitor, and 1 mM iodoacetamide) using a Tri-R homogenizer at setting 7. To prepare crude membranes, the homogenates were centrifuged at 2100 rpm for 5 min and the supernatants were subsequently centrifuged at 42,000 rpm for 60 min in a Type 65 rotor. Proteins were separated on SDS-polyacrylamide gels containing the indicated concentrations of acrylamide, transferred to nitrocellulose, and processed for immunoblotting as described previously(14) . Detection was with enhanced chemiluminescence (Amersham), horseradish peroxidase, or alkaline phosphatase as described(15) .
Two different antibodies specific for the
subunit, Card I and Card C (Fig. 1A), were developed and used to visualize the
expressed protein on immunoblots. The
subunit was
detected as a single large molecular species of
240 kDa, which
migrated more slowly than the myosin (205 kDa) molecular mass marker (Fig. 1B). The observed size of the
subunit is close to the cDNA-predicted size of the full-length
subunit (242,771 daltons; (12) ). The size
of the expressed protein and its reactivity with the Card C antibody,
which is directed at 14 of the last 16 amino acids in the C terminus,
suggested that the expressed protein was a full-length protein with an
intact C terminus. In addition, a single immunoreactive protein of the
same electrophoretic mobility was detected by the Card I antibody (Fig. 1B), which reacts with the II-III cytoplasmic
linker of the cardiac
subunit, thus providing further
evidence that the expressed
subunit is an intact
full-length protein. In agreement with this conclusion, we also
observed expression of a similarly sized protein using recombinant
baculovirus to direct the expression of the
subunit
in insect Sf9 cells.
The baculovirus-expressed protein
additionally contained an epitope tag in its N terminus, which allowed
us to confirm that the expressed protein was full-length. The size of
the expressed
subunit was larger than that of the
subunit in L-type channel preparations previously
purified from cardiac tissue(17) . This suggests that the
purified cardiac protein may have been proteolyzed during the isolation
procedure, or alternatively, that tissue-specific modification of the
subunits occurs. Previous observations have shown
that C-terminal truncation of other
subunits occurs
in native tissues such as skeletal muscle (14, 18) and
brain(19, 20) .
Figure 2:
Multiple species and
pulse-chase analysis of subunit synthesis in
transiently transfected HEK cells. A, higher resolution of the
protein on a 6% polyacrylamide, 24-cm gel reveals
multiple immunoreactive species on a Western blot, ranging from 68 to
72 kDa in size (lane 2). A phosphorimage of the
subunit
immunoprecipitated from cells metabolically labeled with
[
S]methionine revealed at least three labeled
bands ranging from 68 to 72 kDa (lane 1). Comparison of
immunoreactive staining of membranes from
cells with
membranes from cells expressing the epitope-tagged
subunit (lane 3), which contains an additional 8 amino
acid residues, suggests that the relative molecular masses of the lower
doublet are 68 and 69 kDa. The arrow marked 80 kDa represents the position of the prestained albumin molecular mass
marker. B, the
antisera and the monoclonal KT
antibody had the same immunoreactive profile against membrane
particulate fractions from large T-transformed HEK (tsA201) cells
expressing the epitope-tagged
subunit. The
KT
antibody also reacted with the 82-kDa large T antigen,
which contains the KT
epitope. C, labeling of
transiently transfected
cells with
[
S]methionine, followed by solubilization and
immunoprecipitation with the anti-
antibody, revealed at least
three distinct isoforms of the
subunit. Shown are
S-phosphorimages of the
subunit
immunoprecipitated from metabolically labeled
(top panel) and
/
(bottom panel) cells. The
subunit was
initially synthesized as a 68-kDa nascent protein, which was
subsequently modified to at least two higher molecular mass isoforms
indicated by the filled arrows.
Pulse-chase experiments using [S]methionine
were performed using transfected
cells to determine
whether the higher molecular mass isoforms were the result of
post-translational modification. The results indicated that the
protein was initially synthesized as a 68-kDa
protein (Fig. 2C), the size predicted from the cDNA
sequence. As time elapsed, higher molecular mass bands of
69 and
72 kDa appeared on the
S image, suggesting that the higher
molecular mass isoforms of the
subunit arose due to
multiple post-translational modifications of the 68-kDa form. Similar
results were observed in cells expressing either
alone (Fig. 2C, upper panel) or both
and
(Fig. 2C, lower panel). The modification was not due to phosphorylation
of the protein (
)and the predicted primary structure lacks
signature sequences, in the proper context, for myristoylation or
isoprenylation. The modification took place on a relatively slow time
scale; the change in distribution of isoforms was not observed until
several hours after the initial synthesis of the protein. Co-expression
with
did not appear to affect either the rate or
extent of the post-translational modification. Further studies will be
necessary to reveal the nature of these modifications.
The
subunit isoforms, measured collectively, appeared to
turn over relatively slowly with no discernible difference between
and
/
cells.
When the total immunoreactive
subunit bands were
analyzed collectively, the estimated half-life was >8 h (data not
shown). However, this is a crude estimate, because the change in
distribution of label between the multiple forms, as well as the
possibility that the different isoforms may turnover at different
rates, did not allow an accurate analysis of the half-life.
Figure 3:
Cellular distribution of the subunit expressed in transiently transfected HEK cells. A, levels of the
protein were evaluated in
membrane particulate fractions (lanes 1 and 2) and
cytosolic fractions (lanes 3 and 4). The
protein was localized to membrane particulate fractions both in
transiently transfected
cells (lanes 1 and 3) and
/
cells (lanes
2 and 4). B, solubilization of the
protein in detergents and in salt as described under
``Experimental Procedures.'' Shown are immunoblots of
supernatants (top panel) and pellets (bottom panel)
following solubilization of 100 µg of membrane particulate fraction
in various detergents and NaCl.
To further
characterize the cellular location of the expressed subunit,
cells transiently expressing the
subunit were fixed,
permeabilized, stained with the
antisera, and viewed
using confocal microscopy (Fig. 4). In these experiments,
staining of the
subunit clearly was present at the
periphery of the cells, suggesting a membrane localization (Fig. 4A). In addition, diffuse staining was observed
in intracellular compartments. No specific immunoreactivity was seen
when preimmune antisera was used (data not shown). Based on the
cellular fractionation data, the intracellular pools of the
subunit must reflect
in intracellular membranes associated with
protein processing rather than ``soluble'' protein. Indeed,
when cells were pretreated with cycloheximide 2 h prior to fixing and
staining, almost all of the
-subunit immunofluorescence was seen
in dense patches at the plasma membrane, with very little staining
inside the cell (Fig. 4C). Presumably, cycloheximide
treatment inhibited new synthesis of the
subunit and allowed the
protein that was already made to be processed and transported to the
plasma membrane. These results strongly suggest that the
subunit,
which has been referred to as ``the cytosolic subunit,'' is
actually localized to membranes. The
subunit was
also membrane-localized when expressed in mammalian baby hamster kidney
cell (
)and in insect Sf9 cells from a recombinant
baculovirus,
indicating that this is not a cell-specific
phenomenon.
Figure 4:
Whole-cell immunofluorescence of
transiently transfected cells. Cells were
transiently transfected with
subunit and fixed with
paraformaldehyde at 40 h post-transfection. Shown are confocal
microscopy (A and C) and phase-contrast (B and D) views of cells stained with the
antibody. For panels C and D, cells were
pretreated with cycloheximide, as described in the text. Strong
immunoreactivity to the
antibody was detected at the
plasma membrane with diffuse cytosolic staining. Treatment with
cycloheximide eliminates the diffuse cytosolic staining, further
enhancing the visualization of
staining at the plasma
membrane.
Figure 5:
Cellular distribution of the subunit in transiently transfected HEK cells. A,
relative levels of expression of the
protein in
transiently transfected
and
/
cells. Inset shows
immunoreactivity in whole-cell lysates (inset, lane
1) and membrane particulate fractions (inset, lane
2), and no immunoreactivity in cytosolic fractions (inset, lane 3). The results are the average of five
independent experiments. B and C, whole-cell
immunofluorescence of transiently transfected
(B) and
/
cells (C). Shown are phase-contrast and confocal microscopy views of
cells stained with the Card C antibody. In B, note that the
staining is predominantly intracellular and perinuclear. Upon
co-transfection with
, staining of
becomes localized to the plasma membrane (C). Arrows are included for reference.
Figure 8:
Effects of the subunit on the
appearance of Ca
currents as a function of time
post-transfection. A, representative traces from transiently
transfected
and
/
cells at different times post-transfection. Trace a (15
h) is from an
cell, while traces b (15 h), c (42 h), d (70 h), and e (95 h) are from
/
cells. Pulse protocol is shown on
top with voltages indicated in millivolts. B, peak current
density in
(closed bars) and
/
(open bars) cells at
specific times post-transfection, obtained using whole-cell
patch-clamp. Each bar represents an average of 3-4
cells or 5-8
/
cells,
measured during 2-h intervals beginning at the following times
post-transfection: 15, 23, 33, 38, 44, 50, 70, and 95 h. C,
representative immunoblots of membranes from transiently transfected
cells, analyzed for subunit expression at specific times
post-transfection using subunit-specific antibodies. No significant
difference in
levels was detected between
and
/
cells (Fig. 5). Note that the staining patterns of the blot cannot be
used to measure the absolute amount of either expressed protein (i.e. the data cannot be analyzed to determine the ratios of
subunit protein levels).
While the subunit did not
increase the total
protein detected (Fig. 5A), the
subunit did affect the cellular
localization of the
subunit. Cells transfected with
alone and stained with the Card C antibody yielded
immunostaining that was largely perinuclear, and very little staining
of the plasma membrane was evident (Fig. 5B). Only a
few percent (no more than 5%) of the cells exhibited weak, diffuse
cytosolic or membrane staining in cells transfected with
alone (data not shown). In marked contrast, when cells were
transiently transfected with both the
and
subunits, there was significant membrane staining of
(Fig. 5C) suggesting that
co-expression of
and
subunits led
to increased localization of the
subunit to the
plasma membrane. These results provide evidence that interactions
between the
and
subunits may be critical in
channel formation and/or channel targeting to the membrane.
In order
to determine if the and
subunits
were co-localized in
/
cells, cells
were co-transfected with
and
,
co-stained with the Card C and KT
antibodies, and
visualized using FITC- and TRITC-conjugated secondary antibodies,
respectively (Fig. 6). The patterns of fluorescein staining of
the
subunit (Fig. 6, left panels)
and rhodamine staining of the
subunit (Fig. 6, center panels) are strikingly similar. In both
cells, there is strong, punctate staining of the cell periphery, as
well as some intracellular staining. When the fluorescein and rhodamine
images were overlaid (Fig. 6, right panels), most of
the membrane staining became yellow, indicating that the majority of
the
and
subunits in the membrane were
co-localized. Clear areas of intracellular staining of each subunit
alone, particularly the
subunit, were also visible.
In addition, small areas of membrane staining of
only or
only were apparent, suggesting that a small
percentage of each subunit might be present separately in the plasma
membrane.
Figure 6:
Co-localization of and
protein in transiently transfected
/
cells. Cells were co-transfected
with
and the epitope-tagged
and subsequently co-stained with Card C and anti-KT
antibodies. Visualization was done using FITC- and
TRITC-conjugated secondary antibodies. Fluorescein staining on the left panels shows the pattern of
expression, while rhodamine staining in the center panels indicates the pattern of
expression. On the right panel, an overlay shows the co-localization of the
staining for both
and
.
Conceivably the subunit might stabilize the
subunit and increase the probability that it
localizes to the plasma membrane. Pulse-chase studies using
[
S]methionine were used to determine the
half-life of the expressed
subunit in transiently
transfected
and
cells. Cells were initially labeled with
[
S]methionine 20-24 h post-transfection,
fractions were collected every 2 h following the addition of the chase
media, and were subsequently immunoprecipitated with subunit-specific
antibodies. In both
and
/
cells, the observed half-life of
the
subunit was about 3 h (Fig. 7, A and B). The total pool of
protein also
was visualized using immunoblotting (data not shown), which
demonstrated that the decline in
S-labeled protein was not
due to declines in total levels of the protein. From these results, it
appeared that the
subunit did not change the rate of turnover of
the
protein. However, because this analysis cannot
differentiate between
subunit present intracellularly
and in the plasma membrane, we cannot rule out the possibility that the
subunit may specifically stabilize the membrane-localized
protein.
Figure 7:
Pulse-chase analysis of
protein turnover in transiently transfected HEK cells. A,
cells were labeled with
S and analyzed in post chase
studies as described under ``Experimental Procedures.'' The
graph depicts the turnover of the
protein with
respect to time in
(closed circles) and
/
(open circles) cells.
Data were obtained by quantifying the
S signal using a
phosphorimager. B, natural log of radioactive decay of the
protein in
and
/
cells. Data from A were
fitted to a single exponential term by linear least-squares regression
analysis, corresponding to a predicted t
of 3.45
h in cells expressing
alone (r =
0.96) and 3.12 h in cells expressing both
and
(r = 0.98). Data for
cells represent averages of two experiments ± S.D., while
data for
/
cells represents the
average of three experiments ± S.E.
In addition to
increasing peak current density, co-expression of with
increased the number of patched cells
expressing detectable Ca
currents between 24-60
h post-transfection. Only 3-5% of
cells
exhibited low density Ca
currents, while 50-80%
of
/
cells exhibited high density
Ca
currents. These data are consistent with the
finding that co-expression of the
subunit is necessary for the
acquisition of high affinity DHP binding and membrane immunostaining of
the
subunit.
The combined biochemical, immunochemical, and
electrophysiological analyses described here revealed heretofore
unappreciated characteristics of the subunit of
Ca
channels and subunit interactions in mammalian
cells. Overall, the results argue that the
subunit acts to
increase and/or stabilize the population of functional channel
complexes in the membrane in addition to modulating properties of
channel conduction. From previous studies in Xenopus oocytes(3, 4, 5, 6, 7, 8) and
mammalian cells(9) , it is clear that
subunits can
enhance peak current amplitudes and modulate aspects of activation and
inactivation. In addition, in mammalian cells,
subunits have been
shown to enhance DHP or
-conotoxin binding to either L-type or
N-type
subunits,
respectively(5, 8, 9, 28, 29, 30, 31) .
Additionally, recent studies have identified sites that are involved in
direct interactions between the
and
subunits(32, 33, 34) . While these results
collectively support a model of a multimeric channel complex whose
properties are modulated by the
subunits, little has been
reported about the biochemical events that may be involved in these
processes.
An unexpected finding here was that the
subunit itself appears to behave as a membrane
protein. Since none of the cloned
subunit cDNAs predict that any
of these proteins have transmembrane-spanning domains, and since the
proteins are predicted to be extremely hydrophilic, it might have been
expected that the
subunit would be cytosolic in the absence of
other channel subunits. However, in the present studies we found no
evidence for the
subunit in ``cytosolic''
fractions (supernatants after high speed centrifugation), and the
immunocytochemistry clearly demonstrated staining of the
subunit in the membrane periphery. Conceivably the
subunit associates with other membrane proteins or with
cytoskeletal proteins, to become membrane localized. This hypothesis is
consistent with the relatively poor solubility of
in
Triton X-100, since insolubility in this detergent has long been a
characteristic of cytoskeletally associated proteins. A cyoskeletal
localization for the
subunits is attractive in view of the
potential role of the cytoskeleton in the inactivation and
``clustering'' of Ca
channels(35, 36, 37) . We considered
the possibility that the membrane localization of the
subunit might be conferred via an association with other
Ca
channel subunits that might be endogenously
expressed in HEK cells. However, this seems unlikely since
Ca
channel proteins are typically of very low density
and it is unlikely that the HEK cells would express sufficient amounts
of endogenous Ca
channel subunits to allow for the
substantial immunostaining we observed with the relatively
``overexpressed''
subunit at the periphery. In support
of this view, electrophysiological studies have revealed that HEK cells
possess very low or undetectable endogenous Ca
currents (data not shown; 9, 30).
Another unexpected finding
was that the subunit was expressed as multiple
forms, due to unidentified post-translational modifications. It is
unclear whether the multiple isoforms represent sequential
modifications of the nascent 68-kDa protein, or whether these isoforms
are differential modifications of the 68-kDa protein. The differential
solubility of
in salt and detergent suggests that a
post-translational modification might lead to tighter association of
some forms of
with the membrane. Of particular
interest was that the co-expression of the
subunit promoted the
appearance of the
subunit at the plasma membrane.
This correlated with the acquisition of high affinity DHP binding and
appearance of large Ca
currents. In the absence of
the
subunit, the
subunit was largely
intracellular and no saturable ligand binding could be quantified.
Corresponding Ca
currents were of a low density and
could only be observed in a very few percentage of cells. In contrast,
in
/
cells,
localized to the membrane, resulting in saturable ligand binding
as well as a higher percentage of patched cells exhibiting high density
calcium currents. The data from the biochemical and
electrophysiological analyses are consistent with the hypothesis that
the production of large numbers of functional channels is limited by
the
subunit copy number. The interaction with the
subunit
could help target the
subunit to the membrane, and/or
stabilize the
subunit, thus increasing the
probability of membrane insertion. With regard to the latter
possibility, while the half-life of the
subunit was
not altered by the
subunit, we were unable to measure whether the
half-life of the
subunit in the plasma membrane versus that in intracellular compartments might be different.
The oligomerization of the
and
subunits may occur co-translationally, facilitating the insertion
of functional channel complexes into the membrane and/or the formation,
stabilization, and membrane targeting of channel complexes.
Our
conclusion that the subunit increases the amount of
subunit at the plasma membrane is in contrast to findings in Xenopus oocytes, where analysis of charge movement was used to
determine that co-expression of the
subunit did not result in
increased numbers of channels at the cell surface(11) . In
addition, recent studies have demonstrated that the
subunit did
not cause increased immunoreactive
in oocyte plasma
membranes(40) . However, Marban and colleagues have recently
found that in transiently transfected HEK cells, co-expression of the
subunit increases the number of
channels at
the membrane as measured by charge movement(
); this
observation is consistent with the results of this paper. It is likely
that differences between Xenopus and mammalian cell expression
systems may account for the differences in results obtained regarding
the effect of
in these systems.
The present results and those
of others (38) with regard to an inability to detect saturable
DHP binding to cells expressing the -subunit alone
differs somewhat from previous results which reported that the
expressed
subunit alone bound DHPs with high
affinity; however, the level of binding reported was at the lower limit
of detection of specific binding with this
ligand(5, 8, 9, 28, 31, 39) .
Notably, others demonstrated a very rapid dissociation of ligand from
the
subunit in the absence of the
subunit when
expressed in COS cells(38) . This effect appeared to explain
the lack of saturable ligand binding that was observed (38) and
may also explain the results obtained in this study, as well as the
very low levels of DHP binding reported from previous
studies(28, 31, 39) . The increased targeting
of
to the membrane by the
subunit provides an
explanation for the increase in functional channel at the plasma
membrane, as measured by whole-cell currents and intact-cell ligand
binding, in the absence of any increase in
protein
levels.
In summary, the results described here suggest multiple
roles for the subunit in the processing and functioning of the
L-type channel. In addition, further studies on the functional effects
of co-expressing the
and
subunits
in transiently transfected HEK cells have revealed diverse effects on
peak current amplitude and Ca
-dependent inactivation
which are dependent upon levels of
expression. (
)The new information presented here argues that the
subunit can localize to the membrane alone and that
interactions between
and
occur
prior to channel targeting to the plasma membrane. These interactions
appear to be critical for the proper transport and/or targeting of
oligomerized subunits to the plasma membrane, and may also be important
for conformational stabilization of the channel complex.