Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611
Received for publication, August 31, 2000, and in revised form, March 22, 2001
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ABSTRACT |
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L-type Ca2+ channels in
native tissues have been found to contain a pore-forming
The voltage-activated L-type Ca2+ channels are
heteromeric proteins minimally composed of a pore-forming
A puzzling observation that has been made in several laboratories is
that the C terminus of several L-type Ca2+ channels appears
to be truncated in many native tissues. For example, when the
Materials--
All reagents were obtained from general sources
unless otherwise stated. Antibodies Card I, Card C (15), and
CT11,2
as well as the expression vectors pCR3 Antibody Preparation--
To generate an additional antibody
that would recognize C-terminal fragments of the Expression of Channel Subunits and C-terminal Fragments of the
Expression of C-terminal Fragments in Bacteria and GST Fusion
Protein Pull-down Assays--
To express C-terminal fragments of
Co-immunoprecipitation of C-terminal Fragments with the Channel
Complexes--
TsA201 cells were transiently co-transfected with a
combination of the channel subunits and different C-terminal fusion
proteins. Approximately 40 h post-transfection, whole cell lysates
were prepared in lysis buffer (20 mM
Na2HPO4, pH 7.4, 150 mM NaCl, 1%
Triton X-100 plus protease inhibitors (15)). The whole cell lysates
were diluted 1:3 with lysis buffer without Triton X-100 so that the
final concentration of Triton X-100 was 0.3%. The diluted cell lysates
were incubated with either one of the
In experiments where cross-linking was performed before
immunoprecipitation, cells were lysed in lysis buffer, and
cross-linking was performed in the presence of a 20 mM
phosphate and 0.1 mM of the nickel (Ni(II)) complex of the
tripeptide NH2-Gly-Gly-His-COOH (GGH-Ni(II)) (23). The
nickel-peptide complex was formed by mixing a 1:1 molar ratio of nickel
acetate and GGH in water. After a 5-min equilibration, the solution was
added to cell lysates to a final volume of 0.4 ml. The cross-linking
reactions were initiated by the addition of magnesium
monoperoxyphthalic acid hexahydrate (0.1 mM) and incubated
at room temperature for 10 min. The reactions were quenched by the
addition of 1 µl of 0.02 M thiourea. The cell lysates
were then diluted and subjected to immunoprecipitation with the Card I
antibody as described above.
Electrophysiological Assays--
Ba2+ currents were
obtained at room temperature (~22 ± 1 °C) from the
transfected human embryonic kidney cells using the whole cell patch
voltage clamp technique. The currents were recorded utilizing an EPC-7
patch clamp amplifier (List) whose analog output signal was low
pass-filtered at 3 kHz and then digitally sampled at 5 kHz using an
ISO-2 data acquisition system (MFK, Frankfurt/Main, Germany). The input
series resistance was ~70% electronically compensated by adjusting
the circuit to a point just below that which would begin to cause
ringing. Patch pipette electrodes having resistances of 2-5 megaohms
when filled with internal solution were manufactured from borosilicate
glass capillary tubing (Warner Instrument Corp, #GC150F-10) using a
P-97 micropipette puller (Sutter Instrument Co.); the pipette tips were
polished using a Narashige MF-83 pipette polisher. The internal
solution used to fill the pipette electrodes contained 60 mM CsCl, 5 mM HEPES, 1 mM
MgCl2, 5 mM MgATP, 0.1 mM MgGTP, 10 mM ethylene glycol-bis(
Immediately before attempting to obtain whole cell patches, anti-CD8
antibody-coated Dynabeads (Dynal, Oslo, Norway) were added to the bath
chamber to identify which cells had been successfully transfected cells
(21). For some experiments, the internal (pipette) solutions also
contained one of several indicated peptides at a concentration of 1 µg of protein/ml, and whole cell patch Ba2+ current was
recorded either in the presence or absence of the particular peptide.
Data depicting the effect of each of the several peptides to modify the
size of the peak of the initially recorded whole cell Ba2+
current evoked in response to 50-ms depolarizing pulses to +10 mV at
0.1 Hz from a holding potential of Progressive C-terminal Deletions of the Full-length
Cytosolic Application of The Distal Portion of the C-terminal Tail Contains a Channel
Inhibitory Domain--
We next attempted to define smaller regions of
the C terminus that might be responsible for channel inhibition. We
focused initially on fragments CT4 and CT7, which were approximately
equivalent to the fragments deleted from
Similar experiments were performed with GST-CT7. Surprisingly, GST-CT7
was without effect on currents from cells expressing either
We also tested the effects of other peptides derived from the C
terminus of Channels Containing the
To test the above possibilities, we determined whether constructs
containing a "tethered" CT4 or CT7 domain might exhibit the
inhibited currents. Two additional constructs were made that contained
internal deletions within the C terminus but intact CT4 or CT7
segments: These constructs, Co-immunoprecipitation of the C-terminal Fusion Proteins with the
Channel Subunits--
We performed biochemical studies to determine
that CT4 and CT7 could associate with
In other experiments CT7 was co-expressed with either wild type
Interactions of CT7 with the C Terminus of
In summary, the present study has demonstrated that C-terminal
fragments derived from the
Many studies demonstrate that the
It appears that the C terminus of the
Other important proteins involved in signaling are known to undergo
regulated proteolysis (27). Examples include the sterol regulatory
element-binding proteins, the amyloid precursor protein that is linked
to Alzheimer's disease, and Notch, a protein that is important in
developmental signaling (27). Future studies will be required to
unravel the events associated with the processing of the L-type
Ca2+ channels and to further understand how the processing
regulates channel activity.
1 subunit that is often truncated at the C terminus.
However, the C terminus contains many important domains that regulate
channel function. To test the hypothesis that C-terminal fragments may
associate with and regulate C-terminal-truncated
1C
(CaV1.2) subunits, we performed electrophysiological and
biochemical experiments. In tsA201 cells expressing either wild type or
C-terminal-truncated
1C subunits in combination with a
2a subunit, truncation of the
1C subunit
by as little as 147 amino acids led to a 10-15-fold increase in
currents compared with those obtained from control, full-length
1C subunits. Dialysis of cells expressing the truncated
1C subunits with C-terminal fragments applied through
the patch pipette reconstituted the inhibition of the channels seen
with full-length
1C subunits. In addition, C-terminal
deletion mutants containing a tethered C terminus also exhibited the
C-terminal-induced inhibition. Immunoprecipitation assays demonstrated
the association of the C-terminal fragments with truncated
1C subunits. In addition, glutathione
S-transferase pull-down assays demonstrated that the C-terminal inhibitory fragment could associate with at least two domains within the C terminus. The results support the hypothesis the
C- terminal fragments of the
1C subunit can associate
with C-terminal-truncated
1C subunits and inhibit the
currents through L-type Ca2+ channels.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 subunit and accessory
2
and
subunits (1, 2). Each
1 subunit contains four repeated
domains containing a total of 24 membrane-spanning domains as well as a
long hydrophilic C terminus which contains important regulatory domains
that contribute to channel regulation. For example, the C terminus of
the
1C subunit constitutes ~30% of the total mass of
the
1C subunit (3) and is critical for membrane targeting of the channels (4), the regulation of the channels by
protein phosphorylation (5), and the binding of
Ca2+-binding proteins such as calmodulin and sorcin (6-8).
In addition, the C terminus of
1C appears to contain
inhibitory domains because deletion of up to ~70% of the C-terminal
665 amino acids leads to increased currents (9).
1C subunit was isolated from cardiac myocytes, only
10-15% of the total protein was a full-length 240-kDa
1C subunit, whereas the majority migrated on SDS gels as
a ~190-kDa protein that was lacking the distal ~50 kDa of the C
terminus (10). Similar observations have been made for the
1C subunit expressed in brain (11) and the
1S subunit isolated from skeletal muscle (12, 13). If
the C termini of these proteins were truly absent, this would have
major implications for channel regulation. Although many protease
inhibitors have been used to try to prevent this truncation, none has
altered the proportion of full-length to truncated protein (14). In
marked contrast to what has been observed in native systems,
full-length
1C subunits have been observed in
heterologous expression systems (5, 15, 16). Despite the fact that
~90% of the
1C subunits appeared to be truncated when
isolated from cardiac myocytes, immunofluorescence imaging suggested
that the C terminus of the
1C subunit was present in a
1:1 ratio with the
1C subunit and co-localized with
channel subunits in cardiac myocytes (10). This finding led us to
consider the possibility that the processing of the
1C
subunit might have physiological importance and that the C-terminal
fragments might remain functionally associated with the channels (17).
Such a scenario would allow for maintenance of the important regulatory functions ascribed to the cleaved C terminus. As a first test of this
hypothesis, we demonstrated that an exogenous protease, chymotrypsin,
cleaved the full-length 240-kDa expressed
1C subunit in vitro into a "body" of 190 kDa, similar to what is
observed in native systems (10), and C-terminal fragments of 30-50 kDa that remained associated with the membrane (17). Here we report studies
in which we have tested the ability of C-terminal fragments to
associate with and regulate the conductance of C-terminally cleaved
1C subunits expressed in intact cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1C,
pCR3
1C
2024, pCR3
1C
1905, pCR3
1C
1733, pCR3
1C
1623,
pCR3
1C
1733-1905, and pCR3
2a (4) were
described previously. The vectors pCR3
1C
1733-2024
and pCR3
1C
1733-1905
2024 were prepared using
strategies similar to those described earlier (4). A schematic diagram
depicting the various constructs used in this study is shown in Fig.
1.
1C
subunit, a fusion protein encoding amino acid residues 1907-2171
(termed CT4, see Fig. 1B) in the C terminus of the
1C subunit was produced. The sequence in the CT4 region
was subcloned into an expression vector pQE32 (Qiagen), resulting in an
in-frame fusion of the 6×-His tag to the CT4 residues. The 6×-His-CT4
was produced and purified under native conditions from bacteria (15).
Purified CT4 fusion proteins were injected into a rabbit, and
polyclonal antibodies were prepared at Bethyl Laboratories
(Montgomery, TX). The specificity of this antibody was tested using the
wild type
1C subunit, the
1C
1905
subunit that lacks the CT4 fragment (Fig. 1), and CT4 expressed in
tsA201 cells (see Fig. 1C).
1C Subunit in Mammalian Cells--
Various C-terminal
fragments of the
1C subunit (see Fig. 1) were expressed
in tsA201 cells (HEK293 cells transformed with large T-antigen (18))
using the following strategies. The C-terminal domain (CT) containing
amino acids 1623-2171 of
1C was excised as a
BglII/BamHI fragment. The CT4 (containing amino
acids 1907-2171) and CT23 (containing amino acids 1623-1905)
fragments were derived from pCR3
1C and
pCR3
1C
1905 constructs as
BamHI/BamHI and BglII/BamHI fragments, respectively. The CT7 fragment containing amino acids 2026-2171 was derived from pCR3
1C
2024 construct as a
BamHI/BamHI fragment. A fusion protein expression
vector, pCR3His/Myc, was derived from pCR3 (Invitrogen) by inserting a
6×-His tag and six copies of the Myc epitope into the multiclonal
sites of the original vector to allow expression of a protein with both
6×-His- and Myc tag fused to the N terminus. To create expression
constructs encoding different regions of the C terminus of the
1C subunit, the cDNA fragments of CT, CT4, CT7, and
CT23 were subcloned into the BamHI-digested pCR3His/Myc
vector, resulting in in-frame fusions of the C-terminal fragments to
the 6×-His and six copies of Myc tags on the vector. TsA201 cells were
maintained in Dulbecco's modified Eagle's medium (Life Technologies,
Inc. containing 10% fetal bovine serum (Life Technologies, Inc. and
1% penicillin/streptomycin at 37 °C in 5% CO2.
Transient expression of the Myc-tagged C-terminal fusion proteins in
tsA201 cells along with wild type or mutant rabbit
1C
(CaV1.2) subunits (3) and the rat
2a subunit
(19) was carried out using a total of 30-40 µg of plasmid DNA/100-mm plate and either the calcium phosphate precipitation method or the
transfection reagent Effectene from Qiagen as described (15, 20). For
electrophysiological experiments, tsA201 cells were plated onto 6-cm
culture dishes to achieve ~40-60% confluency. The indicated
1C subunit construct cDNAs were co-transfected with
2a subunit cDNA at 1.5 µg each along with 0.2 µg
of CD8 cDNA (as an expression indicator (21)). On the day of the
experiment transfected cells were washed with phosphate-buffered
saline, dissociated using trypsin-EDTA (Life Technologies, Inc.) and
transferred onto either 35-mm culture dishes or 12-mm glass coverslips,
each previously coated with rat tail collagen type VII (1 µg/ml) to achieve ~40% confluency. Cells were allowed to settle on the plate for 2 h before electrophysiological recordings.
1C in bacteria as GST fusion proteins, the cDNAs
encoding CT, CT4, CT7, CT8, CT12, CT14, or CT23 (see Fig. 1) were
subcloned into the BamHI site of the GST fusion protein
expression vector pGEX-5X-2 (Amersham Pharmacia Biotech). The
GST-tagged C-terminal fusion proteins were expressed in
Escherichia coli BL21 and purified using glutathione-agarose after standard procedures (Amersham Pharmacia Biotech). CT7 and CT8
also were expressed and purified as 6×-His-tagged fusion proteins using pQE32-CT7 and pQE30-CT8 and standard procedures. The fusion proteins were used in electrophysiological assays and in GST pull-down experiments. The GST pull-down experiments were performed using various
GST-C-terminal fusion proteins containing CT, CT4, CT7, CT8, CT12,
CT14, CT23 (see Fig. 1B) as well as NT (N-terminal amino
acids 1-154), L1 (loop between conserved domains I and II, amino acids
437-554), and L2 (loop between conserved domains I and II, amino acids
785-930). In each reaction, purified 6×-His-CT7 was diluted into 0.5 ml of binding buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, plus protease inhibitors (15)) and added to 50 µl
of glutathione-Sepharose beads precoupled to either GST alone (control)
or to the various GST-C-terminal, N-terminal, or loop constructs.
Incubations were carried out for 4-6 h at 4 °C with agitation. The
GST beads were washed 4 times with 1 ml of washing buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.4), and after the
last wash, 1/5 volume of SDS-polyacrylamide gel electrophoresis Laemmli
buffer (22) was added to each reaction. The proteins were separated by
SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose, and the bound 6×-His-CT7 was detected by
immunoblotting with the CT4 antibody.
1C subunit-specific antibodies, Card I or CI2, coupled to Ultra-link protein G (Pierce) at 4 °C overnight. The immunoprecipitates were washed with wash buffer (20 mM
Na2HPO4, pH 7.4, 150 mM NaCl, and 0.1% Triton X-100, 3×1 ml) and analyzed using SDS-polyacrylamide gel
electrophoresis and immunoblotting. The detection of the
1C subunits was with the Card I or CI2 antibodies, as
specified in the figure legends. Detection of co-immunoprecipitated
fusion proteins was with the CT4 or anti-Myc antibodies.
-aminoethyl ether)
N,N,N',N' tetraacetic acid
(EGTA), 78 mM methane sulfonic acid, and 78 mM
n-methyl-D-glucamine (corrected to pH 7.3 with CsOH; if necessary, adjusted to 330 mosmol with methanesulfonate N-methyl-D-glucamine). To perform
electrophysiological experiments, the cells were placed in a bath
chamber that was perfused constantly with a solution containing 30 mM NaCl, 10 mM BaCl, 5 mM HEPES, 20 mM CsCl, 1 mM MgCl2, 78 mM methanesulfonic acid, and 78 mM N-methyl-D-glucamine. The pH was corrected to
7.4 with NaOH, and osmolarity was adjusted to 330 mosmol.
90 mV were recorded and are
presented as the peak inward current plotted on the ordinate versus time starting from when the cell was patched and
intracellular access was first obtained on the ordinate. Peak currents
were analyzed using the ISO-2 (MFK, Frankfurt/Main, Germany) analysis software and normalized to the whole cell capacitance. The data for
each condition were pooled and expressed as the mean ± S.E. All
experiments were repeated at least four times.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1C L-type Ca2+ Channel Results in
Potentiated Channel Currents--
Previous studies demonstrate that
truncation of 307-472 amino acids from the C terminus of
1C (in mutants
1C
1856,
1C
1733, and
1C
1700) produced
increased channel currents in Xenopus oocytes (9),
suggesting that the C terminus contains inhibitory elements. To test
the hypothesis that C-terminal fragments of the
1C
subunit might associate with and regulate the conductance of
C-terminal-truncated
1C subunits, we determined if
expressed C-terminal fragments could reconstitute the inhibition of
currents when co-expressed with C-terminal-truncated mutants of
1C. To do so, we first prepared and analyzed currents
from several different mutants. Using 10 mM
Ba2+ as a charge carrier, whole cell current density was
compared from channels containing the full-length
1C
subunit or from the C-terminal deletion mutants
1C
2024,
1C
1905, and
1C
1733 (see Fig. 1).
Each construct was transiently expressed in tsA-201 cells, and all
channel constructs were co-expressed with the rat
2a subunit (19). The current-voltage relationships of channels containing
full-length
1C,
1C
2024,
1C
1905, or
1C
1733 were determined
(Fig. 2). The full-length channel
displayed a characteristic L-type current-voltage profile with the
maximal peak IBa at 0 mV and a mean current
density of 4.97 ± 1.3 (mean ± S.E., n = 4)
pA/pF. In marked contrast, all three truncation mutants displayed significantly larger currents at all voltages between
20 and +40 mV.
The current-voltage (I-V) relationship obtained from cells expressing
the
1C
2024 subunit displayed maximal current at +10 mV with an average peak IBa of 60.1 ± 19.2 pA/pF (n = 5). Similarly, currents from cells
expressing the
1C
1905 or
1C
1733
subunits also exhibited peak currents between 0 to +10 mV with current densities of 59.3 ± 12.9 pA/pF (n = 4) and
44.7 ± 13.4 pA/pF (n = 6), respectively. These
results demonstrated that expression of any of the three truncation
mutants gave rise to markedly larger currents in mammalian cells in a
manner similar to that previously described for the truncation mutants
1C
1856,
1C
1733, and
1C
1700 that were expressed in Xenopus
oocytes. More importantly, the results demonstrated that a C-terminal
truncation of the
1C subunit by as little as 147 amino
acids (
1C
2024) displayed markedly enhanced currents
when compared with the intact full-length channel. This suggested that
the inhibitory motif might be contained between amino acids 2025 and
2171.
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Fig. 1.
C-terminal fusion proteins of the
1C subunit and the
1C subunit constructs used. A
schematic map of the
1C subunit constructs
(A) and the C-terminal constructs (B) used in
this study is shown. I, II, III, and IV refer to the four repeated
domains of the
1C subunit. The C terminus of each
construct is depicted with the starting and ending sites labeled. The
beginning of the C terminus is amino acid residue 1507, and the last
amino acid is 2171. WT, wild type. C, Western
blots depicting the C-terminal fusion proteins used in the
electrophysiological assays. The left side of the blot
depicts the specificity of the CT4 antibody. TsA201 cells were
transfected with either the wild type
1C and
2 subunits (lanes 1 and 4) or the
1C
1905 and
2 subunits (lanes
2 and 5) or with 6×-His-CT4 only. Lysates from cells
containing channel subunits were immunoprecipitated with the Card I
antibody (lanes 1, 2, 4, and
5). Lysates from cells expressing CT4 were enriched for CT4
by concentration on nickel resin (lanes 3 and 6).
Western blotting was performed with the CT4 antiserum (lanes
1-3) or with pre-immune sera (lanes 4-6). Note the
reactivity of the CT4 antiserum with the wild type
1C
subunit (lane 1) but not with the
1C
1905
subunit (lane 2), which lacks the CT4 fragment (see
panel A). The right side of the figure is a
Western blot of the C-terminal fragments used in the
electrophysiological assays. The lanes contained purified
GST-CT (lane 1), GST-CT4 (lane 2), 6×-His-CT8
(lane 3), 6×-His-CT7 (lane 4), and GST-CT23
(lane 5). The blot was probed with the CT4 antibody
(lanes 1-4) or with the CT1 antibody (lane
5).
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Fig. 2.
Enhanced IBa current in
1C truncation mutants. Wild type
or C-terminal-truncated
1C subunits (refer to Fig. 1)
were expressed in tsA201 cells along with the
2a
subunit. Barium currents were measured as described under
"Experimental Procedures." Cells were maintained at a
90-mV holding potential and depolarized for 50 ms at indicated
potentials (pulse applied at 10-s intervals). Currents were normalized
to cell capacitance. The results shown are the means ± S.E. The
numbers in parentheses indicate the number of
experiments performed for each construct; data were obtained from a
minimum of three individual transfections.
1C C-terminal Fragments to
Cells Expressing Truncated
1C Channels Resulted in
Time-dependent Reduction of IBa--
We next
tested if application of C-terminal fragments would inhibit the
C-terminal-truncated
1C subunits. We first tested if the
CT fragment, corresponding to amino acids 1622-2171 (Fig. 1), would
result in inhibition of IBa from
1C
2024 and
1C
1905. GST-CT was
applied through the patch clamp pipette at a peptide concentration of 1 µg/ml pipette solution. During recordings, cells were maintained at
90 mV holding potential and depolarized to +10 mV for 50 ms at 10-s
intervals. In the control cells, currents in both the
1C
1905 (Fig. 3)- and
1C
2024 (data not shown)-expressing cells exhibited a
time-dependent increase in currents that began immediately
upon patching the cells. Peak currents were achieved within ~3 min
after access to the cell was achieved and were maintained with minimal
decrease (<10%) during the 15-min recording period (Fig. 3). In cells
exposed to CT, peptide currents also showed an initial increase in
current density (Fig. 4). However, a
reduced peak, compared with control, was attained within 2 min of open access to the cytosol (Fig. 4). Current continued to decline such that
IBa was markedly reduced compared with control
after a 6-10-min exposure of either
1C
2024 or
1C
1905 to CT (Fig. 4, A and B, and Table I). The inhibitory effect of CT
was essentially abolished when the CT peptide was boiled for 5 min
before addition to pipette solution (Table I). These data demonstrated
that the inhibition of channels could be reconstituted by applying the
CT peptide to cells expressing the C-terminal-truncated mutants,
1C
2024 and
1C
1905. Furthermore the
structural integrity of the peptide was necessary for the peptide to
exert its inhibitory effect. These data provided initial support for
the hypothesis that C-terminal fragments of
1C can
associate with and regulate channel activity in intact cells.
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Fig. 3.
Current traces and time course of
1C
1905 +
2a channels expressed in tsA 201 cells. Cells were held at a
90-mV potential and depolarized for
50 ms at +10 mV at 10-s intervals. Upper panel,
representative traces taken upon open access to the cell (0 min) and at
subsequent 5-min intervals in cells expressing
1C
1905 +
2a subunits. Lower panel, time course
plot of peak current depicted above. Results are shown from a
representative experiment.
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Fig. 4.
Cytosolic application of CT peptide results
in time-dependent attenuation of peak
IBa. Time course of currents from cells
expressing 1C
1905 +
2a (A)
or
1C
2024 +
2a subunits (B)
or in the presence or absence of GST-CT (1 µg/ml) applied via the
patch clamp pipette solution. Results shown are the means ± S.E.
The numbers in parentheses indicate the number of
experiments performed for each construct.
Effects of C-terminal fragments on IBa in cells expressing
1C
1905 or
1C
2024 channel subunits
1C
1905 and
1C
2024, respectively (see Fig. 1). Application of the
GST-CT4 peptide to cells expressing either
1C
2024 or
1C
1905 caused a marked inhibition of currents in a
manner similar to the CT peptide (Fig. 5
and Table I). An early and reduced current peak was observed within 4 min of application of CT4; current density was markedly reduced
compared with control in
1C
2024- and
1C
1905-expressing cells from 4 to 10 min after the
start of the perfusion (Fig. 5, A and B, Table
I). Boiling the CT4 peptide again abolished the inhibitory effect
(Table I).
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Fig. 5.
Inhibition of IBa
currents by application of CT4. Time course of
peak currents from cells expressing either the 1C
1905 +
2a (A) or
1C
2024 +
2a subunits (B) in the presence or absence of
GST-CT4 (1 µg peptide/ml pipette solution) that was dialyzed into the
cell via the patch clamp pipette. Results shown are the means ± S.E., with the number of experiments shown in
parentheses.
1C
2024 or
1C
1905. However, since
truncation of the CT7 fragment in the
1C
2024 mutant
resulted in a loss of inhibition (Fig. 2), we next asked if a different
type of CT7 fusion protein, 6×-His-CT7, could inhibit the
C-terminal-truncated mutants. Conceivably, the GST construct may have
hampered the presentation of CT7 to the channels. Application of
6×-His-CT7 to cells expressing
1C
1905 caused a
marked reduction from control within 6-8 min after access to the
cytosol (Fig. 6A, Table I).
The 6×-His-CT7 also caused inhibition of currents from
1C
2024 (Fig. 6B, Table I). The effects of
CT7 were similar to those of CT and CT4, although the inhibition
appeared to be less robust than that caused by CT4. In particular, the
inhibition of currents from
1C
2024 by CT7 appeared to
develop more slowly and to a smaller extent that that caused by CT4
(compare Figs. 5 and 6, Table I). Nevertheless, these results
demonstrated that application of CT7 could reconstitute inhibition of
currents from either
1C
1905 or
1C
2024. The effects of CT7 were resistant to boiling.
It is well known that the activity of many small proteins, such as
calmodulin, can be resistant to denaturation (24), and thus, the lack
of inhibition of the Ba2+ current during intracellular
dialysis with the small, boiled 6×-His-CT7 was not surprising.
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Fig. 6.
Inhibition of IBa
currents by application of CT7. Conditions were
as in Fig. 5 except that 6×-His-CT7 (1 µg peptide/ml pipette
solution) was added through the pipette. Results shown are the
means ± S.E., with the number of experiments shown in
parentheses.
1C. As a control, we tested GST alone and
found it had no effects on the currents (Fig.
7, A and B, Table
I). Peptide GST-CT23, corresponding to amino acids 1622-1905, also had
no effect on channel currents obtained from either
1C
2024 and
1C
1905 (Fig. 7,
A and B, Table I). Since CT4 appeared to be more
efficacious than CT7 (the C-terminal half of CT4), we tested whether
CT8, which corresponded to the N-terminal half of CT4 (amino acids
1905-2024, Fig. 1), had any inhibitory activity. Conceivably CT4 might
be a better inhibitor because an additional inhibitory domain might be
contained within the CT8 fragment. However, neither GST-CT8 (data not
shown) nor 6×-His-CT8 (Fig. 7, A and B, Table I)
had any effect on channel currents. Taken together, these results
indicated that the inhibition of channel currents by the C terminus
could be reconstituted by applying fragments as small as CT7
(corresponding to the most distal 144 amino acids of the C terminus) to
cells expressing truncated
1C subunits. The more
effective inhibition of currents by CT4 compared with CT7 may have been
due to a more effective association of CT4 with the truncated channels
(see below).
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Fig. 7.
Effect of other C-terminal peptides on
channel current. Cells expressing the 1C
1905
(A, B, and C) or
1C
2024 (D, E, and F)
subunits were presented with GST alone (A and D),
GST-CT23 (B and E), or 6×-His-CT8 (C
and F) (refer to Fig. 1). All fusion proteins were applied
at 1 µg peptide/ml pipette solution, and results shown are the
means ± S.E. with the number of experiments shown in
parentheses.
1C
1733 Subunit Were
Insensitive to Inhibitory Peptides--
The CT7 and CT4 peptides
induced functional inhibition of currents generated through
1C
2024 and
1C
1905. We asked whether the inhibition could be produced with an
1C subunit that
was truncated farther upstream, such as the
1C
1733
truncation mutant. Interestingly, application of CT4 to cells
expressing the
1C
1733 truncation mutant caused a
modest inhibition, but this was not significantly different from
control (Fig. 8, Table I). Similarly, CT7, both at the concentration (1 µg/ml, data not shown) that caused
inhibition of
1C
2024 and
1C
1905 and
at a 4-fold higher concentration (Fig. 8, Table I) caused little or no
inhibition of currents from
1C
1733. These results
suggested that the section of the C terminus between amino acids 1733 and 1905 was necessary for the inhibition caused by pipette application
of either CT4 or CT7. Conceivably amino acids 1733-1905 might be the
"receptor" for CT7 that allows for channel inhibition.
Alternatively, CT7 might interact with another domain to cause
inhibition, and amino acids 1733-1905 might play a role in helping to
present CT7 to the inhibitory receptor. As such, amino acids 1733-1905
might act to anchor or stabilize the inhibitory domain within CT7.
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Fig. 8.
Lack of effect of CT4 and CT7 on current
density from channels containing
1C
1733.
Conditions were as described in Fig. 5, except currents were from
channels containing
1C
1733. Effects of GST-CT4 (1 µg peptide/ml pipette solution, A) and 6×-His-CT7 (at 4 µg peptide/ml pipette solution, B) were tested. The
results shown are the means ± S.E., with the number of
experiments shown in parentheses. Neither CT4 nor CT7
produced statistically significant effects. Ctrl,
control.
1C
1733-1905 and
1C
1733-2024, were essentially the
1C
1733 construct containing a tethered CT4 or CT7,
respectively (see Fig. 1). Currents from these constructs were recorded
and compared with those from
1C
1733. The
current-voltage relationships demonstrated that
1C
1733-1905 and
1C
1733-2024 each
had current profiles that were similar to
1C
1733, but
the peak currents were drastically reduced compared with those from the
1C
1733 subunit (Fig. 9,
Table II). In contrast, both
1C
1733-1905 and
1C
1733-2024
produced peak currents that were comparable with those of the "fully
inhibited" wild type
1C subunit (compare Fig. 9 with
Fig. 2). These results were consistent with the idea that a tethered
CT4 or CT7 could inhibit channels lacking the fragment corresponding to
amino acids 1733-1905 or 1733-2024. To further test the concept that
the tethered CT7 was responsible for the inhibition of the channels, we
created an additional construct,
1C
1733-1905
2024,
which was similar to
1C
1733-1905 but in addition
contained a deletion of CT7. Thus, this construct was predicted to
produce the large currents seen with other constructs lacking CT7.
Indeed, the currents from
1C
1733-1905
2024 were large and comparable to those obtained from
1C
1733
and much greater that those from the parent construct
1C
1733-1905 (Fig. 9, Table II). These results
confirmed that the tethered CT7 in
1C
1733-1905 was
responsible for causing channel inhibition, as the removal of CT7 in
1C
1733-1905
2024 gave rise to large, "uninhibited" currents. That the tethered CT4 or CT7 could cause inhibition in constructs lacking amino acids 1733-1905 suggested that
CT7 interacted with something other than these amino acids to cause
inhibition of the channels. However, since CT7 could not inhibit
1C
1733, which lacks these amino acids, the data are
consistent with the idea that amino acids 1733-1905 play an important
role in presenting CT7 to the inhibitory receptor. This concept
suggests that CT7 might have multiple binding sites.
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Fig. 9.
Inhibition of currents from channels
containing
1C
1733 by a
tethered CT4 or CT7. Currents were recorded from channels
containing mutant
1C subunits containing a deletion of
amino acids 1733-1905 or 1733-2024 (
1C
1733-1905
and
1C
1733-2024, respectively) or a deletion of
amino acids 1733-1905 and 2024-2171
(
1C
1733-1905
2024) (see Fig. 1). Current-voltage
relationships were determined as described in the legend to Fig. 2.
Results are the means ± S.E. with the number of experiments shown
in parentheses.
Peak currents from mutants containing or lacking tethered C-terminal
fragments
1C subunits in tsA
cells. First, CT4 and CT7 in the pCR3His/Myc vector were transiently
transfected into tsA201 cells, and expression was ascertained by
SDS-polyacrylamide gel electrophoresis and immunoblotting. Staining
with the anti-Myc or CT4 antibodies revealed expression of CT4 and CT7
(see Figs. 10 and 11). Next, we tested
whether the C-terminal fragments of the
1C subunit could
directly associate with the channel subunits. C-terminal fragments were
co-expressed with full-length wild type
1C or
1C
2024,
1C
1905,
1C
1733 subunits and the
2a subunits. Whole cell lysates were prepared from the transfected cells and immunoprecipitated with the CI2 antibody. When the channel subunits were co-expressed with CT4 and immunoprecipitated with the CI2 antibody, which is directed against the II-III loop of
1C and does not recognize CT4, CT4 was
co-immunoprecipitated with the
1C subunits (Fig. 10).
The
1C subunits in the immunoprecipitates were detected
on the blot using the Card I antibody (Fig. 10, upper panel), and the co-immunoprecipitated CT4 was detected by the anti-myc antibody (Fig. 10, lower panel). As a negative
control, the CI2 antibody did not immunoprecipitate the CT4 fusion
protein in the absence of the
1C subunits (Fig. 10).
Surprisingly, CT4 co-immunoprecipitated not only with
1C
2024 and
1C
1905 but also with
1C
1733 (Fig. 10), which was not inhibited by CT4
(Fig. 8). However, more CT4 was present in the immunoprecipitates with wild type
1C and
1C
2024 or
1C
1905 compared with those containing
1C
1733. These results suggested that there might be
multiple interaction sites for CT4 and that
1C
1733
lacks an interaction site that is necessary for channel inhibition. The
results are consistent with the concept that amino acids 1733-1905 may
be critical for allowing inhibition by CT4. The observation that CT4
could bind to full-length
1C suggested that the
interaction sites for CT4 were available in this protein.
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Fig. 10.
Co-immunoprecipitation of CT4 with the
channel subunits. TsA201 cells were co-transfected with CT4, the
rat 2a subunit, and wild type or mutant
1C subunits as indicated. Whole cell lysates were
prepared from the transfected cells, and the channel subunits were
immunoprecipitated with the CI2 antibody. For cells expressing CT4
alone, CT4 was concentrated by absorption onto a nickel resin. The
immunoprecipitates (lanes 1-4) or concentrated lysates
(lane 5) were electrophoresed on a 5-15% gradient
(acrylamide) SDS gel and transferred to a filter for immunoblotting.
The filter was cut in half, and the immunoprecipitated
1C subunits (wild-type or deletion mutants) were
detected on the top portion of the immunoblot with the Card I antibody,
whereas the co-immunoprecipitated CT4 fusion proteins were detected on
the bottom portion using the anti-Myc antibody. The lane
marked CT4 alone represents the concentrated CT4 from cells
transfected with only this vector.
1C
2a or with the various C-terminal
deletion mutants of
1C in combination with
2a subunits in tsA cells. In contrast to CT4, the CT7
fusion protein did not co-immunoprecipitate with either the wild type
or the mutant
1C subunits (data not shown). However,
because CT7 was effective in inhibiting channel activity but appeared
less robust in causing inhibition than CT4 (Figs. 4 and 5; Table I), we
reasoned that the association of CT7 might have been weaker and
disrupted during the detergent solubilization and repeated washings of
the immunoprecipitates. Thus, we asked if inclusion of a cross-linking
agent during the immunoprecipitation might allow for detection of the
association of CT7 with the channels. For these studies we used the
cross-linking agent Ni(II) complex of the tripeptide
NH2-Gly-Gly-His-COOH, which has been shown to be highly
specific in that only proteins that specifically associated could be
cross-linked (23). The lysates from cells expressing CT7 and mutant or
wild type
1C subunits were incubated with the cross-linking agent for 10 min at room temperature, and the
reactions were quenched with thiourea. When the channels were
immunoprecipitated with the CI2 (data not shown) or Card I antibodies
(Fig. 11), both of which are directed
against the internal II-III linker of
1C and do not
recognize CT7, CT7 was co-immunoprecipitated (Fig. 11). Taken together,
these results demonstrated that the CT7 could directly associate with
the channels in intact cells. In addition, the results supported the
observations that, although the inhibitory domain appeared to be
contained within CT7, CT4 appeared to associate with the channels more
effectively.
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Fig. 11.
Co-immunoprecipitation of CT7 with the
channel subunits. TsA201 cells were co-transfected with CT7, the
rat 2a subunit, and wild type or mutant
1C subunits as indicated. Whole cell lysates were
prepared from the transfected cells and subjected to cross-linking with
the Ni(II)-GGH complex as described under "Experimental
Procedures." The lysates were diluted, and the channel subunits were
immunoprecipitated with the Card I antibody. For cells expressing CT7
alone, CT7 was concentrated by absorption onto a nickel resin. Other
conditions were as in Fig. 10. The Western blot was cut in half, and
the immunoprecipitated
1C subunits (wild type or
deletion mutants) were detected on the top portion of the immunoblot
with the CI2 antibody, whereas the co-immunoprecipitated CT7 fusion
proteins were detected on the bottom portion using the CT4 antibody.
The lane marked CT7 alone represents the
concentrated CT7 from cells transfected with only this vector.
1C in GST
Pull-down Assays--
Since CT7 appeared to contain the inhibitory
domain, it was of interest to identify the binding sites for CT7. GST
pull-down assays were performed with bacterially expressed
6×-His-tagged CT7 and GST constructs derived from the intracellular
domain of
1C. CT7 bound to GST constructs of CT, CT4,
CT12, CT23, and CT14 but not to CT8, NT, L1, L2, or GST alone (Fig.
12). A common site shared by CT, CT4,
CT12, and CT23 is contained within amino acids 1733-1905. These data
along with those obtained from the electrophysiological and
co-immunoprecipitation data suggest that one interaction site for CT7
is amino acids 1733-1905. However, CT7 also bound to CT14 (amino acids
1623-1733), which is upstream of 1733-1905. This is consistent with
the idea that there are multiple interaction sites for CT7. That the
CT14 site is upstream of amino acid 1733 is consistent with the result
that CT7 associates with
1C
1733 but more poorly than
to other
1C constructs that contain both the CT14 site
and amino acids 1733-1905. In addition, since CT7 can bind to
1C
1733 but cannot inhibit channel activity, it
suggests that binding to CT14 is not sufficient for channel inhibition. It may be that binding of CT7 to amino acids 1733-1905 is important for presenting CT7 to sites that are necessary to cause inhibition, whereas in the constructs such as
1C
1733-1905 and
1C
1733-2024, the tethering of CT7 is sufficient to
allow it to interact with the inhibitory receptor.
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Fig. 12.
GST pull-down assays depicting interaction
of CT7 with C-terminal fragments. GST pull-down assays were
performed as described under "Experimental Procedures" by applying
purified 6×-His-CT7 to glutathione-Sepharose beads containing
immobilized GST-CT (lane 1), GST-CT4 (lane 2),
GST-CT8 (lane 3), GST-CT12 (lane 4), GST-CT14
(lane 5), GST-CT23 (lane 6), GST-NT (lane
7), GST alone (lane 8), GST-L1 (lane 9), or
GST-L2 (lane 10). The reactions were electrophoresed on a
15% acrylamide SDS gel and transferred to a filter for immunoblotting.
The blot was probed for the presence of bound CT7 with the CT4
antibody.
1C subunit can associate with
and regulate the activity of L-type Ca2+ channels
containing a truncated
1C subunit. Truncation of the
1C subunit by 147 amino acids, as in the
1C
2024 subunit, was sufficient to relieve the
inhibition caused by the presence of a full-length C terminus. In
addition, application of the fragment corresponding to the deleted
amino acids, CT7, to cells expressing channels containing an
1C subunit truncated at either position 2024 or 1905 reconstituted channel inhibition. Amino acids 1733-1905 appeared to be
important to allow for this reconstituted inhibition by the CT7 or CT4
fragments, as neither CT7 nor CT4 were able to effectively inhibit
channels truncated at position 1733. However, amino acids 1733-1905
did not appear to be the only receptor for CT7, as the tethered CT7 in
mutants
1C
1733-1905 and
1C
1733-2024 appeared to be capable of inhibiting
currents in the absence of amino acids 1733-1905. It is likely that
these amino acids help to position CT7 to a receptor that allows for
the inhibitory effects to be expressed.
1C subunit appears to
be truncated at the C terminus to a protein of ~190 kDa in native tissues including heart and brain (10, 11, 25, 26). In contrast, in
heterologous expression systems, the
1C subunit has been
found to be a full-length protein (e.g. Refs. 5, 15, and
16). It was not obvious if the truncation observed in the native
systems was an artifact that occurred upon channel isolation or the
result of a physiological processing event. However, earlier findings
demonstrated that the C-terminal domain could be visualized in intact
cardiac myocytes using an immunocytochemical approach (10). In
addition, we found that exogenous chymotrypsin can cleave full-length
expressed
1C subunits into a 190-kDa body that is very
similar to the ~190-kDa fragment observed in native tissues and
C-terminal fragments of 35-50 kDa (17). Interestingly, the C-terminal
fragments remained associated with the membrane after cleavage (17). A
proline-rich domain was identified between amino acids 1974 and 2000 and found to be important for the tethering of the C-terminal fragments
to the membrane (17). Here we have presented complimentary findings
that demonstrated that the C-terminal fragments associated with
C-terminal-truncated channels and regulated channel activity. The
domain termed CT7 was found to contain an inhibitory domain; however
CT4, which contained the CT7 sequence, appeared to interact with the
1C subunit more effectively. Conceivably the difference
in abilities of CT7 and CT4 to associate with the channels was due to
the proline-rich domain. CT7 lacks the proline-rich domain (17),
whereas CT4 contains this motif.
1C subunit is a
complex and important component of this protein. The present results together with those of an earlier study (17) support the hypothesis that the
1C subunit may undergo a physiologically
important processing event in native systems and that the C-terminal
fragments may remain associated with the channels to allow for channel
regulation. A simplified version of our view of how the C terminus of
the channel functions to regulate channel conductance is depicted in
the schematic presented in Fig. 13. An
extension of these conclusions is that if the endogenous C-terminal
fragments do remain associated with the channels, one might predict
that injection of the exogenous C-terminal peptides into cardiac
myocytes would have little or no effect. Indeed, in preliminary
experiments, we tested the effects of CT4 on L-type currents from adult
rabbit cardiac myocytes and found that the exogenously applied CT4 did
not inhibit their whole cell L-type Ca2+ current (data not
shown). Further experiments will be necessary to define the point of
cleavage and the nature of the C-terminal fragments in native
systems.
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Fig. 13.
Schematic depicting how distal C terminus
(DCT) of the L-type Ca2+
channel regulates channel conductance. The C
terminus of the full-length 1C subunit contains several
regulatory domains within the C terminus that are identified and
located schematically. Our previous work indicates that the C terminus
of the majority of the full-length channel is cleaved proximal to the
1900-amino acid region into both proximal and distal portions. The
present data (obtained using the CT4 and CT7 peptides) suggest that the
portion of the distal C terminus between amino acid 2024 and 2171 (corresponding to CT7) serves as an inhibitory domain. The efficacy for
inhibition is enhanced by the peptide region 1909-2024 (included in
CT4 but absent from CT7). Inhibition mediated by the cleaved peptide
inhibitory domain requires the peptide region between 1733 and 1905 on
the proximal portion of the C terminus, and therefore, we suggest that
this region contains a distal C terminus binding region. In this way
the distal C terminus functions to inhibit and thus regulate channel
conductance through and in response to an as yet undefined mechanism.
Also, shown for completeness are several other known regulatory domains
including the EF-hand, calmodulin binding domain (CBD) (7),
the proline-rich domain (PRD) (17), and the Ser-1928 residue
(the only site capable of in vivo phosphorylation)
(5).
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL23306 (to M. M. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by National Research Service Award Training Grant
Fellowships T32-DK07169.
§ Supported by Deutsche Forschungsgemeinschaft Postdoctoral Fellowship BU1133/1/1.
¶ To whom correspondence should be addressed: Dept. of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave. S215, Chicago, IL 60611. Tel.: 312-503-8286; Fax: 312-503-5349; E-mail: r-teneick@northwestern.edu
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M008000200
1 T., Gao and M. M. Hosey, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: CT, C-terminal domain; GST, glutathione S-transferase.
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