From the Department of Physiology and Pharmacology, Sackler School
of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel
 |
INTRODUCTION |
In the heart, Ca2+ current via the
voltage-dependent L-type channels
(dihydropyridine-sensitive) underlies the plateau of the action
potential and provides calcium ions necessary for initiation of cardiac
cell contraction (2). Similar channels are found in smooth muscle,
where they play a major role in regulation of tonus and contraction (3,
4), and in the nervous system (5, 6). L-type channels are composed of
the following three subunits: the main, pore-forming
1C,
the cytosolic
2, and the
2
subunit which is mostly
extracellular (5, 7-11).
1C contains four homologous
membrane domains numbered I-IV, each one with six transmembrane
segments and a re-entrant P-loop that forms the pore lining; N- and
C-terminal domains and the linkers connecting the domains I-II,
II-II, and II-IV are cytosolic (see Ref. 7 for review, and see Fig.
6A for a scheme). The C terminus was implicated in
Ca2+- and voltage-dependent inactivation
(12-15) and modulation by protein kinase A (16-19); linker I-II
contains the binding site for the
subunit (20, 21).
Cardiac and smooth muscle L-type channels are tightly regulated by
hormonal and neuronal signals via G proteins and protein kinases (22,
23). Protein kinase C (PKC)1
is one of such regulators; its actions appear to be tissue- and species-specific. PKC activators, such as phorbol esters and
diacylglycerols, increase Ca2+ channel currents in cardiac
and smooth muscle cells of various mammals (24-33), and PKC has been
implicated in mediating the stimulation of Ca2+ channels by
intracellular ATP (34), angiotensin II (26), glucocorticoids (28),
pituitary adenylate cyclase-activating polypeptide (33), and
arginine-vasopressin (32). PKC up-regulation results from changes in
channel gating because it is accompanied by an increase in single
channel open probability, Po (30, 35, 36). In
many cases, a biphasic effect of PKC activators has been described,
with an increase followed by a later decrease (25, 27, 30), and some
preparations such as adult guinea pig heart cells (37, 38) respond to
phorbol esters only by a decrease in Ca2+ currents, an
effect that may not be mediated by PKC (38). The biphasic response to
PKC stimulators is fully reconstituted when expression of L-type
channels in Xenopus oocytes is directed by RNA extracted
from rat heart (39, 40) or cRNA of rabbit cardiac
1C
subunit (39). Increase of Ca2+ channel activity by phorbol
esters has also been observed in a mammalian cell line (baby hamster
kidney) expressing the rabbit cardiac
1C (36). The
potentiation by phorbol esters of Ca2+ channels expressed
in the oocytes is mediated by PKC because it is mimicked by
diacylglycerols and blocked by specific PKC inhibitors (39, 40).
Both
1C and
are substrates for PKC-catalyzed
phosphorylation (Ref. 41 and references therein).
1C
subunit has been recognized as the target for the Ca2+
channel enhancement caused by PKC, since coexpression of the auxiliary
subunits was not necessary to reproduce the effect of phorbol esters;
on the contrary, coexpression of the
subunit weakened the
enhancement suggesting a modulatory effect for this subunit (39).
However, it is not known which part of
1C is involved in
the PKC action.
1C isoforms cloned from rat brain (42)
and human heart (43) are not up-regulated by PKC (43, 44), suggesting
that the site of PKC action lies in one of the variable regions. More
specifically, Bouron et al. (43) proposed that
phosphorylation of the initial segment of the N terminus of the rabbit
heart isoform may account for PKC potentiation, but this hypothesis has
not been tested. It was unclear how this part of
1C can
affect the function of the channel, because in a recent publication Wei
et al. (1) reported that deletion of up to 120 initial
N-terminal amino acids strongly increased the whole cell
Ca2+ channel current and, proportionally, the total gating
charge movement but did not affect the voltage dependence of the charge movement or of the macroscopic current activation. It has been proposed
(1) that the N-terminal deletion causes an increase in the amount of
functional channels (hence the increase in total gating charge
movement) but does not affect channel function.
In the beginning of this study we set out to test which part of
1C accounts for the PKC-induced enhancement of the
rabbit heart L-type channel, using deletion and single-site mutagenesis and expression in Xenopus oocytes. We found that, as
predicted by Bouron et al. (43), deletion of the first
46 a.a. (which are thought to be unique to the rabbit heart
isoform) eliminates the PKC-induced enhancement. To understand whether
and how the N terminus affects the function of the channel, we have
undertaken a more elaborate study of the properties of N-terminal
deletion mutants and GST fusion proteins. Immunochemical and single
channel measurements demonstrated that N-terminal deletions do not
increase channel expression but rather enhance activation on single
channel level. We find evidence for an interplay between N terminus,
PKC, and the
subunit, although we could not detect any direct
binding between N terminus and
. Our results point to the
possibility that potentiation of L-type Ca2+ channels by
PKC, and part of the enhancement caused by the
subunit, may result
from attenuation of a tonic inhibitory control exerted by the N
terminus. Furthermore, since the enhancing effect of PKC on L-type
Ca2+ channels is widespread among mammalian species, our
data suggest that
1C isoforms with N termini of the
"rabbit heart" type must also be widespread.
 |
EXPERIMENTAL PROCEDURES |
DNA Constructs and mRNA--
cDNAs of rabbit heart
1C
(pCAH), rabbit heart
2A, and skeletal muscle
2/
subunits were
prepared and used as described previously (45, 46). The cDNAs of
the following mutants of
1C were made within the
original pCAH construct:
N88-139; st1665; S533I; S1575A
(16). A PCR-based approach was designed for engineering all the other
constructs used in this work. In an attempt to improve the expression
of channels composed of
1C alone, we subcloned the
coding sequence of
1C into a high expression vector,
pGEM-SB, derived from pGEM-HE which contains a 50-base 5'-untranslated
region and a 300-base 3'-untranslated region from Xenopus
-globin (47). pGEM-SB was produced by a standard PCR procedure used
to extend the polylinker which now includes the following restriction
sites: SmaI, BamHI, SalI,
ClaI, BstEII, EcoRI, XbaI,
and HindIII. By using standard PCR procedures, a SalI site was created immediately preceding the initial ATG
of
1C, and a HindIII site was created
following the termination codon. After a series of intermediate
subcloning and ligation steps, the coding sequence of
1C
was inserted between SalI and HindIII sites of
pGEM-SB. The resulting cDNA was termed
1C-SB; for
synthesis of sense RNA with T7 polymerase, it was linearized with
NheI. This procedure did not significantly improve the
currents directed by the expression of
1C RNA alone, as
compared with pCAH, but provided a convenient tool for the creation of
N-terminal deletion mutants. The cDNA of the neuronal
1C isoform, rbC-II, was kindly provided by T. P. Snutch (University of British Columbia; see Ref. 42); this DNA was
injected directly into the nuclei.
To create
1C N-terminal truncations, PCR amplification
with Vent polymerase (New England Biolabs) was performed using 100 ng
of WT
1C cDNA as template for 25 cycles of 30 s
at 95 °C, 1 min at 55 °C, and 2 min at 72 °C. For each
deletion mutant unique forward and reverse primers were used, with a
forward primer creating a SalI site followed by an
initiation codon and then by the original WT
1C sequence
starting from the desired base. For
N2-46, the forward
primer was 5'-AT GTC GAC TAA ACC ATG G330GT TCC
AAC TAT GG-3'. The reverse primer was 5'-GCT AAG GCC ACA CAA
TTG GC-3', which is complementary to nucleotides 687-706 and includes an endogenous unique MfeI site. The restriction
sites SalI and MfeI are underlined, respectively.
To create
N2-139, the forward primer was 5'-TAG CCG
GTC GAC ATG A609AG AAC CCC ATC CGG A-3'.
Reverse primer sequence was 5'-AG CTC AAT TTT CTC CTC CTT GGC CTC-3',
complementary to nucleotides 2673-2698 and is close but downstream
from the endogenous unique EcoRI site (the superscript
number in the oligonucleotide sequence indicates the corresponding
position in the rabbit cardiac
1C sequence) (48). The
amplified fragments were used to replace the corresponding fragment in
1C-SB DNA. The net result was the deletion from the second amino acid residue through the number indicated in the name of
construct.
DNAs of
1C fragments designed to create glutathione
S-transferase (GST) fusion proteins were constructed using a
similar PCR strategy, with primers containing the desired restriction sites. These fragments were cloned into pGEX-4T-1 (Amersham Pharmacia Biotech). The N-terminal cDNA fragment (N, encoding a.a. 1-154) and three C-terminal cDNA fragments (C, encoding a.a. 1505-2171, C1, encoding a.a. 1664-1845, and C2, encoding
a.a. 1821-2171) were inserted into EcoRI and
NotI restriction sites of pGEX-4T-1. GST-LI-II,
encoding a.a. 438-550, was inserted into EcoRI and XhoI sites of pGEX-4T-1. The C1 fragment was
additionally subcloned into pGEM-HE vector at the same restriction
sites, and two more N-terminal fragments, N1-139 and
N88-139, were created by a similar PCR procedure and also
inserted into pGEM-HE between EcoRI and HindIII
for in vitro transcription and expression in oocytes. All
PCR products were sequenced at the Tel Aviv University Sequencing
Facility. Capped mRNAs were synthesized in vitro using the suitable RNA polymerases, as described (49). When WT and one of the
mutant channels have been compared in electrophysiological experiments,
care was taken always to use RNAs derived from the same cDNA
vector. Materials and enzymes for molecular biology were purchased from
Boehringer-Mannheim, Promega, or MBI Fermentas.
Oocyte Culture and Electrophysiology--
Xenopus
laevis frogs were maintained and operated, and oocytes were
collected, defolliculated, and injected with RNA as described (49, 50).
In each experiment, oocytes were injected with equal amounts (by
weight) of the mRNAs of the various channel subunits in the desired
combinations and with RNAs of additional proteins as detailed in the
figure legends. Oocytes were incubated at 20-22 °C in ND96 solution
(96 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 5 mM HEPES, pH 7.5) supplemented with 1 mM CaCl2, 2.5 mM sodium pyruvate, and 50 µg/ml gentamycin. For patch clamp experiments, the vitelline membrane was removed with fine forceps after ~5 min incubation in the
bathing solution, as described (49). Whole cell currents were recorded
using two-electrode voltage clamp as described (45), in a solution
containing 40 mM Ba(OH)2, 50 mM
NaOH, 2 mM KOH, and 5 mM HEPES, titrated to pH
7.5 with methanesulfonic acid. Usually, the concentrations of
1C RNA of WT and N-terminal deletion mutants were not
equal but chosen in such a way that the amplitudes of the expressed
currents were below 3 µA, to avoid artifacts introduced by series
resistance and by Ba2+-activated Cl
currents
when larger currents are measured (51). Net Ba2+ currents
were obtained by a standard leak subtraction procedure or, when
1C subunit alone was expressed, by subtraction of
currents measured after inhibiting all Ca2+ channel
currents by 100 µM Cd2+. Absence of
contribution of the endogenous currents of the oocyte was verified by
inhibiting IBa with 10 µM nifedipine. Single
channel recordings were done in the cell-attached mode as described
(50), using Axopatch 200 amplifier (Axon Instruments, Foster City, CA). Pipettes contained 110 mM BaCl2, 10 mM HEPES/NaOH, pH 7.5. The oocytes were bathed in a
solution containing 130 mM KCl, 1 mM MgCl2, 10 mM HEPES/KOH, pH 7.5. Currents were
filtered at 2 kHz (4-pole Bessel) and sampled at 10 or 5 kHz. Voltage
steps from
80 to 10 mV lasting 140 or 280 ms were delivered every 1 or 2 s. Leak and capacitative currents were subtracted from the
traces using blank sweeps during the analysis session. Data acquisition and analysis were done with pCLAMP (Axon Instruments, Foster City, CA).
Immunochemistry--
This was performed as described (50, 52).
Oocytes were injected with mRNAs and incubated in NDE solution
containing 0.5 mCi/ml [35S]methionine/cysteine (Amersham
Pharmacia Biotech) for 3-4 days at 22 °C. Plasma membranes together
with the vitelline membranes (extracellular collagen-like matrix) were
removed manually with fine forceps after a 5-15-min incubation in a
low osmolarity solution. The remainder of the cell (internal fraction)
was processed separately. 10-30 plasma membranes and 10 internal
fractions were solubilized in 100 µl of buffer (4% SDS, 10 mM EDTA, 50 mM Tris, pH 7.5, 1 mM
phenylmethanesulfonyl fluoride, 1 mM pepstatin, and 1 mM 1,10-phenanthroline) and heated to 100 °C for 2 min.
Following the addition of 100 µl of H2O and 800 µl of
the immunoprecipitation buffer (190 mM NaCl, 6 mM EDTA, 50 mM Tris, pH 7.5, and 2.5% Triton
X-100), homogenates were centrifuged for 10 min at 1000 × g at 4 °C. The supernatant was incubated for 16 h
with the Card-I polyclonal antibody (53), incubated for 1 h at
4 °C with protein A-Sepharose, and pelleted. Immunoprecipitates were
washed 3 times with immunowash buffer (150 mM NaCl, 6 mM EDTA, 50 mM Tris-HCl, pH 7.5, 0.1% Triton
X-100, and 0.02% SDS). Samples were boiled in SDS-gel loading buffer and electrophoresed on 3-8% SDS-polyacrylamide gradient gel together with standard molecular mass markers (45-205 kDa). Gels were dried and
placed in PhosphorImager (Molecular Dynamics) cassette for up to 3 days. The protein bands of the image were estimated quantitatively using the software ImageQuant, as described (50, 54).
Binding of the GST Fusion Proteins to 35S-Labeled
Proteins--
This was done essentially as described (21).
[35S]Met/Cys-labeled
2A was translated on
the template of in vitro synthesized RNA using a translation
rabbit reticulocyte kit (Promega) according to manufacturer's
instructions. The fusion proteins were synthesized and extracted from
Escherichia coli according to pGEX-4T-1 manufacturer's instructions (Amersham Pharmacia Biotech). The protein concentration was estimated using the Bio-Rad protein assay kit (Munchen, Germany). Purified GST fusion proteins (5-10 µg) or purified GST (~10 µg) were incubated with 5 µl of the lysate containing the
35S-labeled
2A in 500 µl of
phosphate-buffered saline with 0.05% Tween 20, for 2 h at room
temperature, with gentle rocking. Then the GST fusion protein was
immobilized on glutathione-Sepharose beads (Amersham Pharmacia Biotech;
30-µl beads were added) for 30 min at 4 °C and washed four times
in 1 ml of phosphate-buffered saline with 0.05% Tween 20. (In some
experiments, the 35S-labeled proteins were incubated with
GST fusion proteins already immobilized on the glutathione-Sepharose
beads.) Following washing, GST fusion proteins were eluted with 20 mM reduced glutathione in elution buffer (120 mM NaCl, 100 mM Tris-HCl, pH 8, 30 µl) and
analyzed by SDS-PAGE.
Data Presentation and Statistical Analysis--
The results are
always presented as means ± S.E. Multiple group comparisons have
been done by one-way analysis of variance test followed by Dunnett's
test. Two-group comparisons were done using Student's t
test.
 |
RESULTS |
N-terminal Deletions Increase Ca2+ Channel Current but
Not Protein Expression--
To study the role of the first 46 amino
acids of the N terminus, we created a deletion mutant of
1C in which these amino acids, except the initial
methionine, have been deleted (
1C
N2-46; see Fig. 6 for a scheme of the channel to help localize the deletions). Ba2+ currents via the expressed Ca2+ channels
were measured using the two-electrode voltage clamp technique. The
subunit composition of the channels used in this study was varied
according to the specific questions asked. The expression of wild-type
(WT)
1C subunit alone was rarely employed because it
resulted in very small currents, usually below 20 nA (cf.
Ref. 45). In most cases,
1
2
combination was used, because the
subunit was found to interfere or
interact with the modulatory effects of N-terminal deletions and of PKC
(see below).
In agreement with Wei et al. (1), the whole cell
Ca2+ channel currents carried by Ba2+
(IBa) via channels containing the mutant
1C
N2-46 subunit were 5-10-fold larger
than with the wild-type
1C in all subunit combinations
tested as follows:
1C alone,
1
2
, or
1
2
(e.g. Fig.
1A). Even when the oocytes
were injected with twice as much WT
1C RNA, the
N2-46 mutant still gave ~4-fold larger currents (Fig.
1B). The kinetics of the current (Fig. 1A; see also Figs. 3 and 5) and the voltage dependence of activation were not
altered; the latter is demonstrated by the similarity of the normalized
current-voltage (I-V) curves (Fig. 1C). An additional deletion mutant missing most of the N terminus,
1C
N2-139, increased the current about
~10-fold compared with WT (Fig. 1D) and did not shift the
I-V curve (data not shown). However, we noticed differences in
voltage-dependent inactivation of
1C
N2-139 and the WT channels. In the
1
2
composition, when compared with the
WT, the steady-state inactivation curve of the mutant was shifted to
more positive potentials; the slope of the curve was increased, and the
proportion of non-inactivating current was reduced (Fig.
1E). Coexpression of
2A subunit shifted the
inactivation curve of the WT type channel to negative potentials and
increased the slope (compare data shown by solid circles in
Fig. 1, E and F), as reported previously (45, 55,
56). In the full subunit composition, the deletion of the 139-a.a.
again caused a decrease in the non-inactivating fraction and an
increase in the slope. However, unlike in the
1
2
channels, in the
1
2
channels the 139-a.a. deletion
caused a leftward shift in the curve. The inactivation kinetics were
unaffected. With 3-min long depolarizing pulses to +30 mV, after an
initial decay the
1
2/
channels' current reached a steady-state level (after ~2.4 min) of 61 ± 2% of peak in WT (n = 10) and 57 ± 3% of peak
in
N2-139. Although the functional significance of the
changes in voltage dependence of inactivation is unclear, they indicate
that the deletion of N-terminal amino acids may affect gating of the
channel. Since the presence of the
subunit modified the effect of
the N-terminal deletion, a cross-talk between N terminus and the
subunit is possible.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 1.
Effects of N-terminal deletions on whole cell
Ca2+ channel current and on voltage dependence of
activation and steady state inactivation of IBa.
A, Ca2+ channel currents from representative
X. laevis oocytes of one batch, injected with RNAs of
1C of WT or N2-46 deletion mutant, in
combination with 2 subunit. Currents were elicited by
test pulses to +20 mV from a holding potential of 80 mV.
B, mean currents of in groups of oocytes injected as
explained in A, obtained from three batches of oocytes. 5 ng
of 1CWT and 2.5 ng of
1C N2-46 cRNAs were injected per oocyte,
with an equal amount of 2 RNA. Currents were measured
6-8 days after RNA injection. Numbers above bars indicate
the number of cells assayed; numbers in parentheses indicate
the number of donors (oocyte batches). C, normalized I-V
curves recorded in oocytes of one batch. Currents elicited at each test
potential were normalized to the maximal amplitude in the same oocyte.
Averaged currents (mean ± S.E.) are plotted as a function of test
potential. 1CWT + 2 , ,
n = 5; 1C N2-46+
2 , , n = 6. D,
Ca2+ channel currents measured 3 days after the injection
of equal amounts (2.5 ng per oocyte) of cRNAs of 1CWT or
1C N2-139, in combination with
2 . E and F, averaged
steady-state inactivation curves in 1 2
(E) or 1 2 (F)
channels containing either WT ( ) or N2-139 ( )
1C. The currents were examined with two-pulse protocol
as follows: a 3-s prepulse to different voltages (starting from 80
mV, with 10 mV increments) followed by a test pulse to +20 mV. Data
were obtained from two different batches of oocytes including at least
five cells in each group. Results are represented as means ± S.E.
The averaged data were fitted to the Bolzmann equation:
IBa/Imax = f + 1/{1 + exp((Vprepulse V1/2)/Ki)}, where
Imax is the current obtained by the step from 80 to 20 mV, V1/2 is the half-inactivation voltage,
Ki is a slope factor, and f is the non-inactivating
fraction. The solid lines were drawn with the following
values (WT is given first, N2-139 second): in
E, Ki was 19.6 mV in WT and 17 mV in
N2-139; V1/2, 19 and 38 mV; f, 0.45 and 0.33; in F, Ki was 15.9 mV in WT and
13.2 mV in N2-139; V1/2, 6 and 3.6
mV; f, 0.47 and 0.38.
|
|
The increase in whole cell Ca2+ channel currents by the
N-terminal deletions might be due to an increase in the amount of
1C protein in the plasma membrane. Xenopus
oocytes present a convenient experimental system to examine this
question, since a very clean preparation of the plasma membrane can be
obtained by mechanical separation from the rest of the cell
("internal fraction," Refs. 52 and 57). Newly synthesized proteins
are metabolically labeled with 35S by incubating the
oocytes in [35S]methionine/cysteine for 3-4 days
following the RNA injection, immunoprecipitated, and subjected to
SDS-PAGE, and the relative amount of protein is quantified using an
imaging procedure (50, 52). This allows high precision measurements of
changes in the content of protein in whole oocytes and in plasma
membrane (50, 52, 54).
The channels were expressed in the
1
2
composition.
1 subunit was immunoprecipitated (50) with
the Card-I antibody directed against part of the II-III linker (53)
and analyzed as explained above. Fig.
2A illustrates the results of
a representative experiment that demonstrated comparable expression of
channels containing either WT or
N2-46
1C in the internal fraction (right panel, lanes
2 and 3) and in the plasma membrane (Fig. 2A,
left panel, compare lanes 5 and 6). Oocytes
uninjected with RNA gave no signal (Fig. 2A, lanes 1 and
4). Fig. 2B summarizes the results of the
quantitative analysis of the experiments in all three oocyte batches
tested. It can be seen that the total cellular amount of
N2-46
1C was only about half that of the
WT, whereas the amounts of WT and
N2-46
1C in the plasma membrane were roughly equal (the
~30% reduction in the mutant protein was not statistically
significant). These data suggest that the vast increase in the whole
cell Ca2+ channel current caused by the 46-a.a. deletion is
not caused by an increase in the level of expression of the channel
protein.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 2.
Comparison of the expression level of the WT
and N2-46 1C protein in plasma membrane
and internal fractions of Xenopus oocytes. A,
digitized PhosphorImager scan of SDS-PAGE analysis of the
1C and 1C N2-46 proteins
(coexpressed with 2 subunit) in oocytes metabolically
labeled with [35S]Met/Cys. Radiolabeled proteins were
immunoprecipitated from plasma membranes (lanes 1-3) and
internal fractions (lanes 4-6) separately. In each lane,
immunoprecipitates from 30 plasma membranes and 10 internal fractions
were loaded. Lanes 1 and 4 represent
immunoprecipitates from native oocytes that have not been injected with
cRNAs. B, relative amounts of the
1C N2-46 detected in plasma membrane and
internal fraction, calculated as percent of the expressed
1CWT protein in the same batch of oocytes, recalculated
per single oocyte. Total protein is the sum of both fractions. Band
intensities were measured using PhosphorImager. Data were averaged from
three different batches of oocytes and presented as means ± S.E.
|
|
The N-terminal Deletions Modify Channel Gating--
If the
N-terminal deletion does not alter the amount of channels in the plasma
membrane, then the increase in whole cell current must result from an
increase in the activity of each channel (which can be measured using
the patch clamp technique). In other words, the open probability of a
single channel must be higher in
N2-46
1C than in the WT
1C, whereas the number
of channels in patches of similar sizes must be comparable. To address
this question, an accurate estimate of the amount of channels in the
patch (N) is imperative (58). Unfortunately,
Po of the L-type channels is low (<1%), making
such estimate extremely difficult. However, Po
increases substantially in the presence of dihydropyridine agonists
such as (
)-BayK 8644 (reviewed in Ref. 8). In Xenopus oocytes expressing L-type Ca2+ channels, in the presence of
this drug, N can be reliably estimated from the number of
overlapping openings in a long series of depolarizing voltage steps,
provided that N <3 (50, 59). Before recording single
channel activity, we have verified that (
)-BayK 8644 increases the
whole cell IBa via WT and
N2-46 channels by
the same factor at all voltages (Fig. 3.
To avoid possible series resistance errors, the amounts of WT and
mutant RNAs were adjusted to produce currents of similar amplitudes.).
Thus, in the presence of (
)-BayK 8644, the differences between the WT
and
N2-46 channels appear to be preserved.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
( )-BayK 8644-induced enhancement of
IBa in oocytes expressing WT or N2-46
1C in combination with 2 . In the
control group, WT 1C and 2/ RNAs were
injected at 2.5 ng/oocyte; in the test group, 1C
N2-46 and 2/ RNAs were injected at
0.5 ng/oocyte. A and B show the typical effect of
( )-BayK 8644 on the currents elicited by depolarization step from
80 mV to +10 mV. C summarizes the increase in
IBa induced by application of 1 µM ( )-BayK
8644 at different voltages. Data were averaged from three batches of
oocytes (n >15 in all groups). , WT; ,
N2-46.
|
|
Single channel recordings were performed in cell-attached configuration
with 110 mM Ba2+ in the pipette, and in the
presence of 1 or 2 µM (
)-BayK 8644 in the bath.
Ca2+ channels (
1
2
composition) were activated by depolarizing pulses from
80 to +10 mV.
Our first observation was a similarity of the number of channels in
membrane patches in oocytes expressing WT or
N2-46
1C. For instance, with 0.6 or 1.2 ng of RNA of each
subunit per oocyte, and with pipettes of similar resistances (3.5-4.5
megaohms), the average number of channels in a patch was 1.3 ± 0.5 (n = 11) in WT and 1.1 ± 0.3 (n = 21) in
N2-46
1
2
channels.
Fig. 4A exemplifies records of
channel activity in oocytes expressing WT or
N2-46
channels (n = 2 in both cases). It appears that the
mutant channels spend more time in the open state than the WT ones.
Indeed, as shown in Fig. 4B, Po was
~10-fold higher for the
N2-46 channels (WT, 10 patches;
N2-46, 8 patches; p < 0.01). Open time distribution was fitted by two exponents with time
constants (
1 and
2) of about 0.4 and 2 ms for both channel types (Fig. 4, C and D, and
Table I), but the fraction of time
contributed by the longer openings (f2) was significantly higher in the
N2-46 than in WT channels (Table I). The increase in the proportion of long open times may at least partially account for the total increase in Po caused by
the N-terminal deletion. By visual examination, another prominent
difference was the prevalence of very long bursts of channel activity
in the mutant channels; such bursts were rare in the WT channels. A
rigorous burst analysis will require one-channel recordings which were
rare in this study. Whatever the main factor contributing to the
increase in Po, it is evident that the
N-terminal 46-a.a. deletion causes a major change in the gating
properties of the cardiac L-type Ca2+ channel, at least in
the presence of (
)-BayK 8644. However, we must add a reservation: in
a few oocytes where Po was measured both before
and after the addition of (
)-BayK 8644, the increase in
Po was much stronger than in whole cell
recordings, ranging from 5- to 180-fold. The reason for this phenomenon
is unknown, but it warrants caution in extrapolating the findings
obtained in the presence of this drug to the characteristics of
naïve channels.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 4.
Comparison of the single channel behavior of
WT and N2-46 1C (coexpressed with
2 ) in presence of 1 µM ( )-BayK 8644. A, representative consecutive traces of WT and mutant
channel activity at 10 mV. B, averaged
Po from 10 patches for WT and 8 patches for
N2-46 channels. C and D, typical
open time histograms. For each of the channels, both brief and long
open events were present. The smooth curves represent best
fits with two exponentials. The values of the time constants
( 1 and 2) and the fractions of total open
time spent in each of the open states (f1 and
f2) are shown in the insets.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Parameters of single channel activity of WT and N2-46
1C 2 channels
To calculate Po (the single channel open
probability), the number of channels was determined from the maximal
number of overlapping openings. Only patches with no more than three
channels have been analyzed. Open time histograms of the first level
openings were fitted with two exponents. 1 and 2
are the short and the long time constants, and f1 and
f2 are the fraction of total open time contributed by openings
corresponding to 1 or 2, respectively.
|
|
To account for the above observations and for the finding that removal
of proximal N terminus increases gating charge movement (1), we put
forward a working hypothesis: the N terminus hinders activation
(e.g. by obstructing the movement of the voltage sensor), therefore its deletion improves activation. It is notable that coexpression of the
subunit also improves activation and alters gating charge movement (60, 61), although the details differ (see
"Discussion"). Therefore, we assumed that the N terminus and the
subunit may affect a common mechanism and thus they may interact
with each other (as also suggested by the changes in
voltage-dependent inactivation; see above). This was tested by studying the effect of coexpression of the
subunit with channels containing either WT or one of the deletion mutants of
1C (
N2-46 or
N2-139).
Coexpression of
2A with
1
2
increased the whole cell
IBa both in WT (Fig.
5A, a) and in
N2-139 (Fig. 5A, b), but the increase caused
by coexpression of
2A was significantly (p < 0.01) smaller in
N2-139 than in
WT, at all voltages (Fig. 5B). The results with
N2-46 were similar (in this case, we tested the effect
of coexpression of
2A on channels composed of
1C alone).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
The effect of coexpression of the
2A subunit on the amplitude of currents via
channels containing 1C of WT,
N2-139, and N2-46 type.
A, currents recorded in representative oocytes of one batch
by depolarization from 80 to 20 mV. The oocytes were injected with
the subunit combinations indicated near the traces. a, RNAs
of all subunits were injected at 2.5 ng/oocyte. b, RNAs of
all subunits were injected at 1 ng/oocyte. B, summary of the
effects of 2A coexpression on channels containing WT
( ) and N2-139 ( ) 1C (2 batches; 10 oocytes in each group). In all groups, 2 was also
expressed. In each oocyte IBa was expressed as percent of
the mean amplitude of the current in the control group of oocytes from
the same donor. These normalized values were averaged across all oocyte
batches tested. Data are shown as mean ± S.E. C,
summary of the effects of 2A coexpression on channels
with WT ( ) and N2-46 ( ) 1C (two
batches; n = 18 in each group). In these experiments,
2 was not expressed; thus, channels composed of
1 alone versus 1 were
tested. Averaging of data was done as explained in B.
|
|
To probe for a possible physical interaction between the
subunit and the N terminus of
1C, we have measured
in vitro binding of GST fusion proteins corresponding to
some of the intracellular parts of
1C, to
2A synthesized in reticulocyte lysate and labeled with
[35S]methionine/cysteine (see Ref. 20). The following GST
fusion proteins were used: GST-N, corresponding to amino acids (a.a.) 1-154, i.e. the whole-length N terminus;
GST-LI-II, corresponding to most of the intracellular
linker between domains I and II (a.a. 438-550); and GST-C1
(a.a. 1664-1845) and GST-C2 (a.a. 1821-2171), corresponding to two parts of the C terminus. The scheme of the
1C subunit in Fig.
6A illustrates the positions
of the different pieces. As expected (20, 21),
2A bound
to GST-LI-II, but we could not detect binding to any one of
the other fusion proteins tested (Fig. 6B). (Note that the
amounts of all GST fusion proteins loaded on the gel were similar, as
demonstrated in Fig. 6C.)

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 6.
Interaction of in vitro
synthesized 2A with GST fusion proteins of
1C fragments. A, schematic presentation of
the 1C subunit of the L-type Ca2+ channel
(rabbit heart isoform numbering). B, interaction of
2A with GST fusion proteins corresponding to parts of
1C. The proteins were separated by SDS-15% PAGE and
monitored using PhosphorImager. N denotes GST-N (the whole N
terminus); L denotes GST-LI-II (a.a. 438-550);
C2 denotes GST-C2 (a.a.
1821-2171(end); C1 denotes GST-C1
(a.a. 1664-1845). Similar results were obtained in two additional
experiments. C, Coomassie Blue staining of the gel shown in
B. Arrowheads indicate the predicted positions of
the fusion proteins.
|
|
If the N terminus obstructs activation, then artificial proteins
corresponding to fragments of N terminus may be expected to reduce
IBa. We constructed DNAs encoding proteins corresponding to
N-terminal a.a. 1-139 of
1C (N1-139),
N-terminal a.a. 88-139 (N88-139), and C-terminal a.a.
1664-1845 (C1664-1845; denoted as C in Fig.
7). The corresponding RNAs directed the
expression of proteins of correct size in reticulocyte lysate (data not
shown). Coexpression of RNAs encoding N1-138 and
N88-139 proteins with channels containing a truncated
1C (either
N2-46 or
N2-139) reduced IBa, whereas
C1665-1845 was without effect (Fig. 7). The reduction was
stronger when the channels contained the
N2-139
truncation than
N2-46, possibly because in the
N2-46
1C the presence of the remaining part of N terminus hindered the access of the exogenous proteins to a
target site.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 7.
Inhibitory effect of coexpression of
N-terminal fragments on IBa via channels containing
N2-139 (A) or N2-46
1C (B). Top, summaries of all
experiments with N2-139 (A, a) and
N2-46 (B, a). Channels were expressed in the
1 2 composition, without (control) or
with the addition of RNA of the desired fragment, as indicated
below the bars. In each oocyte, IBa
was expressed as percent of the mean amplitude of the current in the
control group of oocytes from the same donor. These normalized values
were averaged across all oocyte batches tested. Data represent the
means ± S.E. Numbers above bars indicate the number of
cells assayed, and numbers in parentheses indicate the
number of donors (oocyte batches). The decrease in IBa
caused by both N-terminal fragments tested was statistically
significant in all cases (p < 0.05).
Bottom, normalized I-V curves recorded in the oocytes of
the same groups. , control; , N1-139; ,
N88-139; , C.
|
|
The First 46 Amino Acids Are Essential for PKC-induced Increase in
IBa--
Fig. 8A
shows diaries of representative experiments in which the PKC activator,
PMA, was added to the extracellular solution at t = 0. In agreement with our previous report (39), PMA caused a biphasic
change in IBa, an increase within several minutes was followed by a later decrease; the increase was stronger in
1
2
channels than in any combination
containing
2A. Fig. 8B summarizes the
measurements done in two oocyte batches in which we examined the
differences in response to PMA among channels of
1
2
,
1
, and
1
2
composition (increase in
IBa 15 min after PMA addition is shown). Clearly,
coexpression of
2A significantly reduced the extent of
current potentiation caused by PMA.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 8.
Importance of the subunit and of the
unique N-terminal part of 1C in modulation of the
channel activity by -PMA-induced stimulation of PKC. A,
changes in IBa induced by 10 nM -PMA as a
function of the time in representative oocytes injected with the
indicated RNAs. 200-ms test pulses from a holding potential of 80 mV
to a test potential of +20 mV were applied every 30 s. Peak
current amplitudes were normalized to the current in control
conditions, whose stabilization was verified for at least 5 min before
PMA application. B, attenuation of the -PMA effect by
coexpressed 2A subunit. The right bar shows
the effect of -PMA on channels containing the
1C(st1665) truncation mutant. In each oocyte,
IBa was expressed as percent of the current amplitude
before application of -PMA. These normalized values were averaged
across all oocytes in the batches tested. Numbers above bars
indicate the number of cells assayed, and numbers in
parentheses indicate the number of batches. Asterisks
indicate statistically significant difference (p < 0.05) from WT 1 2 , obtained by
two-tailed t-test. C, summary of the effects of
-PMA on different 1 mutants compared with the WT. The
bar denoted neuronal corresponds to the neuronal rbC-II
1C isoform. Data analysis and presentation as in
B. Asterisks indicate statistically significant
difference (p < 0.05) from WT examined in the same
batches of oocytes. D, alignment of the N-terminal sequences
of three isoforms of L-type Ca2+ channel 1C
subunits (see definitions in the text). Asterisks indicate
identity in all three sequences, x indicates identity in two
out of three sequences.
|
|
The C terminus of
1C contains a large number of putative
PKC and protein kinase A phosphorylation sites. To examine whether these sites play a role in PKC modulation, we expressed channels based
on a truncation mutant,
1C(st1665), in which part of the C terminus beyond a.a. 1665 is missing (16). Full subunit combination,
1
2
, was tested, because the
1C(st1665) mutant usually gave rather small currents
when expressed without
. Fig. 8B (right column) shows that the effect of PMA was not altered by this
truncation (compare with the results obtained with WT
1
2
).
In the following experiments,
1
2
combination was used to allow a better visualization of PMA-induced
enhancement of IBa. The effects of PMA varied among oocyte
batches; therefore, mutant and WT channels were always compared in the
same batch(es) of oocytes. Fig. 8C summarizes the results of
this series of experiments and shows that the deletion of the first 46 N-terminal amino acids completely eliminated the PMA-induced increase
in IBa, leaving the reduction phase intact (a
representative experiment diary is shown in Fig. 8A,
triangles). A rat brain
1C isoform with a variant N
terminus (see below) did not show an increase in IBa in
response to PMA, in agreement with a previous report (44). The PMA
effect remained intact in all other mutants tested, among them
1CS533I (a putative PKC site in linker I-II),
1CS1575A (a C-terminal site preceding the st1665
truncation), and an N-terminal deletion
N88-139.
Fig. 8D compares a.a. sequences (deduced from the
corresponding cDNA sequences) of the initial N-terminal segment of
three most widely tested variants of
1C as follows:
rabbit heart
1C (48) used in this study (RH);
rat brain
1C isoform, rbC-II (RB; Ref. 42);
and a human heart isoform (HH; Ref. 43). The latter two
isoforms are not up-regulated by PKC activators, and their N termini
vary from that of the RH
1C. N termini of two additional isoforms cloned from lung (62) and rat brain (rbC-I; Ref.
42) are identical to that of RB in Fig. 8D. N
termini of all isoforms are essentially identical beyond a.a. 46 (numbering by RH
1C). A closer examination
shows that there is an additional region between a.a. 6 and 20, in
which RB and HH are identical to each other and
also show significant homology (33% identity) to RH
1C. The correlation between PMA effects (or their
absence) and the primary structure of the initial
1C
N-terminal segments of these isoforms supports the idea that the unique
initial 46 amino acids of RH
1C are essential
for PKC modulation.
 |
DISCUSSION |
N Terminus Modulates L-type Channel Gating--
Our results
demonstrate the functional importance of the N terminus of
1C subunit in L-type Ca2+ channel function
and modulation. Deletion of the initial 46 amino acids of the N
terminus, which are unique to rabbit heart isoform, increases the whole
cell Ca2+ channel current (see also Ref. 1) but does not
increase the expression of the channel, as testified by the unchanged
plasma membrane content of
1C protein monitored by an
immunochemical method, and similar density of functional channels
detected by patch clamp methodology. Our data strongly suggest that
this deletion alters the gating of the channel. First of all, it
enhances the activity of single Ca2+ channels as testified
by the ~10-fold increase in Po. This change in
channel gating alone is sufficient to account for the increase in whole
cell Ca2+ channel current caused by this and probably by
the other deletions tested (a.a. 2-139). An alteration of channel
gating by the N terminus is further supported by differences in voltage
dependence of inactivation in WT and
N2-139 channels,
and by a decrease in whole cell current amplitude by coexpression of
proteins corresponding to N-terminal a.a. 1-139 or 88-139, but not by
a C-terminal protein. The results of the latter experiments imply that,
in addition to the first 46 amino acids, other parts of the N terminus
participate in its effect; however, a more detailed study will be
necessary to scrutinize this hypothesis. We propose that, in L-type
channels containing the rabbit heart isoform of
1C, the
N terminus imposes a tonic inhibitory control which is relieved in the
truncation mutants tested. This mechanism is, to some extent, similar
to that proposed to explain the increase in Ca2+ channel
current caused by C-terminal deletions and by protein kinase A
phosphorylation (12, 13).
In expression studies, changes in total gating charge movement
(Qmax) caused by coexpression of
Ca2+ channels
or
2
subunits (60, 61,
63, 64) usually correlate well with the amount of
1C
protein detected in the membrane by immunochemical methods (50). How
can our results be accommodated with the fact that deletions of initial
40-120 a.a. of
1C increase Qmax
without changing its voltage dependence (1)? We claim that, in general,
a change in Qmax does not necessarily report a
change in the number of functional channels. In various
voltage-dependent channels, Qmax can
be altered by drugs, toxins, or by fatty acids. For instance,
Qmax in Na+ channels is decreased by
Anthopleurin-A toxin (65), fatty acids (66), and lidocaine (67); in
L-type Ca2+ channels, Qmax is
decreased by dihydropyridines (68, 69). Thus, the N terminus might
decrease Qmax by directly or allosterically interfering with the movement of the voltage sensor of the channel; removal of the N terminus would increase
Qmax.
Subunit and PKC Interact with the N Terminus--
Our data
suggest a cross-talk between the N terminus of
1C and
the
2A subunit. It appears that the presence of the
subunit attenuates the inhibitory effect of the N terminus. This is
supported by the following observations. (i) Coexpression of
2A enhances the whole cell currents more efficiently
when the N terminus is intact than when a.a. 1-46 or 1-139 are
removed. It is possible that part of the
-induced channel
enhancement is due to a weakening of the inhibitory effect of the N
terminus; this explains why, in the absence of the latter, the
augmentation caused by
subunit is less pronounced. (ii)
subunit
counteracts the effect of PKC which is mediated via an interaction with
the N terminus (see below). A cross-talk between
2A and
the N terminus of
1C is also supported by the
observation that
N2-139 deletion-induced changes in
voltage-dependent inactivation properties are different in
the absence and presence of
2A. The interaction does not
appear to be a direct one, since
2A does not bind a GST
fusion protein of the first 154 a.a. (Of the GST fusion proteins
tested, the only
2A-binding protein was that of the
I-II domain linker, which contains a
2A-binding site
conserved in all known
1 subunits (20, 21, 70, 71); the
results of Fig. 6 suggest that, unlike
1E (71),
1C does not seem to have a C-terminal
subunit binding site.) Thus,
subunit interacts with the N terminus
allosterically ("at distance"). The mechanism is unclear and seems
to involve the voltage sensing machinery. It would be an
oversimplification to assume that the action of
subunit is
mechanistically analogous to removal of the N terminus, because of the
differences in the effects of
-coexpression and of N-terminal
deletions on charge movement; the former alters the voltage dependence
of current activation without changing Qmax, and
the latter increases Qmax (60, 61, 70). In this
respect, the enhancement of channel activity by C- and N-terminal
deletions is also mechanistically different, since C-terminal
truncations do not affect Qmax and have been
proposed to improve the coupling between voltage sensor movement and
pore opening (12).
We have identified the initial 46 a.a. of the
1C as
a site indispensable for the potentiating action of PKC on the channel, since the removal of this segment fully eliminates the current increase
caused by PKC activation. The decrease caused by PMA must be mediated
by an action on another site. It is not clear whether the enhancing
effect of PKC is caused by a direct phosphorylation of one of the amino
acid residues in this region of the channel. Theoretically,
phosphorylation may occur on another part of
1C or even
at an unknown protein (present in the oocytes) that modulates the
channel via an interaction with the N terminus. Identification of the
site of phosphorylation remains an important challenge for the
future.
The biophysical mechanism by which PKC enhances the activity of the
channel is unknown; one possibility is that it weakens the inhibitory
control exerted by the N terminus. This assumption is in line with the
observation that the PKC-induced increase in open probability of the
channel is accompanied by an increase in the proportion of long
openings (30, 36), like the N-terminal deletion (Table I). It is also
compatible with the fact that PKC-elicited increase in Ca2+
channel current is attenuated by the
subunit; according to our
hypothesis, the inhibition imposed by the N terminus is already weakened when
is present, and there is less room for a further improvement of channel activity (an occlusion mechanism). A cross-talk between the
subunit and PKC has also been proposed for the neuronal
1B (N-type) channels (44, 72). However, the details of
the proposed interaction differ significantly; in the N-type channel, PKC phosphorylates the I-II domain linker and thus counteracts an
inhibitory effect of the G protein 
subunit (G
) which binds to the same loop; the Ca2+ channel
subunit also binds
to the same loop, reducing the inhibition caused by G
and thus
occluding the PKC effect (72). In the L-type channel, no modulation by
G
has been reported; I-II linker is the site of
subunit
binding but it is not phosphorylated by
PKC2;
appears to interact
with the PKC target site allosterically rather than sterically.
1C Subunits with Rabbit Heart-type N Terminus
Should Be Widespread--
Alternative splicing products of
1C are found in various tissues; they have been proposed
to play an important role in generating a diversity of
electrophysiological properties (14, 42, 62, 73, 75). Alternative
splicing was also proposed to take place in the N terminus, and
"rabbit heart," "rat brain," and "lung" cDNAs have been
assumed to be splice variants of the same gene product (42, 62).
Recently, the genomic structure of human L-type Ca2+
channel has been characterized and shown to contain at least 44 invariant and 6 alternative exons (74). The unique stretch between
Ser21 and Gly47 present in rabbit heart isoform
(RH
1C numbering; Fig. 8D) appears to be missing from the human genomic
1C RNA (74),
casting doubt on the biological relevance of the isoform cloned from
rabbit heart. In fact, however, the details of splicing at the
beginning of the human
1C N terminus are unclear. The
incomplete sequencing of putative intron 1 that separates between exon
1 ending with Gln16 (
1C HH
numbering; Fig. 8D) and exon 2 starting with
Gly17 (corresponds to Gly47 of rabbit heart
1C) left open the possibility of an additional exon(s)
in this region (74). In view of the commonality of PKC-induced enhancement of Ca2+ channel activity in heart and smooth
muscle cells of many mammalian species, it is probable that an
additional variable exon encoding an N-terminal sequence similar or
identical to the rabbit heart isoform may exist. The presence of an
isoform containing this sequence in a particular cell type may be
predictive of an enhancing PKC effect, and vice versa.
We thank I. Lotan for many discussions and
for a critical reading of the manuscript; M. Hosey for the gift of the
Card-I antibody; and T. P. Snutch for the gift of the rbC-II
cDNA.