From the Centre de Recherche de Biochimie Macromoléculaire,
CNRS Unité Propre de Recherche 1086, 1919 Route de Mende, BP
5051, F34033 Montpellier, France
 |
INTRODUCTION |
Beyond their role in membrane excitability, voltage-gated
Ca2+ channels are of particular interest due to the
fundamental importance of the biological processes regulated by
incoming Ca2+ ions (for review, see Ref. 1). Several
different types of Ca2+ channel have been identified in
neurons, where they are crucial for secretion, synaptic transmission,
or gene expression (2-4). Ca2+ entry through L-, N-, T-,
or P-type channels is primarily regulated by the level of membrane
depolarization. However, the frequency of these depolarizations is also
an important regulatory factor that has been implicated in the
regulation of synaptic transmission and that may be of importance for
long-term potentiation. Accordingly, repetitive stimuli or strong
pre-depolarization can up-regulate the activity of different types of
Ca2+ channel, giving rise to the so-called Ca2+
current facilitation (for review, see Ref. 5). Although a similar
increase is recorded for the various Ca2+ channels,
different mechanisms, however, have been proposed. Experimental
evidence firmly correlates a given mechanism with a particular
Ca2+ channel type, as is the case for N-type
Ca2+ channels, which are sensitive to a direct,
voltage-dependent G-protein block.
L-type Ca2+ channel facilitation has been
described using various preparations, and different mechanisms leading
to current potentiation have been proposed. Strong pre-depolarizations
can induce a change in gating mode in cardiac L-type
Ca2+ channels, whereas the same protocol appears to lead,
in skeletal and vascular muscle, to a voltage-dependent
phosphorylation of the channel by protein kinase A and calmodulin
kinase II, respectively (6-8). At the molecular level, these effects
seem to be related to different isoforms of the L-type
Ca2+ channel (
1C,
1D, or
1S), different splice variants, and/or different
cellular preparations. Current facilitation of the neuronal L-type Ca2+ channel (9) has been recorded,
using the appropriate protocol, in isolated neurons (10, 11) or after
expression of the
1C subunit in Xenopus
oocytes (12, 13). This type of facilitation can be distinguished from
cardiac, skeletal, or vascular L-type Ca2+
channel facilitation (6-8, 14-18) by its insensitivity to phosphatase inhibitors and permeating ions (12), suggesting a specific mechanism for neuronal facilitation. As opposed to N-type Ca2+
channels, coexpression of specific
subunits (
1,
3, or
4) is absolutely necessary for the
development of
1C subunit facilitation (12, 19). The
inhibitory effect of the
2 subunit (13) and the
insensitivity to G-protein stimulation or inhibition (12) are also
distinctive features. Altogether, these data suggest that the molecular
mechanisms of L- and N-type Ca2+ channel facilitation are
different processes. In this work, using deleted and chimeric subunits
constructed from permissive and nonpermissive
subunits, we
demonstrate the existence of a conserved sequence in the
subunits
necessary for L-type Ca2+ channel facilitation.
We also identify a short autoinhibitory segment on the N-terminal tail
of the
2 subunit partly responsible for the
insensitivity of the
1C +
2 subunit
combination to prepulse facilitation. Expression of individual
subunits could therefore reveal latent properties that are central for
neuronal plasticity.
 |
EXPERIMENTAL PROCEDURES |
Preparation of Truncated, Chimeric, and Mutated Forms of the
Subunits and Xenopus Oocyte Injection--
The following calcium
channel subunits were used:
1C and
2-
(9),
1b (20), and
2a (21). All these
subunit cDNAs were inserted into the pMT2 expression vector under
the control of an SV40 promoter (9).
Deleted Mutants of the
Subunit--
1-TF1-4
and
2-TF1-4 were constructed by
PCR.1 The sense primer was
engineered to possess an EcoRI site and a start codon (boldface) when necessary. In the reverse primer, an XbaI
site (preceded by a stop codon in the case of the TF4 subunits) was added at the 5'-end. Numbers in parentheses correspond to the position
of the primer in the sequence. GenBankTM accession numbers
were X61394 for the
1b subunit and M80545 for the
2a subunit. The primers used were as follows:
1-TF1: sense, EcoRI-ATG-(235-257
1b), and antisense, XbaI-(1837-1857
1b);
1-TF2: sense,
EcoRI-ATG-(559-579
1b), and
antisense, identical to
1-TF1;
1-TF3:
sense, EcoRI-ATG-(706-726
1b),
and antisense, identical to
1-TF1;
1-TF4:
sense, EcoRI-(64-84
1b), and antisense,
XbaI-TCA-(1298-1317
1b);
2-TF1: sense, EcoRI-ATG-(424-444
2a), and antisense,
XbaI-TCA-(2168-2190
2a);
2-TF2: sense, EcoRI-ATG-(748-769
2a), and antisense, identical to
2-TF1;
2-TF3: sense,
EcoRI-ATG-(1009-1031
2a), and
antisense, identical to
2-TF1; and
2-TF4:
sense, EcoRI-(115-133
2a), and antisense,
XbaI-TCA-(1600-1623
2a). The PCR product was checked and purified on agarose gel.
Subunit Chimeras--
The chimeras were obtained by a PCR
strategy as described (22) using the Expand High Fidelity PCR system
(Boehringer Mannheim). The N-terminal fragments were amplified using
the following primers:
CH1: sense, identical to
2-TF4, and antisense, (559-576
1b + 731-747
2a);
CH2: sense, identical to
2-TF4, and antisense, (706-723
1b + 878-894
2a);
CH3: sense, identical to
2-TF4, and antisense, (706-724
1b + 993-1008
2a); and
CH4: sense, identical to
2-TF4; and antisense, (235-249
1b + 409-423
2a). The C-terminal fragments were amplified
using the following primers:
CH1: sense, (731-747
2a + 559-576
1b), and antisense, identical to
1-TF1;
CH2: sense, (878-894
2a + 706-723
1b), and antisense, identical to
1-TF1;
CH3: sense, (993-1008
2a + 706-724
1b), and antisense, identical to
1-TF1;
CH4: sense, (407-423
2a + 235-249
1b), and antisense, identical to
1-TF1.
All PCR products were separated on 1% agarose gel, cut out, and
purified. A second PCR was performed using these products to produce
the final chimeras.
-Subunit truncated forms and chimeras were
finally digested using EcoRI and XbaI, subcloned
into pBluescript (Stratagene) for sequencing (DiDeoxy Terminator
technology, Applied Biosystems) and in vitro translation
(TNT® coupled reticulocyte lysate systems, Promega), and subsequently
into pMT2 for injection and expression.
2M123 and
2/
1-CH4M123 Constructions--
The
mutants
2M123 and
1/
2-CH4M123 were obtained by a PCR
strategy as described (22) using the Expand High Fidelity PCR system.
The N-terminal fragments were amplified using the following primers:
2M123 and
2/
1-CH4M123:
sense, identical to
2-TF4, and antisense,
5'-GGACACCCGTACTGCCGCGGCATGTACCAGCCCG-3' ((388-420
2a), excepted the mutated sequence is underlined), using
2 as a matrix. The C-terminal fragments were amplified
using the following primers:
2M123: sense,
5'-GGGCTGGTACATGCCGCGGCAGTACGGGTGTCC-3' (388-420
2a, excepted mutated sequence underlined), and
antisense, identical to
2-TF1, using
2 as
a matrix; and
2/
1-CH4M123: sense,
identical to
2M123, and antisense, identical to
1-TF1, using
2/
1-CH4 as a
matrix. The complete sequence was obtained as described for
chimeras.
All the constructions of the
subunits used in this paper were
checked for correct translation and molecular weight by in vitro translation using the T7 or T3 TNT coupled reticulocyte lysate system (Promega). All these constructions were able to increase
Ba2+ current amplitude when co-injected with the
1C subunit, demonstrating association with the
1C subunit (respective current amplitudes for
1C alone,
368 ± 48 nA (n = 5);
for
1,
1-TF1,
1-TF2,
1-TF3, and
1-TF4,
813 ± 306 nA
(n = 20),
702 ± 235 nA (n = 6),
687 ± 273 nA (n = 14),
757 ± 414 nA
(n = 10), and
411 ± 100 nA (n = 5), respectively; for
2,
2-TF1,
2-TF2,
2-TF3, and
2-TF4,
815 ± 403 nA (n = 9),
1220 ± 440 nA
(n = 11),
1035 ± 399 nA (n = 17),
527 ± 200 nA (n = 5), and
1447 ± 678 nA (n = 6), respectively; and for
2/
1-CH1,
2/
1-CH2,
2/
1-CH3, and
2/
1-CH4,
930 ± 424 nA
(n = 8),
1200 ± 540 nA (n = 9),
1633 ± 823 nA (n = 3), and
1650 ± 644 nA (n = 18), respectively).
Deleted Mutants of the
1C
Subunit--
1C
N and
1C
C were
generated by PCR using the Expand Long Template PCR system (Boehringer
Mannheim). An N-terminal fragment was generated to introduce the
untranslated avian myeloblastosis virus sequence just before the
sequence of the
1C subunit (the antisense primers used
were 5'-CCTCGTGTTTTCATTGACCATGGTGGAAGTATTTGAAAGAAA-3' for
1C
C (nucleotides 290-310;
GenBankTM accession number M67515), and
5'-GCAGCTGCATTGGCATTCATGGTGGAAGTATTTGAAAGAAA-3' for
1C
N (nucleotides 377-396)). This fragment was then
used as a megaprimer to generate the final mutated forms of
1C with an antisense primer
(5'-GTTTTCCTTTTGCGGCCGCGTACAGGCCTCCAGCCCTCCTGAAGATGTC-3' for
1C
C (NotI, stop codon in
boldface, nucleotides 5378-5404) and
5'-ATAGTTTAGCGGCCGCCTACAGGTTGCTGACATAGGACC-3' for
1C
N (NotI, nucleotides 6699-6721)). The
mutated forms were subcloned into the pMT2 expression vector.
Xenopus oocyte preparation and injection (5-10 nl of
1 +
2-
+
subunit cDNAs at
0.3 ng/nl) were performed as described elsewhere (13). Oocytes were
then incubated for 2-7 days at 19 °C under gentle agitation before
recording.
Electrophysiological Recordings--
Whole cell Ba2+
currents were recorded under two-electrode voltage clamp using the
GeneClamp 500 amplifier (Axon Instruments, Inc., Foster City, CA).
Current and voltage electrodes (<1 megaohms) were filled with 2.8 M CsCl and 10 mM BAPTA, pH 7.2, with CsOH. Ba2+ current recordings were performed after injection of
BAPTA (one or two 40-70-ms injection at 1 bar of 100 mM
BAPTA-free acid (Sigma), 10 mM CsOH, and 10 mM
HEPES, pH 7.2). The recording solution had the following composition:
10 mM BaOH, 20 mM tetraethyl ammonium hydroxide, 50 mM
N-methyl-D-glucamine, 2 mM CsOH, and
10 mM HEPES, pH 7.2, with methanesulfonic acid. Only
oocytes expressing Ba2+ currents with amplitudes in the
range of 0.5-5 µA were analyzed to ensure sufficient resolution and
to avoid voltage-clamp problems. Currents were filtered and digitized
using a DMA-Tecmar Labmaster and subsequently stored on an IPC 486 personal computer using pClamp software (Version 6.02, Axon
Instruments, Inc.). Ba2+ currents were recorded during a
typical test pulse from
80 to +10 mV of 0.4-s duration. Current
facilitation was elicited by applying a 200- or 400-ms depolarization
(pre-depolarization) to +60 to +140 mV, 50 ms prior the test pulse.
Current amplitudes were measured using Clampfit (pClamp Version 6.02, Axon Instruments, Inc.). Facilitation was quantified by dividing the
current amplitude recorded after pre-depolarization by the control
current amplitude (without pre-depolarization). All values are
presented as means ± S.D. The significance of the difference
between two means was tested using Student's t test
(p < 0.05).
 |
RESULTS |
We have previously shown that the activity of the
1C subunit L-type Ca2+ channel,
recorded during a single voltage step at +10 mV, could be up-regulated
by applying strong positive pre-depolarizations (12). This type of
current facilitation was sensitive to the expression of a
subunit
(12), but was not affected by the omission of the
2-
subunit. We have further identified the existence of permissive and
nonpermissive
subunits for this facilitation (13).
Fig. 1 shows that application of a
+120-mV pre-depolarization to an oocyte expressing the
1C and
1 subunits (see protocol shown on
top of the figure) increases the Ba2+ current recorded
during a subsequent depolarization (in the range of
20 to +30 mV).
The same pre-depolarization was without effect when applied to oocytes
expressing the
1C and
2 subunits (Fig. 1A). This lack of facilitation was recorded independently of
the amplitude of the test pulse, as exemplified for voltage steps from
20 to +30 mV (Fig. 1, A and B). It was not due
to a
2 subunit-induced shift in the voltage dependence
of facilitation since stronger (or longer) pre-depolarizations were
also inefficient in producing
1C +
2
subunit current potentiation (13). Given that the
1C subunit, when expressed alone, was not sensitive to pre-depolarization, we concluded that the
1,
3, or
4 subunit carried specific sequences able to produce
novel properties when expressed with the
1C subunit. We
used the
2 subunit sequence as a matrix for chimeric
subunits to identify these sequences.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1.
Regulation of L-type Ca2+ channel
facilitation by different subunits. A, Ba2+
currents recorded from oocytes injected with cDNA coding for the
1C and 1 or 2 subunits.
Current facilitation ( ) was recorded during a typical 400-ms-long
depolarizing pulse to (from top to bottom) 20, 10, 0, +10, +20, or
+30 mV by applying a conditioning depolarization to +120 mV during 200 ms. Note the lack of facilitation when the 2 subunit was
expressed. B, current voltage curves obtained from the
oocytes shown in A and recorded during test pulses preceded,
or not, by a 400-ms-long conditioning depolarization to +120 mV. Note
that expression of the 2 subunit prevented facilitation
for all these pulses.
|
|
The four
subunit genes identified so far are organized in two
conserved domains (C1 and C2, 60-80% homology; white
boxes in Fig. 2A),
surrounded by variable regions where homology is lower (V1-3;
black boxes) (see also Ref. 23). The sequence
responsible for the interaction with the
1 subunit (the
subunit interaction domain) has been localized to the second
conserved domain (C2; see arrow in Fig. 2A), and
recent studies suggest that important deletions outside this region
have only slight effects on the capacity of this subunit to affect
calcium channel function (24). Keeping these results in mind, we
constructed and expressed a series of N- and C-terminal deletions of
the
1 and
2 subunits in which this
subunit interaction domain was preserved and four chimeric
subunits, between the permissive
1 and nonpermissive
2 subunits, in which each of the five domains was
exchanged (Fig. 2B).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 2.
Schematic drawing of the different subunits used in this study. A: amino acid similarity among
the four subunits. The white boxes represent
the two conserved regions where homology is high (>90% between the
1 and 2 subunits). B:
left panel, representation of the different truncated and
chimeric forms of the 1 and 2 subunits.
The precise location of the deletion is depicted on top of each
schematic. Note that for each truncated subunit, the second
conserved region, which includes the 1 subunit-binding
site (24), is preserved to allow association with the pore-forming
subunit (see "Materials and Methods"). All chimeras are composed of
a C-terminal half from the 1 subunit and an N-terminal
half from the 2 subunit. In
2/ 1-CH2, a central insertion
(black boxes in B and C)
present in the 2 subunit (amino acids 170-203) and in
2/ 1-CH3 has been removed. In
2/ 1-CH4, the first 57 amino acids of the
1 subunit were replaced by the first 16 amino acids of
the 2 subunit. Right panel: autoradiograms of
SDS-polyacrylamide gels of in vitro translated,
[35S]methionine-labeled, truncated and chimeric subunits. Films were exposed overnight.
|
|
Fig. 3 shows the effects of N- and
C-terminal deletions of the
1 subunit on current
facilitation. Prepulse facilitation was elicited using a 200-ms-long
prepulse at +100 mV. Under these conditions, the average current
potentiation recorded by expressing the full-length
1
subunit with the
1C subunit was 47 ± 18%
(n = 17) larger than the control current, recorded
without pre-depolarization. Deletion of the first variable region (V1,
first 58 amino acids,
1-TF1) of the
1
subunit did not affect its capacity to induce facilitation since the
increase in current recorded after the pre-depolarization was 54 ± 16% (n = 5) (Fig. 3). Subsequent deletions of the
N-terminal region of the
1 subunit including the first conserved domain and the second variable region (first 166 and 215 amino acids, respectively;
1-TF2 and
1-TF3) were also without effect on facilitation
(respective increases of 66 ± 6% (n = 7) and
48 ± 9% (n = 9)) (Fig. 3). In all cases, the
increase in current following pre-depolarization was accompanied by an
acceleration of inactivation, as already noted for class C
L-type Ca2+ channel facilitation (12).
Similarly, the C-terminal deletion of the last variable domain (amino
acids 418-597) (Fig. 2) produced a truncated
1 subunit
that was able, when expressed with the
1C subunit, to
generate Ca2+ channels sensitive to pre-depolarization
(43 ± 13% (n = 5) of current increase; see
trace labeled
1-TF4 in Fig. 3). Altogether, these data suggested that the sequences necessary to induce
L-type current facilitation are located in the second
conserved domain of the
1 subunit (C2, amino acids
215-418). Since this domain is highly conserved between the
1 and
2 subunits (92% homology), the
2 subunit should also be able to produce facilitation.
Under these conditions, the lack of facilitation recorded with the
2 subunit may be due to some inhibitory processes
instead of a missing facilitatory sequence.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 3.
L-type current facilitation recorded with
truncated forms of the 1 subunit. Ba2+
currents were recorded during a depolarization to +10 mV from a holding
potential of 80 mV in Xenopus oocytes injected with the
1C subunit and different truncated forms of the
1 subunit. Current facilitation was recorded, without
significant differences upon coexpression of each of these truncated
forms, when the appropriate conditioning depolarization was applied
(200 ms in duration from 80 to +100 mV; traces labeled *).
Left panel, constructions used; middle
panel, current traces (scale bars = 1 µA, except for 1-TF4, 200 nA); right
panel, averaged facilitation, quantified as the ratio of the
current amplitude after pre-depolarization divided by the control
current amplitude.
|
|
We have tested this idea by constructing a series of four truncated
2 subunits. In
2-TF1, the first 16 amino
acids of the
2 subunit were removed. As shown in Fig.
4, Ba2+ currents recorded
from oocytes expressing this subunit with the
1C subunit
were not sensitive to pre-depolarization since their amplitude was not
increased by the conditioning voltage step (5 ± 2%,
n = 11). The subsequent deletion of the first conserved domain (C1) gave rise to
2-TF2 (Fig. 4). Surprisingly,
application of pre-depolarization to oocytes injected with this
subunit, in conjunction with the
1C subunit,
significantly increased the Ba2+ current recorded during a
subsequent test pulse to +10 mV (38 ± 6% (n = 8)
compared with 5 ± 6% (n = 9) for the full-length
2 subunit). The facilitated current displayed the
typical inactivating phase usually recorded with this paradigm (compare
traces labeled
2-TF2 in Fig. 4 and
1 in Fig. 1). A larger deletion including the central
variable region V3 (amino acids 1-212,
2-TF3 in Fig. 4)
had the same effect and transformed this subunit into a permissive subunit for facilitation (average increase of 42 ± 13%,
n = 3). Deletion of V3, the C-terminal variable
sequence of the
2 subunit (amino acids 416-604,
2-TF4 in Fig. 4), however, had no effect, and currents
recorded with or without pre-depolarization were almost
indistinguishable (7 ± 3%, n = 6).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 4.
L-type current facilitation recorded with
truncated forms of the 2 subunit. The same
recording conditions were used as those described in the legend to Fig.
3, but using truncated 2 subunits. Left
panel, constructions used; middle
panel, current traces (scale bars = 1 µA, except for 2-TF3, 500 nA); right
panel, averaged facilitation, quantified as the ratio of the
current amplitude with pre-depolarization divided by the control
current amplitude. #, significantly different from the 2
subunit.
|
|
The voltage dependence of facilitation of these truncated subunits was
characterized by using depolarization in the range of +60 to +140 mV.
Such depolarizations induced an increase in Ba2+ currents
recorded during a subsequent test pulse for
1C subunit Ca2+ channels containing
1,
1-TF1,
1-TF3, or
2-TF2. As
shown in Fig. 5 (A and
B) for typical recordings, this increase rose with the
amplitude of the conditioning depolarization, reaching a plateau for
amplitude greater than +100 mV. The voltage dependence of this increase
was similar for all these constructions (e fold changes for
1,
1-TF1,
1-TF3, and
2-TF2 were 45 ± 12 mV (n = 3),
23 ± 15 mV (n = 4), 52 ± 26 mV
(n = 5), and 47 ± 27 (n = 3),
respectively). Depolarization of the same amplitude was, however, without effect when the
1C subunit was expressed with
either
2 or
2-TF4 (Fig. 5B).
The induction of facilitation by deletions of the N-terminal tail (V1 + C1 domains) of the
2 subunit strongly suggests (i) the
existence of a conserved facilitatory sequence in the C2 domain of the
subunit, able to induce facilitation by both the
1
and
2 subunits and (ii) the presence, in the full-length
2 subunit, of an inhibitory sequence located in the
first 128 amino acids. To test this hypothesis more directly, we
constructed four chimeras based on the N-terminal truncated
1 subunits
1-TF1,
1-TF2,
and
1-TF3, which were all able to induce facilitation (Fig. 3). The results are shown in Fig.
6. The average facilitation, recorded
using the standard protocol, was 47 and 5% for the
1 and
2 subunits, respectively (Figs. 3, 4, and 6). In the
first construct, the missing sequence of
1-TF3 (V1 + C1 + V2 domains) was replaced with the corresponding sequence of the
2 subunit (amino acids 1-212) (Fig. 2). Expression of
this
2/
1-CH3 chimera with the
1C subunit prevented the promotion of facilitation
normally recorded with
1-TF3 (9 ± 1%,
n = 3) (Fig. 6). The lack of facilitation recorded with
the
2 subunit can therefore be transferred to the
1 subunit by addition of these amino acids, which
confirmed the existence of an inhibitory sequence. In
2/
1-CH2,
2/
1-CH1, and
2/
1-CH4, the contribution of the
2 subunit to the total sequence of the chimera was
further reduced.
2/
1-CH2 was deleted of
an insert specifically found in the V2 domain of the
2
subunit (amino acids 170-212, black box in Fig.
6).
2/
1-CH1 and
2/
1-CH4 kept only the V1 + C1 and V1
sequences, respectively, from the
2 subunit, with the
remaining sequence coming from the
1 subunit (Fig. 2).
As shown in Fig. 6, none of these constructs was able to promote
current facilitation in response to pre-depolarization. The extreme
case is the
2/
1-CH4 chimera, where
addition of the first 16 amino acids of
2 to
1 was able to completely block current facilitation
(4 ± 5%, n = 10; not significantly different from the
2 subunit; see bottom of Fig. 6), suggesting
that an inhibitory sequence was indeed present in these few amino
acids.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Voltage dependence of 1C
subunit current facilitation recorded with truncated 1
and 2 subunits. Current facilitation,
quantified as described in the legend to Fig. 3, was recorded with
prepulse (PP) amplitude varying from +60 to +140 mV (the
protocol is shown on top of the figure). A, oocytes were
injected with the 1C and 2- subunits
and 1, 1-TF1, or 1-TF3.
B, oocytes were injected with the 1C and
2- subunits and 2,
2-TF2, or 2-TF4. Note that facilitation
of increasing amplitude was recorded with expression of
1, 1-TF1, 1-TF3, or
2-TF2, whereas prepulses of various amplitude were
without effect on Ba2+ currents recorded upon coexpression
of 1C, 2- , and 2 or
2-TF4.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
L-type current facilitation
recorded with chimeras between the 1 and
2 subunits. The same recording conditions were used
as those described in the legend to Fig. 3, but using chimeras between
subunits. Left panel, chimeric subunits
used; middle panel, current traces
(scale bars = 1 µA); right
panel, averaged facilitation, quantified as the ratio of the
current amplitude with pre-depolarization divided by the control
current amplitude. Facilitation was not observed when the first 16 amino acids of the 2 subunit were present
( 2/ 1-CH4). #, significantly different
from the 1 subunit.
|
|
The primary sequence of this inhibitory segment of the
2
subunit is shown in Fig. 7
(top). The sequence is characterized by the presence of a
short stretch of positively charged amino acids (boldface
letters). To test the possible involvement of these charges
in the inhibition of facilitation, we mutated these three arginines of
the
2/
1-CH4 chimera to alanines. The
mutated subunit,
2/
1-CH4M123, was then
coexpressed with the
1C subunit. As shown in Fig. 7,
Ba2+ currents recorded from oocytes expressing this
combination of mutated subunits could be increased by the application
of conditioning depolarization (28 ± 5%, n = 7),
reversing the effect of the insertion of the N-terminal tail of the
2 subunit (compare traces recorded with the
non-mutated and mutated
2/
1-CH4 chimeras
in Fig. 7; difference was statistically significant).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 7.
Involvement of positive charges of
2/ 1-CH4 in inhibition of current
facilitation. Top, schematic showing the N-terminal
sequences of the 2 subunit incorporated in the
2/ 1-CH4 chimera. The three arginines that
are mutated to alanine in 2/ 1-CH4M123 are
in boldface. Middle, current traces recorded when
the non-mutated (left) and mutated (right)
2/ 1-CH4 subunits were coexpressed with
the 1C subunit. Note that the replacement of the
arginines allowed prepulse-induced facilitation. Facilitation is
normally not observed with this chimera (scale
bars = 1 µA). Bottom, average current
facilitation recorded with 2/ 1-CH4 and
2/ 1-CH4M123. The prepulse lasted 200 ms
and had an amplitude of +100 mV (test pulse of 400 ms at +10 mV). #,
significantly different from
2/ 1-CH4.
|
|
Although facilitation was a property specifically carried by the
subunit, participation of the
1 subunit appeared also to
be essential since this type of current potentiation was recorded only
with the
1C pore-forming subunit. In a first attempt to identify critical amino acids of the
1C subunit involved
in this regulation, we constructed two deletions in the
1C subunit that have been reported to increase
Ca2+ current amplitude (25, 26).
1C
N had
its first 29 amino acids removed, corresponding to the deletion made in
the rabbit
1C subunit (first 60 amino acids) (26).
Similarly,
1C
C had a deletion of amino acids
1706-2143, corresponding to deletion of the homologous residues
1733-2171 of the rabbit subunit (25). Our prediction was that these
sequences could be involved in a tonic block of the Ca2+
channel activity that could be relieved either by deletion (25, 26) or
by voltage-dependent interactions with
subunits, thus inducing current facilitation. We thus expressed these two truncated forms of the
1C subunit with either the
1
or
2 subunit and tested their capacity to respond to
conditioning pre-depolarization (Fig. 8).
As expected, the amplitudes of the Ba2+ currents recorded
with both
1C
N and
1C
C were
systematically larger than those recorded with the full-length
1C subunit (data not shown). However, deletion in
neither the N-terminal nor C-terminal tail affected the response of
these subunits to pre-depolarizations with the
1 or
2 subunit. Respective increases in Ba2+
current recorded with the
1 and
2
subunits were 80 ± 30% (n = 9) and 11 ± 11% (n = 11) for
1C
N and 63 ± 36% (n = 3) and 11 ± 3% (n = 2)
(9 and 14%) for
1C
C. We therefore conclude that these two sequences, despite their role in modulating current amplitude, were not directly involved in the regulation of
1C subunit voltage-dependent
facilitation.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 8.
N- and C-terminal deletions of the
1C subunit have no effect on current facilitation.
The same recording conditions were used as those described in the
legend to Fig. 3, but using truncated 1C subunits.
Left panel: constructions used; middle
panel, current traces (scale bars = 1 µA); right panel, averaged facilitation
recorded with these deleted forms coexpressed with the 1
or 2 subunit. 1C N was made by removing
the first 29 amino acids, whereas 1C C had the last
437 amino acids removed (amino acids 1706-2143).
|
|
 |
DISCUSSION |
Functional Diversity of Facilitation--
Our results show that
neuronal L-type Ca2+ channel facilitation can
be promoted or blocked by distinct short sequences of the ancillary
subunit. The lack of effect of acute injection of okadaic acid, the
insensitivity to incoming Ca2+ ions (12), and the
requirement for permissive
1
3, and
4 subunits (13) clearly distinguished this type of
facilitation from L-type facilitations previously
characterized in cardiac, skeletal, and vascular cells (6-8,14-18)
and suggest that different underlying mechanisms are involved. The
presence or absence of these different types of facilitation with
related L-type Ca2+ channels, such as, for
example, the cardiac L-type Ca2+ channel, may
depend not only on the type of auxiliary subunit associated with the
1 subunit in a particular cell type, but also on
specific kinase activities (6, 8, 17, 27) or expression of other
associated proteins such as ryanodine receptors (for the L-type
Ca2+ channel) (28, 29) or syntaxine (for the N-type
Ca2+ channel) (30). All of these elements have been shown
to be crucial for the normal development of specific forms of
facilitation. Our results, however, emphasize the importance of the
auxiliary
subunits for neuronal L-type facilitation and
can explain the diversity of the response of the same
1C
subunit to conditioning depolarizations when recorded in cardiac and
neuronal cells. Differentially spliced variants of the
2
subunit have been described in rat, rabbit, and human (23). At least
three display important variations in their N-terminal V1 domains,
suggesting that functional differences regarding
L-type Ca2+ channel facilitation may exist. One
consequence of the differential expression of these splicing variants
could therefore be to finely tune the level of Ca2+
entry during sustained or repetitive depolarizations.
Molecular Mechanism of Neuronal
1C Subunit
Facilitation--
Although we have previously noted the importance of
normal protein kinase A activity for facilitation (12), a mechanism involving voltage-dependent phosphorylation of the channel
as a key step for the promotion of facilitation can be disregarded for
the following reasons. First, we have demonstrated that ATP
S, AMP-PCP, and okadaic acid cannot stabilize or prevent
L-type Ca2+ channel facilitation (12). Second,
facilitation can be recorded on expression of the
1
subunit, but not the
2/
1-CH4 chimera, which retains the putative protein kinase A and C phosphorylation sites
present in the
1 subunit (and of course in the
1C subunit). Third, cardiac L-type
Ca2+ channels, which are highly sensitive to protein kinase
A phosphorylation (31), are completely resistant to this type of
voltage-dependent facilitation (13). Altogether, these data
suggest that facilitation requires a phosphorylated
1C
subunit, but do not support the existence of a
voltage-dependent phosphorylation step, as described in
skeletal muscle (7), in the pathway leading to current
potentiation.
We have mapped two distinct regions in the sequence of the
Ca2+ channel
subunit as critical for the promotion and
inhibition of L-type Ca2+ channel facilitation
(Fig. 9). The promoting sequence,
localized in the C2 domain of
1, appears to be conserved
on the four
subunits, as shown by the capability of
3 and
4 (12, 13) and
2-TF2
(this work) to promote facilitation. This domain contains the
subunit interaction domain identified by De Waard et al. (32) and shown to be responsible for the
subunit-induced regulation of Ca2+ channel properties. Facilitation is, however, the
first property demonstrated to be carried out by the
subunit,
although it appears to be specific to the
1C subunit.
This suggests that the molecular consequences of
subunit binding
are specific among the
1 subunits. Among possible
molecular mechanisms of facilitation, we have excluded the involvement
of a voltage-dependent block of the channel by the amino-
and carboxyl-terminal tails of the
1C subunit, although their deletions have been shown to increase the L-type current amplitude (25). Another mechanism could be a prepulse-induced reactivation of inactivated channels since the same sequence that is
shown here to block facilitation has already been shown to block
voltage-dependent inactivation (33). Whether or not the three arginine residues that we have mutated and the chimeras used in
this work also affect inactivation remains to be determined. Further
experiments to test the effects of these on channel inactivation will
be useful in clarifying this issue.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 9.
Schematic drawing summarizing the results
presented in this study. Shown is a schematic representation of
the putative secondary structure of the 1C and subunits and their possible implication in L-type Ca2+
channel facilitation. Deletions of the N- and C-terminal tails of
1C subunits have no effect on current facilitation.
However, whereas the second conserved segment of the subunit is
necessary for facilitation (marked as Excitatory), the
N-terminal tail (V1; see sequence on the figure) of the
2 subunit has a unique role in preventing current
potentiation. The sequence of the V1 domain, with charged amino acids
in boldface, is shown on the bottom of the figure.
|
|
The inhibitory sequence is found only in the
2 subunit.
The lack of current facilitation produced by coexpression of the
2/
1-CH4 chimera suggests that specific
interactions with the first few amino acids of the
2
subunit are important for the mechanism by which facilitation is
blocked. Furthermore, the abolition of the effect by the mutation of
the three arginines located in this short sequence suggests that
electrostatic interactions may be of importance for this inhibition.
The absence of facilitation recorded with
2-TF1 is worth
noting and could also suggest the existence of another inhibitory
sequence within the C1 domain of the
2 subunit. At this
point, however, we are not able to precisely locate the inhibitory
binding site(s) of these sequences on the
1C or
2 subunit. On-going experiments, using chimeras between
1 subunits permissive and nonpermissive for
facilitation, should allow us to further define this binding site and
to precisely determine the molecular mechanism of this form of neuronal
L-type current facilitation. Whether the same sequences of the
2 subunit are also important for other types of
Ca2+ channel facilitation, such as
G-protein-dependent N-type Ca2+ channel
facilitation, remains to be determined.
We thank Drs. T. Snutch and E. Perez Reyes
for kindly providing calcium channel
1C and
subunit
cDNAs; Drs. D. Fisher, P. F. Mery, and J. P. Pin for
helpful comments on the manuscript; and Drs. S. Galas, J. C. Labbe, T. Lorca, and N. Morin for technical expertise in in
vitro translation.