Voltage and Calcium Use the Same Molecular Determinants to
Inactivate Calcium Channels*
Thierry
Cens,
Sophie
Restituito,
Simon
Galas, and
Pierre
Charnet
From the Centre de Recherches de Biochimie Macromoléculaire,
CNRS UPR 1086, 1919 Route de Mende, F34293 Montpellier, France
 |
ABSTRACT |
During sustained depolarization, voltage-gated
Ca2+ channels progressively undergo a transition to a
nonconducting, inactivated state, preventing Ca2+ overload
of the cell. This transition can be triggered either by the membrane
potential (voltage-dependent inactivation) or by the
consecutive entry of Ca2+
(Ca2+-dependent inactivation), depending on the
type of Ca2+ channel. These two types of inactivation are
suspected to arise from distinct underlying mechanisms, relying on
specific molecular sequences of the different pore-forming
Ca2+ channel subunits. Here we report that the
voltage-dependent inactivation (of the
1A
Ca2+ channel) and the
Ca2+-dependent inactivation (of the
1C Ca2+ channel) are similarly influenced by
Ca2+ channel
subunits. The same molecular determinants
of the
subunit, and therefore the same subunit interactions,
influence both types of inactivation. These results strongly suggest
that the voltage and the Ca2+-dependent
transitions leading to channel inactivation use homologous structures
of the different
1 subunits and occur through the same
molecular process. A model of inactivation taking into account these
new data is presented.
 |
INTRODUCTION |
Calcium channels formed integral membrane proteins through which
Ca2+ ions can flow into the cells during membrane
depolarization, thereby activating many fundamental physiological
functions such as contraction, synaptic transmission, or gene
activation (1, 2). According to their biophysical and pharmacological
properties, they have been classified into L, N, P, Q, R, and T type
Ca2+ channels (2-6). All these types are able to respond
to membrane depolarization by opening a pore, selective for
Ca2+ and other divalent cations, and thus generating an
inward current into the cells (2, 7). During sustained depolarizations, all of these Ca2+ currents progressively undergo a
voltage-dependent inactivation with specific kinetics,
primarily regulated by the membrane potential (2, 3, 8, 9). Of
particular interest in the case of the L-type voltage-gated
Ca2+ channels is the existence of a second type of
inactivation driven by the intracellular Ca2+ concentration
(10-12). In this case, Ca2+ ions entering into the cell
through calcium channels can bind to a specific site located close to
the inner mouth of the channel and promote the so-called
Ca2+-dependent inactivation (12-15). This type
of inactivation, as opposed to the voltage-dependent
inactivation, is not recorded when Ba2+ or
Sr2+ permeate the channel (2, 4, 11, 12). While
voltage-dependent inactivation has been found in all types
of Ca2+ channels, the
Ca2+-dependent inactivation seems to be
specific to the L-type, dihydropyridine-sensitive, Ca2+
channels and has been suggested to arise from a completely different mechanism (5, 12, 15-19).
It is strongly believed that all types of functionally characterized
Ca2+ channels possess at least three different subunits:
1, the pore-forming subunit, and
2-
and
, two regulatory subunits (20-26). The biophysical and
pharmacological properties of a Ca2+ channel are primarily
driven by the
1 subunit (13, 27-31), for which 10 genes
(named class A, B, C, D, E, F, G, H, I, and S) have been identified
(23, 25, 26, 32-34). The
subunit (four genes identified:
1-4)
and, to a lesser extent, the
2-
subunit (only one
gene) seemed to have only a regulatory role on these properties (8,
35-42). The
1 subunit is an integral membrane protein
organized in four homologous domains, each containing six
-helical
membrane-spanning segments (23, 25, 26). It has been shown that the
differences in the kinetics of the voltage-dependent inactivation between two distinct
1 subunits (encoding
class E and A calcium channels, respectively) were due to the segment S6 of the first domain (I-S6) (30), suggesting a role for important and
delimited molecular structures in the process of inactivation as was
the case for K+ channels (43). On the other hand, a typical
Ca2+ binding site (44) and other regions involved in the
Ca2+-dependent inactivation (45, 46) have
recently been located in the carboxyl-terminal tail of the
1 subunit. The identification of two different
structures regulating voltage and
Ca2+-dependent inactivation supports the
hypothesis of different underlying mechanisms for these two types of
inactivation. However, donation of this Ca2+ binding site
(EF-hand motif) to the
1E subunit gave chimerical Ca2+ channels that did support
Ca2+-dependent inactivation, whereas the
parental
1E subunit did not. The fact that
Ca2+-dependent inactivation can be transplanted
by the sole donation of a Ca2+ binding site presupposes
that the molecular backbone responsible for the closure of the channel
during sustained depolarization may be shared by voltage- and
Ca2+-dependent inactivation and would therefore
be present on all types of Ca2+ channel.
To test this hypothesis, we have compared the regulation of
inactivation of the class A and class C Ca2+ channels by
different
subunits in conditions where pure voltage and
Ca2+-dependent mechanisms drove inactivation,
respectively. We found that voltage and
Ca2+-dependent inactivations were similarly
regulated by the four
subunits. Moreover, deleted or chimerical
forms of the
subunit that altered the regulation of the
voltage-dependent inactivation also modified the
Ca2+-dependent inactivation. We proposed a
scheme for the inactivation mechanism and suggested that common distal
steps shared by Ca2+- and voltage-dependent
inactivations use homologous molecular structures. This scheme may be
useful in elaborating new experiments designed to understand the
molecular mechanisms underlying voltage and
Ca2+-dependent inactivations.
 |
EXPERIMENTAL PROCEDURES |
Preparation of Truncated and Chimerical Forms of the
Subunits--
The following calcium channel subunits were used:
1A (47),
1B (48),
1E (49),
1C (50),
1b (40),
2a (38),
3 (37), and
4 (36). All of these subunit
cDNAs were inserted into the pMT2 expression vector (51). Ndel-
2
was obtained by PCR.1 The
sense primer was engineered to possess an EcoRI site (italic type), a start codon (boldface type), and the 748-769 sequence (underlined, accession number M80545) of the
2a subunit
(5'-GGAATTCATGGAGAACATGAGGCTACAGCAGCATG-3'). In the reverse primer, an XbaI site was added at the 5'-end
of the 2190-2168 sequence of
2a
(5'-GCTCTAGATCATTGGCGGATGTATACATCCC-3'). The PCR
product was checked and purified on agarose gel. The chimera were
obtained by a PCR strategy as described (52). The N-terminal fragments
were amplified using the following primers: for
ch1, sense primer,
5'-GGAATTCAGCCCCCTGAAAGGAGATC-3' (representing
EcoRI plus positions 114-133 of
2a) and
reverse primer, 5'-CAGCAGGCGAAGGCTGTCTAGTTTGACCGGGCTTG-3' (representing positions 576-559 of
1b, accession number
X61394, plus positions 747-731 of
2a); for
ch4, sense primer same as
ch1 sense primer and reverse primer,
5'-GTAGGACTCTGCTGAGCCATAGGACACCCGTACTC-3' (representing positions 235-249 of
1b + positions
407-423 of
2a); for
ch5, sense primer,
5'-GGAATTCATGGTCCAGAAGAGCGGCATG-3' (representing
EcoRI plus positions 67-84 of
1b) and
reverse primer, 5'-TAGGAGTCTGCCGAACCCTGACGGACAAAGCTGTT-3'
(representing positions 424-440 of
2a plus positions
217-234 of
1b). The C-terminal fragments
were amplified using the following primers: for
ch1, sense primer,
5'-CAAGCCCGGTCAAACTAGACAGCCTTCGCCTGCTG-3' (representing positions 731-747 of
2a plus positions
559-576 of
1b) and reverse primer,
5'-GCTCTAGATCAGCGGATGTAGACGCCTTG-3' (representing XbaI plus positions 1857-1837 of
1b); for
ch4, sense primer, 5'-GAGTACGGGTGTCCTATGGCTCAGCAGAGTCCTAC-3' (representing
positions 407-423 of
2a plus positions
235-249 of
1b) and reverse primer same as
the
ch1 reverse primer; for
ch5, sense primer, 5'-AACAGCTTTGTCCGTCAGGGTTCGGCAGACTCCTC-3' (representing
positions 217-234 of
1b plus positions 424-440 of
2a) and reverse primer, 5'-GCTCTAGATCATTGGCGGATGTATACATCCC-3' (representing
XbaI plus positions 2168-2190 of
2a).
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. Ndel-
2 and -
chimera were finally digested
using EcoRI and XbaI, subcloned into pBluescript
(Stratagene) for sequencing (DiDeoxy Terminator technology; Applied
Biosystems), and subsequently subcloned into pMT2 for injection and expression.
I-II and III-IV loops of the
1A and
1C
subunits were produced by PCR, subcloned into pBluescript, and in
vitro transcribed (T7 m-Message m-Machine; Ambion) for oocyte
injection (at an mRNA concentration of 1 µg/µl). Correct
translation and molecular weight of these loops were checked by
in vitro translation (T7-TNT coupled reticulocyte lysate
system from Promega).
Xenopus oocyte preparation and injection (5-10 nl of
1,
1 plus
, or
1 plus
2-
plus
cDNAs at ~0.3 ng/nl) were performed as described elsewhere (42, 53). Oocytes were then incubated for 2-7
days at 19 °C under gentle agitation before recording.
Electrophysiological Recordings--
Whole cell Ba2+
and Ca2+ currents were recorded under two-electrode voltage
clamp using the GeneClamp 500 amplifier (Axon Instruments, Burlingame,
CA). Current and voltage electrodes (less than 1 megaohm) were filled
with 2.8 M CsCl, 10 mM BAPTA, pH 7.2, with
CsOH. Ca2+ and Ba2+ current recordings were
performed after injection of BAPTA (one or two 40-70-ms injections at
1 bar of 100 mM BAPTA free acid (Sigma), 10 mM
CsOH, 10 mM HEPES, pH 7.2, using solutions of the following
composition: 10 mM BaOH/CaOH, 20 mM
tetraethylammonium hydroxide, 50 mM
N-methyl-D-glucamine, 2 mM CsOH, 10 mM HEPES, pH 7.2, with methanesulfonic acid.
Ca2+ and Ba2+ current amplitudes were usually
in the range of 1-5 µA, except for the
1C subunit
expressed alone (70-250 nA) as reported by others (35). Expression of
the
2
subunit was sometimes performed but did not
significantly modify either the kinetics of the two types of
inactivation or the effects of overexpressing intracellular I-II and
III-IV loops. All of the results mentioned here were obtained without
2
.
Currents were filtered and digitized using a DMA-Tecmar labmaster and
subsequently stored on an IPC 486 personal computer by using version
6.02 of the pClamp software (Axon Instruments). Ba2+ or
Ca2+ currents recorded during a typical test pulse from
80 mV to +10 mV of 2.5-s duration were well fitted using a
biexponential function: i(t) = (A1*
exp(
(t
K)/Tau1) + A2 * exp(
(t
K)/Tau2)) + C, where t is
the time; K is the zero time; A1, A2,
Tau1, and Tau2 are the amplitudes and time
constants of the two exponential components; and C is the
fraction of noninactivating current.
Current amplitudes and inactivation time constants were measured using
Clampfit (pClamp version 6.02, Axon Instruments). Pseudo-steady state
inactivation (2.5 s of conditioning depolarization) and current-voltage
curves were fitted using the equations
I/Imax = R + (1
R)/(1 + exp((V
V0.5)/k)) (for the inactivation
curve) and I/Imax = g *
(V
Erev)/(1 + exp((V
V0.5)/k))
(for the current-voltage curve), where g is a normalized
conductance, Erev is the extrapolated reversal
potential for barium, k is the slope factor,
V0.5 is the potential for half-inactivation or
activation, V is the conditioning depolarization
(inactivation curve) or the membrane potential used to record current
(current-voltage curve), and R is the proportion of
noninactivating current. All values are presented as mean ± S.D.
Student's t test was used at the 0.05 confidence level to test the significance of the difference between two means.
 |
RESULTS AND DISCUSSION |
When expressed in Xenopus oocytes, each class of
Ca2+ channels displays only one type of inactivation, which
can be driven either by membrane potential or change in intracellular
Ca2+ concentration (Fig. 1,
A and B). Current kinetics of the
1A,
1B and
1E
Ca2+ channel, co-expressed with the ancillary
Ca2+ channel
1 or
2 subunits,
were identical using either Ca2+ or Ba2+ as
charge carriers, as shown by the superimposed
scaled traces seen in Fig. 1, A and
B. These kinetics were only modulated by the amplitude of
the depolarization, as expected for a voltage-dependent inactivation (not shown) (41, 54, 55). In contrast, Ca2+
currents recorded from oocytes injected with the
1C
subunit inactivated markedly faster than the corresponding
Ba2+ currents (Fig. 1A). This behavior, typical
of the so-called Ca2+-dependent mechanism of
inactivation (13, 56-58), was present whether the
1 or
the
2 subunit was co-expressed. Despite differences in
their initiating events, voltage- and
Ca2+-dependent inactivations were nevertheless
both sensitive to the co-expression of the ancillary
subunits.
Co-expression of the
1A subunit with the
1 or
2 subunit, for example, led to fast or slowly decaying Ba2+ currents, respectively (Fig.
1C, left). These differences in current kinetics
were similar, in the case of the
1A subunit (but also
1B and
1E), using either 10 mM Ca2+ or Ba2+ as the charge
carrier (see Fig. 1, A and B). Fast and slowly inactivating Ca2+ currents were also recorded upon
co-expression of the
1 or
2 subunit with
the
1C subunit (Fig. 1C, right).
In this case, however, the use of 10 mM Ba2+ as
the charge carrier completely blocked current inactivation for these
two subunit combinations, further demonstrating the absolute
requirement of Ca2+ for
1C inactivation
(Fig. 1, A and B). To understand the molecular mechanisms underlying these two different types of inactivation, the
1A and
1C subunits were chosen as
prototype Ca2+ channels with pure voltage and
Ca2+-dependent inactivations, respectively.
Averaged data using long depolarizations and extended to the
co-expression of the four known
subunits with these two
1 subunits are shown in Fig. 2. When compared with the corresponding
1 subunit expressed alone, voltage- and
Ca2+-dependent inactivations were significantly
accelerated upon co-expression of the
1 subunits (Fig.
2,
1A (top left) and
1C (top right); p < 0.05). The same effect was found upon co-expression of the
3 or
4 subunit, without significant
difference when compared with
1A plus
1.
Co-expression of the
2 subunit, however, slowed both the
voltage and the Ca2+-dependent inactivation
when expressed with the
1A or the
1C subunit, respectively (significantly different from
1A
plus
1). A kinetic analysis of these current decays
confirmed and extended this observation. Voltage- as well as
Ca2+-dependent inactivation could be well
approximated by a biexponential decay. The fast time constant
(Tau1, Fig. 2) of inactivation of the
1A or
1C subunits was always accelerated upon co-expression of
each of the
subunits (no statistical differences between the four
subunits). The slow time constant (Tau2, Fig. 2) was either decreased (
1,
3, and
4) or increased (
2), for these two
1 subunits, after co-expression of a
subunit, and it
was thus responsible for the marked slowing of inactivation recorded with the
2 subunit (together with an increase in the
relative amplitude of the slow component in both cases; not shown).
Taken together, these data demonstrate that
subunits modified the same kinetic components of the voltage- and
Ca2+-dependent inactivations and thus suggest
that they affect the same molecular processes on the two
1 subunits. Because protein-protein interactions between
the
1 and the
subunits are the basis of these
regulations (8, 41, 42, 51, 59-63) and because
1 subunits alone are able to undergo both voltage-dependent
(
1A) and Ca2+-dependent
(
1C) inactivation (28-30, 41, 44, 51), we hypothesized that homologous molecular determinants on these different pore-forming subunits are responsible for these distinct modes of inactivation. One
immediate prediction is that structural modifications of the
subunit, modifying the voltage-dependent inactivation,
should also change, in parallel, the
Ca2+-dependent inactivation. We have tested
this hypothesis using four different constructions of the
subunit
(Fig. 3, top). A large
deletion in the amino terminus of the
2 subunit (125 amino acids) strongly accelerated the slow
voltage-dependent inactivation of the
1A
subunit, usually recorded with the full-length
2 subunit (Fig. 3, Ndel-
2, left). This deleted
2 subunit had exactly the same effect on the
1C Ca2+-dependent inactivation,
which became as fast as
1 (see Fig. 3,
Ndel-
2, right). When this deleted sequence
was inserted at a homologous position in the
1 subunit
(
ch1; see Fig. 3, top, for construction and traces),
Ba2+ currents recorded from oocytes expressing
1A plus
ch1 subunits displayed the slow inactivation
kinetics typically recorded with the
2 subunit. A
similar slowing of the Ca2+-dependent
inactivation was recorded when this chimera was expressed with the
1C subunit (Fig. 3,
ch1,
right). These data suggest that the presence of the
amino-terminal end of the
2 subunit is responsible for
the slow current decays in both voltage and Ca2+-dependent inactivation. This was further
confirmed by the use of chimera
ch4 and
ch5 (see Fig. 3,
top, for constructions). Co-expression of
ch4 (where the
1 subunit had its first variable domain (64),
corresponding to amino acids 0-58, replaced by the homologous amino
acids of the
2 subunit) decreased the
voltage-dependent inactivation (
1A) as well
as the Ca2+-dependent inactivation
(
1C). In both cases, inactivation became as slow as in
the case of co-expression of the full-length
2 subunit
(Fig. 3,
ch4 traces). The reverse chimera
ch5 (where the
2 subunit has its first domain,
corresponding to amino acids 1-16, replaced by the homologous domain
of
1) had opposite effects and induced fast-inactivating
currents comparable with those recorded with the
1
subunit (or Ndel-
2), irrespective of the
1 subunit and therefore of the mode of inactivation. All
of these data, summarized at the bottom of Fig. 3, clearly
show that any change in the structure of the
subunit affecting the
voltage-dependent inactivation also modified the
Ca2+-dependent inactivation and suggest that
the same mechanism underlies these two distinct phenomena.

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Fig. 1.
Regulation of voltage- and
Ca2+-dependent inactivation by
subunits. A and B,
Ba2+ and Ca2+ currents recorded from oocytes
injected with the 1A, 1B,
1C, or 1E Ca2+ channel
subunits together with the auxiliary 1 (A) or
2 (B) subunit. Inactivation
(I2/I1) was quantified by dividing the current at the end of
a 400-ms-long depolarization (I2) by the peak current
(I1) and is displayed as a bar graph
for these different subunit combinations in the presence of external
Ba2+ and Ca2+ (10 mM).
Corresponding scaled traces are displayed at the
bottom of each combination. Currents were recorded during
400-ms-long depolarizations to +10 mV from a holding potential of 100
mV: 1A plus 1 (n = 6);
1A plus 2 (n = 7); 1B
plus 1 (n = 5); 1B plus
2 (n = 5); 1C plus
1 (n = 11); 1C plus
2 (n = 10); 1E plus
1 (n = 6); 1B + 2 (n = 6). For a given combination of
subunits, an asterisk indicates when inactivation of
Ca2+ current was significantly faster (p < 0.05) than that of the Ba2+ current. C,
Ba2+ and Ca2+ current traces recorded during
long depolarization of oocytes expressing the 1A or the
1C subunits, respectively, with either the
1 or the 2 subunit. Current traces were
scaled at the same size and superimposed for comparison (depolarization
of 2.5 s to +10 mV from a holding potential of 100 mV).
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Fig. 2.
Effect of the subunits on the kinetics of inactivation of the
1A and
1C Ca2+ channels.
Top, inactivation was quantified as the ratio of the
noninactivating current at the end of the pulse over peak amplitude
(I2/I1). Depolarizations of 2.5 s were used to maximize
measurements of current inactivation. Dashed
lines, superimposed fit of the two exponential components
Tau1 and Tau2 necessary to describe current
inactivation kinetics. I2/I1, Noninactivating currents
calculated for different subunit combinations having either a
voltage-dependent inactivation ( 1A, barium)
or a Ca2+-dependent inactivation
( 1C, calcium). Note that the 2 subunit
decreased both the voltage-dependent ( 1A
subunit recorded in the presence of Ba2+) and the
Ca2+-dependent ( 1C subunit
recorded in the presence of Ca2+) inactivation
(n = 8, 10, 6, 7, and 7 for 1A,
1A plus 1, 1A plus
2, 1A plus 3, and
1A plus 4, respectively;
n = 6, 9, 7, 2, and 4 for 1C,
1C plus 1, 1C plus
2, 1C plus 3, and
1C plus 4, respectively). *,
1 plus 1 significantly different from
1 alone; #, 1 plus 2
significantly different from 1 plus 1
(see "Results and Discussion"). Tau1, fast time
constants of voltage-dependent inactivation
( 1A; recorded using Ba2+) and
Ca2+-dependent inactivation ( 1C;
recorded using Ca2+). Note that, in both cases, the fast
time constant was accelerated by expression of the different subunits. Tau2, only the 2 subunit increased
the slow time constant of both the voltage-dependent
( 1A) and the Ca2+-dependent
( 1A) inactivations. Currents were recorded during
depolarization at +10 mV of 2.5 s (n = 7, 13, 4, 8, and 7 for 1A, 1A plus
1, 1A plus 2,
1A plus 3, and 1A plus
4, respectively; n = 3, 9, 8, 3, and 3 for 1C, 1C plus 1,
1C plus 2, 1C plus
3, and 1C plus 4,
respectively.
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Fig. 3.
Mutations on the subunit have the same effects on the two types of
inactivation. A, schematic representation of the
truncated and chimeric forms of the subunit used to study the
regulation of inactivation. B, effect of these constructions
on voltage and Ca2+-dependent inactivation.
Oocytes were injected with the 1A or the
1C subunits in combination with the 1
(n = 12 and 7, respectively), 2
(n = 6 and 7), Ndel- 2 (n = 4 and 11), ch1 (n = 3 and 9), ch4
(n = 6 and 3), and ch5 (n = 5 and 4)
subunits. Currents were recorded during depolarization at +10 mV during
2.5 s, and traces were scaled. Note that the presence of the
N-terminal sequence of the 2 subunit governed the
kinetics of both types of inactivation. C, bar
graph, averaged ratio I2/I1 of the
noninactivating 1A and 1C currents for
the six combinations of subunit tested. 1 and
2 were the same values as in Fig. 2. Each subunit has
the same effect on the two types of inactivation. An
asterisk represents a statistical difference between
1A plus 2 and the constructs.
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Voltage- as well as Ca2+-dependent
inactivations are known to be intrinsic properties of the
1 subunit. However, until now, only
voltage-dependent inactivation was known to be regulated by
auxiliary
subunits (8, 41, 42, 51, 65). We show here the presence
of two kinetic components of inactivation (characterized by
Tau1 and Tau2) in both
1A- and
1C-directed currents. These two components are both
either voltage-dependent (
1A inactivation) or Ca2+-dependent (
1C
inactivation; see Fig. 2), suggesting that the two types of
inactivation each have two different underlying mechanisms. Our results
emphasize the fact that a given
subunit regulates, in the same way,
each of these two components, independently of the mode of inactivation
(voltage- or Ca2+-dependent), and therefore the
type of
1 subunit. To take into account these new data,
we propose a new scheme of inactivation where voltage and
Ca2+ inactivate these two different channels by using a
"ball and chain" mechanism, with blocking particles and binding
sites encoded by homologous sequences on the
1A and
1C subunits and therefore sensitive to the same
molecular interactions with the
subunit (see Fig.
4). Binding of the particle to its
binding site would ensure channel inactivation, as in the case of
potassium channels (43). In the case of voltage-dependent
inactivation, this binding is not ion-sensitive and can occur with
either Ba2+ or Ca2+ as charge carriers,
voltage-dependence being due to state-dependent changes in
the mobility of the particle and/or the accessibility of the binding
site. The Ca2+-dependent mechanism of
inactivation is essentially the same, except that accessibility to, or
functionality of, the binding site needs the fixation of a
Ca2+ ion to a site located near the inner mouth of the
channel (44) (Fig. 4, right). In our scheme, this fixation
would produce a conformational modification of the binding site into a
high affinity state, therefore allowing binding of the ball and
inactivation of the channel. The sensitivity of this newly formed
inactivated state to the continuous presence of a bound
Ca2+ ion on the
1 subunit might explain the
differences in the immobilization of channel gating charges between
voltage- and Ca2+-dependent inactivation (56).
In this scheme, regulation of inactivation by
subunits occurs
mainly through their N-terminal tail, where essential palmitoylation
sites have just been identified (66-68), and would be due to
modifications in the mobility of the inactivating particle by bound
auxiliary subunits (Fig. 4). Accordingly, this region of the
2 subunit has also been shown to influence voltage-dependent inactivation of the
1A
Ca2+ channel (68-70). Therefore, the I-II loop, connecting
domains I and II of the pore-forming
1 subunit
represents an attractive candidate for the blocking particle, since it
possesses the
1 interaction domain sequence, responsible
for the interaction with the
subunit (60), and multiple mutations
in this domain affect inactivation (71, 72). Accordingly, co-expression
of an excess of free
1A I-II loop with the slowly
inactivating
1A plus
2 Ca2+
channels significantly accelerated voltage-dependent
inactivation (
1A(I-II); Fig.
5, A and B).
Similar effects on the voltage-dependent inactivation of
the
1A plus
2 Ca2+ channels
were also recorded after co-expression of the I-II loop from
1C (
1C(I-II); Fig. 5, A and
B) but not with the III-IV loop from
1A
(
1A(III-IV)), suggesting that the
1C I-II
loop can promote voltage-dependent inactivation of the
1A Ca2+ channel. Indeed, similar changes
were obtained when steady state inactivation was studied, with smaller
noninactivating current and hyperpolarizing shift of the
half-inactivation potentials obtained with I-II loops but not the
III-IV loop (Fig. 5C; mean values for
V0.5 and R as follows:
H2O,
12 ± 7 mV and 0.57 ± 0.08 (n = 12);
1A(III-IV),
13 ± 7 mV
and 0.61 ± 0.13 (n = 15);
1A(I-II),
22 ± 5 mV and 0.35 ± 0.13 (n = 11);
1C(I-II),
16 ± 7 mV
and 0.41 ± 0.09 (n = 7)). Co-expression of these
loops did not affect significantly (or slightly hyperpolarized; see
Fig. 5C) the voltage dependence of activation and the
average current amplitude (not shown). These latter data confirmed a
direct effect on inactivation rather than a displacement of the
2 subunit from
1A by an excess of free
1 interaction domain, which would be expected to
depolarize the current-voltage curve (mean activation potentials were
as follows: H2O,
8 ± 3 mV (n = 12);
1A(III-IV),
11 ± 6 mV (n = 15);
1A(I-II),
15 ± 6 mV (n = 11);
1C(I-II),
7 ± 12 mV (n = 7)) and
depress current amplitude (62). Possible molecular determinants for the
binding site include the carboxyl end of the
1 subunit,
where an EF-hand Ca2+ binding motif and other sequences,
important for Ca2+ dependent inactivation, have been
identified (44-46, 72, 73). Supporting our scheme, transfer of this
domain to the
1E subunit confers
Ca2+-dependent inactivation (44), while
1C chimera subunits harboring a carboxyl-terminal tail
originating from a non-Ca2+-sensitive
1
subunit lost their Ca2+-sensitive inactivation while
preserving voltage-dependent inactivation (72).

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|
Fig. 4.
Proposed mechanism underlying voltage- and
Ca2+-dependent inactivation. The
schematic drawing represents the hypothetical
mechanism underlying voltage- and
Ca2+-dependent inactivation. The blocking site
of the inactivating particle is always present in 1
subunits displaying voltage-dependent inactivation
(top). In the case of Ca2+-dependent
inactivation (bottom), this site needs the binding of
Ca2+ to be functional. The inactivating particle is encoded
by a homologous sequence in both types of Ca2+ channel
1 subunit. subunits regulate Ca2+
channel inactivation by modifying the mobility of the blocking particle
(see "Results and Discussion").
|
|

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Fig. 5.
Overexpression of the
1 subunit I-II loop speeds up
inactivation. A, left, schematic
representation of the two-dimensional structure of the
1A subunit. Loop I-II and loop III-IV represent
connecting loops between domains I-II and domains III-IV of the
1 subunit. Loop I-II and loop III-IV of the
1A and 1C subunits
( 1A(I-II), 1A(III-IV), and
1C(I-II), respectively) were subcloned into pBluescript
and in vitro transcribed for oocyte injection.
Right, autoradiogram of SDS-polyacrylamide gel of in
vitro translated [35S]methionine-labeled
1A(I-II), 1A(III-IV), and
1C(I-II) loops showing the correct production of these
loops from the corresponding synthetic RNA. The film was exposed
overnight. B, left, typical current traces
recorded from oocytes injected with cDNA coding for the
1A and the 2 subunits co-injected with
H2O, 1A I-II loop ( 1A(I-II)),
1C I-II loop ( 1C(I-II)), or
1A III-IV loop ( 1A(III-IV)) RNAs.
Currents were recorded during 2.5-s-long depolarizations to +10 mV from
a holding potential of 100 mV. Right, averaged current
inactivation (I2/I1; see Fig. 1) calculated from
different oocytes: H2O (n = 15);
1A(III-IV) (n = 15);
1A(I-II) (n = 11); and
1C(I-II) (n = 7). Asterisk,
significantly different from control (H2O). C,
steady state (left) and current-voltage (right)
curves recorded from the same oocytes as in C, co-injected
with cDNA coding for the 1A and the 2
subunits and with H2O (square),
1A I-II loop ( 1A(I-II);
circle), 1C I-II loop
( 1C(I-II); triangle), or 1A
III-IV loop ( 1A(III-IV), inverted
triangle) RNAs. The holding potential was 100 mV.
Smooth lines represent the best fit using
equations described under "Experimental Procedures." Calculated
values for half-inactivation potentials and R for these
particular oocytes were as follows: H2O, 11 mV and 0.65;
1A(III-IV), 10 mV and 0.66; 1A(I-II),
17 mV and 0.31; 1C(I-II), 11 mV and 0.51. Half-activation potentials were as follows: H2O, 8 mV;
1A(III-IV), 7 mV; 1A(I-II), 7 mV;
1C(I-II), 5 mV (see "Results and Discussion" for
averaged values).
|
|
The presence of the two kinetic components (Tau1 and
Tau2) can be best explained by the existence of an
additional blocking particle instead of multiple binding sites, since
(i) fast and slow inactivation have distinct regulation by
subunits
and (ii) the two components are either voltage- (
1A) or
Ca2+- (
1C) dependent, as expected for a
unique binding site. Differences in the proportion of each component
between voltage- and Ca2+-dependent
inactivation may be due to sequence variation between the
1 subunits. This scheme also disregards any
participation of phosphatases in the process of
Ca2+-dependent inactivation, since the
intracellular Ca2+ concentration was sufficiently buffered
to inhibit Ca2+-dependent enzyme, and ATP
S,
as well as okadaic acid, had no effect either on Ca2+
current amplitude or on kinetics (74, 75). In conclusion, we show that
voltage and Ca2+ use homologous structure of related
1 subunits to inactivate Ca2+ channels, and
we provide evidence for a direct channel block by the intracellular
I-II loop of the
1 subunit. The use of similar molecular
determinants for the two types of inactivation suggests that
voltage-dependent inactivation has been modified during
evolution to provide the structural basis for the
Ca2+-mediated inactivation, therefore preventing cellular
Ca2+ overload.
 |
ACKNOWLEDGEMENTS |
We thank Dr. T. Snutch and E. Perez-Reyes for
kindly providing calcium channel cDNAs; Dr. T. Lorca, J. C. Labbe, and N. Morin for invaluable technical help during the course of
these experiments; and Drs. D. Pietrobon, D. Yue, P. F. Mery, M. Moris, and D. Fisher for helpful comments on the manuscript.
 |
FOOTNOTES |
*
This work was supported by CNRS, INSERM, MRES, the
Association Française contre les Myopathies, the Association pour
la Recherche contre le Cancer, the Fondation pour la Recherche
Médicale, the Ligue nationale contre le cancer, and NATO.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.
To whom correspondence should be addressed. Tel.: 33-467-61-33-52;
Fax: 33-467-52-15-59; E-mail: charnet{at}crbm.cnrs-mop.fr.
 |
ABBREVIATIONS |
The abbreviations used are:
PCR, polymerase
chain reaction;
ATP
S, adenosine 5'-O-(thiotriphosphate);
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic
acid.
 |
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