From the Centre de Recherche de Biochimie
Macromoléculaire, CNRS Unité Propre de Recherche 1086, Institut Federatif de Recherche 24, 1919 Route de Mende, 34293 Montpellier, France and ¶ Mt. Sinai Hospital, SLRI-860, Toronto,
Ontario M5G 1X5, Canada
Received for publication, September 17, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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Neuronal Ca2+
sensor protein-1 (NCS-1) is a member of the Ca2+ binding
protein family, with three functional Ca2+ binding EF-hands
and an N-terminal myristoylation site. NCS-1 is expressed in brain and
heart during embryonic and postnatal development. In neurons, NCS-1
facilitates neurotransmitter release, but both inhibition and
facilitation of the Ca2+ current amplitude have been
reported. In heart, NCS-1 co-immunoprecipitates with K+
channels and modulates their activity, but the potential effects of
NCS-1 on cardiac Ca2+ channels have not been investigated.
To directly assess the effect of NCS-1 on the various types of
Ca2+ channels we have co-expressed NCS-1 in
Xenopus oocytes, with CaV1.2,
CaV2.1, and CaV2.2 Ca2+ channels,
using various subunit combinations. The major effect of NCS-1 was to
decrease Ca2+ current amplitude, recorded with the three
different types of Ca2+ entry through voltage-gated
Ca2+ channels is essential for various cellular processes
that include muscle contraction, pacemaker activity, synaptic
transmission, or gene expression. Several types of Ca2+
channels have been characterized (T, L, N, P/Q, and R) that appear to
play a specific role in each of these functions. These channels share a
common architecture composed of a major The neuronal Ca2+ sensor protein-1
(NCS-1),1 the mammalian
homologue of frequenin, belongs to a group of small
Ca2+-binding proteins comprising four EF-hand motifs, three
of which are able to bind Ca2+ in the micromolar (EF-2) or
submicromolar (EF-3,4) range (14-16). NCS-1 also contains an
N-terminal myristoylation site (17). NCS-1 has been shown to facilitate
synapse formation, spontaneous and/or evoked neurotransmitter release,
paired-pulse facilitation, and exocytosis in several cell types
(18-22). NCS-1 interacts directly with phosphatidylinositol
4-kinase in yeast (23), COS-7 (24), and chromaffin cells (25),
leading to the hypothesis that part of the effects mediated by NCS-1
could involve modifications in cellular trafficking through regulation
of the phosphatidylinositol signaling pathway, thereby affecting
vesicular transport and recycling, as well as inositol
1,4,5-trisphosphate-sensitive Ca2+ stores (24, 25).
However, a possible involvement of NCS-1 in expression and regulation
of voltage-gated Ca2+ channels has also been proposed.
Overexpression of a dominant-negative mutant of NCS-1, which displays
impaired Ca2+-dependent conformational changes
(19), or direct loading of presynaptic nerve terminals with NCS-1
suggested that voltage-independent inhibition, as well as
activity-dependent facilitation of P/Q-type Ca2+ channels (CaV2.1), could be controlled by
NCS-1, possibly via direct protein-protein interactions (18). Effects
on N-type Ca2+ channel (CaV2.2) properties have
also been reported (20), and the expression of NCS-1 in mammalian
cardiac myocytes and subsequent effect on K+ channel
expression (26) gave rise to the possibility that NCS-1 may regulate
multiple types of Ca2+ channels and other
voltage-dependent ion channels, not only in neurons
(27).
In a first step to explore this possibility, we have co-expressed NCS-1
with three different types of Ca2+ channel,
CaV1.2, CaV2.1, and CaV2.2,
associated with different combinations of auxiliary Materials and Oocyte Preparation--
The following cDNA
were used, and the GenBankTM accession number is provided:
CaV1.2 (
Xenopus laevis oocyte preparation and injection
were performed as described previously (30). Each oocyte was injected
with 5-10 nl of a cDNA mixture containing the
Electrophysiology--
Whole-cell Ba2+ currents were
recorded under two-electrode voltage clamp using the GeneClamp 500 amplifier (Axon Instruments, Union City, CA). Current and voltage
electrodes (less than 1 megohm) were filled with 3 M KCl,
pH 7.2, with KOH. Ba2+ and Ca2+ current
recordings were performed after injection of BAPTA (~50 nl of the
following (in mM): BAPTA-free acid (Sigma), 100; CsOH, 10;
HEPES, 10; pH 7.2 with CsOH) using the following bathing
solution (in mM): BaOH/CaOH, 10; TEAOH, 20; NMDG, 50; CsOH,
2; HEPES, 10; pH 7.2, with methanesulfonic acid. Currents were filtered
and digitized using a DMA-Tecmar Labmaster and subsequently
stored on a Pentium-based personal computer using the pClamp software (version 6.02; Axon Instruments). Ba2+ or Ca2+
currents were recorded during a 400-ms test pulse from
Inactivation kinetics were estimated by fitting Ba2+
current decay with two exponential components using the equation,
I(t) = A1e(
Several independent experiments (N, number of batches of
oocytes injected) were performed, always including a control group without NCS-1 expressed. These experiments always gave the same statistical result, and the total number of recordings (from
n oocytes of these N experiments) are thus
presented. All values are presented as mean ± S.E., and
comparison between groups of oocytes were evaluated using a Student's
t test, with a statistical significance set at the
p value <0.05.
Biochemistry--
Selected oocytes were first homogenized in the
lysis buffer (5 µl/oocyte of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 50 mM NaF, 10 mM
Expression of NCS-1 in XenopusOocytes--
Previous works on
chromaffin, human embryonic kidney 293, and COS-7 cells have shown that
NCS-1 was endogenously expressed at non-negligible levels (24,
28).2 In non-injected
X. laevis oocytes, the level of
expression of the endogenous NCS-1 was barely detectable in
Western-blots and much lower than in human embryonic kidney 293 and
COS-7 cells (see Fig. 1A).
Thus X. laevis oocytes are a system of choice to study the functional effect of NCS-1 on voltage-gated Ca2+
channels. In these oocytes, injection of the cDNA coding for rat
NCS-1 led, as expected, to a massive expression of a protein of a
molecular mass of ~23 kDa, in accordance with the theoretical molecular mass of NCS-1 (21.9 kDa; see Fig. 1B). In the
following experiments, the oocytes used for current recordings were
collected after recordings and submitted to a similar Western blot
analysis to ensure that the NCS-1 protein was properly expressed.
Down-regulation of CaV1.2, CaV2.1, and
CaV2.2 Expression by NCS-1--
In a first set of
experiments, the effects of NCS-1 were tested on Ba2+
currents flowing through L-, N-, and P/Q-type Ca2+
channels. For each Ca2+ channel type, this was done by
co-injecting a mixture of cDNA containing
The Ca2+ channel Effect of NCS-1 on CaV2.1 Properties--
We next
investigated whether co-expression of NCS-1 with the CaV2.1
subunit might have other functional consequences on Ca2+
channel properties. Current-voltage curves and isochronal inactivation curves were constructed from currents recorded in oocytes expressing the CaV2.1 subunit with the
As presented in Table I, in the
presence of Ca2+ ions and in the absence of
We then analyzed the effects of NCS-1 on Ba2+ current
kinetics. CaV2.1 current inactivation could be approximated
by an exponential decaying phase, best described using a fast
(
To get some insight into the possible molecular determinants involved
in NCS-1 effects, the same experiments, with the
Co-expression of either NCS-1E120Q or NCS-1G2A
had the same effect as wild-type NCS-1 on the current-voltage curve of
the CaV2.1+ Recent studies have extended the area of expression of NCS-1 from
the nervous system to neuroendocrine cells and even cardiac myocytes
(21, 26, 33). In these cell types, NCS-1 modulates synaptic
transmission (14, 22, 34), secretion (21, 25), or cellular excitability
(18, 20) via complex processes that include regulation of key enzymes
for membrane transport (15, 17, 23, 24, 35) and specific regulation of
various ion channels (16, 18-20, 28). K+ channels were the
first ion channel target to be characterized in expression systems (26,
36). Co-expression studies described an up-regulation of channel
expression and activity, specifically recorded with the
KV4 K+ channel family (26, 36). Modifications
in the expression and properties of other ion channels have also been
reported. In endocrine cells and neurons P/Q-type and N-type
Ca2+ channels are clearly affected (18-20, 28), but no
effect on L-type channels has been reported so far (20). These results suggest the existence of specific effects among the different voltage-activated Ca2+ channel types or among different
tissues. However, the molecular basis of this specificity, and in
particular the role of the subunit composition of the channel, in the
observed effects remains unknown.
In the present work, using heterologous expression of defined
Ca2+ channel subunits, we show that NCS-1 has two major
effects on high voltage-activated Ca2+ channels: (1) a
decrease in the current amplitude that is Effect of NCS-1 on Ca2+ Current Amplitude--
Effects
of NCS-1 on P/Q- and N-type Ca2+ channel current amplitudes
have been documented over the past years but always based on
experiments using either dominant negative mutants or overexpression of
wild-type NCS-1 in native cells constitutively expressing the protein
(18-20). Our study is therefore the first to analyze the effects of
NCS-1 on multiple types of Ca2+ channel in the same
environment, using heterologous expression of defined Ca2+
channel subunits. In neurons, NCS-1 has been shown to clearly increase
N-type Ca2+ channel expression (20), with significant
modifications of the electrophysiological parameters. The opposite
effects have been found on P/Q-type Ca2+ channels in bovine
chromaffin cells, where a dominant negative mutant of NCS-1 increased
Ca2+ current amplitude (19) without effect on L-type
Ca2+ channels. Using cDNA injection, we show that all
three channel types are down-regulated by co-expression of NCS-1. Our
experimental conditions and our analysis of the expression level of the
CaV2.1 subunit (Fig. 2C) suggest that this
effect must take place after protein synthesis and could involve
modifications in the correct folding and trafficking of the channel
complex to the plasma membrane and/or regulation of the channel
activity per se, taking place after insertion of the channel
into the plasma membrane. Membrane expression of high voltage-activated
Ca2+ channels is known to rely on a short sequence located
within the intracellular loop connecting the homologous domains I and II of the
Interestingly, NCS-1 had no effect on CaV2.1
Ca2+ channel co-expressed with the
The fact that mutants that prevented Ca2+ binding
(NCS-1E120Q) and protein myristoylation
(NCS-1G2A) both decreased the effects of NCS-1 on channel
expression underlines the requirement for a fully functional NCS-1
protein to record these effects. Mutation of glutamate 120 to glutamine
(NCS-1E120Q mutant) disrupts a high affinity
Ca2+ binding site and impairs
Ca2+-dependent conformational changes (19).
This mutant acts as a dominant negative mutant for the regulation of
the P/Q channel in chromaffin cells (19). In Xenopus oocytes
the NCS-1E120Q mutant appeared also less potent than
wild-type NCS-1. However, a clear and significant decrease in current
amplitude was nevertheless observed, suggesting that the mutation did
not completely suppress NCS-1 activity. These differential effects
constitute another argument in favor of the presence of different
mechanisms working in chromaffin cells or Xenopus oocytes to
regulate Ca2+ channels, i.e. removal of a
voltage-independent inhibition versus down-regulation of
channel trafficking, and suggest that
Ca2+-dependent changes are absolutely necessary
only for the removal of the voltage-independent inhibition of the P/Q
channels (19, 28). Preserved interactions of NCS-1E120Q
with cellular proteins can be an argument for the dominant negative
effect of the mutant, as suggested (19), but can also constitute an
interesting area of investigation to identify preserved interactions
that may be still functional and involved in a
Ca2+-independent down-regulation of the channel activity.
Mutation of the myristoylation site, NCS-1G2A, is
known to affect subcellular localization of NCS-1 (29), with minor
modifications in the protein structure (46). The tendency of the
NCS-1G2A mutation to decrease Ca2+ current
amplitude was not found statistically significant. This may simply
reflect a small decrease in the availability of the NCS-1G2A mutant at early stages of protein assembly,
because of the loss of the perinuclear localization (29) or because of more indirect effects, related to mutation-induced modifications in the
degree of cooperativity in Ca2+ binding (46). The
construction and testing of the double mutant NCS-1G2A/E120Q should help to solve this issue.
Effect of NCS-1 on Ca2+ Current Properties--
Beyond
the decrease in current amplitude, we also noted that NCS-1 could
specifically affect the electrophysiological properties of the
CaV2.1 channel, but only when co-expressed with the
Alternatively, it has also been proposed that NCS-1 could activate the
same targets as calmodulin (35), a regulatory element of the
CaV1.2 and CaV2.1 Ca2+ channels,
responsible for the Ca2+ sensitivity of inactivation and
facilitation (10, 12, 13, 47, 48). The binding site for calmodulin on
these channels is positioned on their intracellular C-terminal tails
and is constituted of multiple, Ca2+-dependent,
or constitutive microsites. The same sites can accommodate CABP1,
another Ca2+-binding protein, which is able to displace
calmodulin from its sites, to induce a Ca2+-independent
positive shift of the current-voltage curve accompanied by an
acceleration of the inactivation kinetics (11). These effects are
reminiscent of the action of NCS-1 on Ca2+ channels in
Xenopus oocytes. However, several questions need to be
answered before one can speculate that NCS-1 and CABP1 share the same
regulatory pathway. For example, CABP1 has only been tested on
CaV2.1 channels containing the
In conclusion, we demonstrate here that NCS-1-dependent
regulation of high threshold Ca2+ channels includes
modifications in the current amplitude and the electrophysiological
properties. These modifications are specifically modulated by the 1 subunit. When expressed with
CaV2.1, the depression of Ca2+ current
amplitude induced by NCS-1 was dependent upon the identity of the
subunit expressed, with no block recorded without
subunit or with
the
3 subunit. Current-voltage and inactivation curves were also slightly modified and displayed a different specificity toward the
subunits. Taken together, these data suggest that NCS-1 is able to modulate cardiac and neuronal voltage-gated
Ca2+ channels in a
subunit specific manner.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 subunit (for which ten genes are known) tightly associated with regulatory subunits
2-
(four different genes),
(four genes), and
possibly
(eight genes) in a functional multimeric complex (1-4).
This molecular diversity, further expanded by the existence of several splice variants for each of these genes, produces a large number of
possible Ca2+ channel subunit combinations with different
pharmacological and biophysical properties and specific cellular and
subcellular localization (5). The precise regulation of the
Ca2+ influx in response to various physiological situations
is further controlled by several regulatory mechanisms, working at
different levels, including channel expression, localization, or
activity, via additional interactions with modulatory proteins. Several Ca2+-dependent feedback mechanisms sense
incoming Ca2+ ions to finely tune channel activity to the
cellular Ca2+ demands and prevent cytotoxic
Ca2+ overload (6). These mechanisms use
Ca2+-sensing proteins and are specific of a given type of
Ca2+ channel. It has been shown, for example, that
Ca2+-dependent inactivation of the L-type
Ca2+ channel (encoded by the CaV1.2
1 subunit) is governed by a Ca2+-driven
interaction between calmodulin and the C-terminal tail of the channel
1 subunit (7-10). A similar functional interaction also
appears to exist on the P/Q-type Ca2+ channel (encoded by
the CaV2.1 subunit), the major channel type involved in synaptic
transmission in the mammalian central nervous system (11-13).
subunit, and
measured the resulting Ba2+ and Ca2+ currents.
These combinations are likely to be expressed in different cell types
where they represent potential targets for NCS-1 effects. Our goal was
to explore the effect of NCS-1 on both Ca2+ channel
expression and properties and to provide a first description of the
molecular requirements necessary for NCS-1 effects on Ca2+
channel that may help in the understanding of the precise mode of
action of this Ca2+-binding protein. We have, however,
focused this study on the P/Q-type (CaV2.1)
Ca2+ channels, which seem to be a primary target in various
cell types (28). Our results show that NCS-1 down-regulates expression of L-, N-, and P/Q-type Ca2+ channels in a
subunit-specific manner and induces minor modifications of the
electrophysiological properties of the channel. We provide evidence of
direct functional effects of NCS-1, in addition to modifications in the
expression level and/or trafficking of the channels to the membrane.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1C), M67515; CaV2.1
(
1A), M64373; CaV2.2 (
1B),
D14157; NCS-1, L27421;
1b, X61394;
2a, M80545;
3, M88751;
4, L02315; and
2-
1, M86621. Mutations NCS-1E120Q and
NCS-1G2A have been described previously (29).
Ca2+ channel subunits were subcloned into the pmt2 vector,
whereas NCS-1 and its mutants were subcloned into pcDNA3 (Invitrogen).
1+
2
+
+NCS-1 cDNAs at ~0.3 ng/nl with a ratio of
1:2:3:1. When one or more of these cDNAs was omitted, cDNA
concentrations were kept constant by addition of the appropriate volume
of deionized water. Oocytes were kept for 2 to 4 days before recordings
at 18 °C and under gentle agitation.
80 to +10 mV.
Current amplitudes were measured at the peak of the current. Comparisons of averaged amplitudes between batches were always made
with amplitudes measured the same day after injection. Comparisons between similar experiments were made by normalizing all averaged amplitudes with respect to the control current amplitude set as 100%.
Isochronal steady-state inactivation curves (2.5 s of conditioning voltage followed by a 400-ms test pulse to +10 mV) were fitted using
the equation, I/Imax = Rin + (1
Rin)/(1 + exp((V
Vin)/k)), where I is the current amplitude measured during the test
pulse at +10 mV for conditioning voltages varying from
80 to +50 mV, Imax is the current amplitude measured during
the test pulse for a conditioning voltage to
80 mV,
Vin is the potential for half-inactivation, V is the voltage, k is the slope factor, and
Rin is the proportion of non-inactivating
current. Current to voltage curves were fitted using the equation,
I/Imax = G*(V
Erev)/(1 + exp((V
Vact)/k)), where I is the current amplitude measured during voltage
steps varying from
80 to +50 mV, Imax is the
peak current amplitude measured at the minimum of the current-voltage
curve, G is the normalized macroscopic conductance,
Erev is the apparent reversal potential,
Vact is the potential for half-activation,
V is the value of the voltage step, and k is a
slope factor.
t/
1) + A2e(
t/
2) + C, where
I is the current amplitude, t is the time,
1,
2, A1, and
A2 represent the time constants and amplitudes
of the two components, and C is a constant. The proportion
of the slow time constant (%
2) is the ratio
A2/(A1+A2).
-glycerophosphate, and 5 mM
Na4P2O7) and centrifuged at 4 °C
and 14000 rpm for 5 min. The upper aqueous phase was collected and
subjected to 10% (see Fig. 2C) or 15% SDS-polyacrylamide
gel electrophoresis (loaded with ~3 oocytes/lane).
Proteins were electrotransferred to nitrocellulose filters. For
immunological detection, the blots were first blocked for 1 h with
8% non-fat milk powder in TBST (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% Tween 20), incubated overnight at 4 °C
with anti-NCS-1 (Zymol at 1/1000 dilution in 0.1% bovine serum albumin
in TBST) or anti-CaV2.1 antibodies (Alomone Laboratories
Jerusalem, Israel), and after five washes, incubated with an
anti-rabbit antibody coupled to horseradish peroxidase. Antibody
binding was detected by chemiluminescence (PerkinElmer Life
Sciences; 1/10000 in 0.1% bovine serum albumin, 0.8% nonfat milk powder in TBST). Correct expression of NCS-1 was always
checked after electrophysiological recordings.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Non-injected oocytes do not express
detectable levels of NCS-1. A, Western blot showing the
low level of endogenous NCS-1 protein expressed in non-injected oocytes
(lane 1) compared with COS-7 or human embryonic kidney 293 cells (lanes 2 and 3, respectively).
B, injection of pcDNA3.1 plasmid encoding NCS-1 led, in
2 to 4 days, to the apparition of a single NCS-1 immunoreactive band of
~23 kDa of molecular mass corresponding to overexpression of NCS-1
(lane 1). Lane 2, control, non-injected oocytes.
Each lane was loaded with ~3 oocytes. Numbers
on the right show the positions of the molecular mass
markers.
2,
2-
, and the appropriate
1
Ca2+ channel subunits (CaV1.2,
CaV2.2, or CaV2.1, respectively) with either
NCS-1 cDNA or water into two different batches of oocytes. After 2 to 4 days of incubation, Ba2+ current amplitudes were
recorded from the two batches of oocytes injected the same day, during
a single 400-ms-long depolarizing step to +10 mV from a holding
potential of
80 mV and compared. Under these conditions, a clear
decrease in the averaged Ba2+ current amplitude was seen
upon co-expression of NCS-1 (see Fig. 2A). This effect was most
pronounced in oocytes expressing the CaV2.2
Ca2+ channel, where the averaged Ba2+ current
amplitude recorded in the batch of oocytes co-injected with rat NCS-1
cDNA (N = 1 experiment, n = 38 oocytes) was only 10% of the control current amplitude recorded from
oocytes co-injected with H2O instead of the NCS-1 cDNA
(N = 1, n = 35). However, a similar
effect was also found when NCS-1 was co-injected with CaV1.2 (N = 3, n = 71; 52%
of the control amplitude n = 70) or CaV2.1
Ca2+ channel subunits (N = 7, n = 206; 46% of the control amplitude n = 162).
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Fig. 2.
Effects of NCS-1 on CaV1.2,
CaV2.1, and CaV2.2 current amplitudes.
A, NCS-1 reduced Ba2+ currents through L-, P/Q-,
and N-type Ca2+ channels. Histogram showing the effects of
NCS-1 on Ba2+ current amplitude recorded in oocytes
injected with the 2-
, the
2, and
CaV1.2, CaV2.1, or CaV2.2
Ca2+ channel subunits, with or without NCS-1 cDNA, are
shown. Peak Ba2+ currents were recorded 2-4 days after
injection during a test pulse to +10 mV from a holding potential of
80 mV and expressed as % of control (averaged current amplitude
recorded with the same subunit combination but without NCS-1
expressed). *, significantly different from control (p < 0.05); n.s., not significantly different. B,
histogram showing the effects of NCS-1 on Ba2+ current
amplitude recorded on oocytes injected with the CaV2.1, the
2-
, and the
1,
2,
3,
4, or no
Ca2+ channel
subunits, with or without NCS-1 cDNA. Peak Ba2+
currents were recorded 2-5 days after injection during a test pulse to
+10 mV from a holding potential of
80 mV. *, significantly different
from control (p < 0.05); n.s., not
significantly different. In A and B, Western blot
analyses of the expression of NCS-1 in the oocytes used for the
recordings are shown at the bottom. Each lane was
loaded with ~3 oocytes. C, Western blot of total proteins
obtained from oocytes injected with, from left to
right, the
CaV2.1+
2-
+
2 cDNAs, the
CaV2.1+
2-
+
2+NCS-1
cDNAs, or water, probed with an anti-CaV2.1 antibody.
Each lane was loaded with ~ 3oocytes. Note the
presence of a specific band at 250 kDa of similar density in oocytes
injected with the CaV2.1 subunit with or without NCS-1. The
cross-reactive band at ~160 kDa, also found in non-injected oocytes,
was used as a control of the loading charge.
subunit is an important determinant of
the final Ca2+ current amplitude observed at the oocyte
surface membrane and regulates many of its electrophysiological
properties (4, 31, 32). Hence, we tested the role of the
subunit on
the effects of NCS-1. Using the same experimental approach, we
co-expressed NCS-1 with the CaV2.1 Ca2+ channel
either with the
2-
subunit alone or with the
2-
and one of the four
subunits
(
1-
4). Again, in each case, the resulting current amplitude was compared with the current amplitude recorded in
oocytes injected with the same combination of Ca2+ channel
subunits but without NCS-1. Interestingly, when NCS-1 was co-expressed
with CaV2.1 without
subunit, almost no effect on
current amplitude was observed (N = 2, n = 32 and 26 for control). A similar result was also
found upon co-expression of the
3 subunit (N = 12, n = 198, with control
n = 198), whereas co-expression of NCS-1 with the
CaV2.1 and
1,
2, or
4 subunit decreased the expressed Ba2+
current amplitude to between 25 and 45% of their respective control values (see Fig. 2B, N = 3, 7, 2 and
n = 100, 206, 71 and n = 65, 162, 84 for controls, respectively). The lack of effect of NCS-1 on the
CaV2.1 Ca2+ channel subunit, expressed alone or
with the
3 subunit, could not be attributed to a deficit
of NCS-1 protein in these oocytes, because a robust expression of NCS-1
was also detected in these oocytes by Western blot (see
bottom of Fig. 2B). On the other hand, to ensure
that the decrease of the Ba2+ current amplitude obtained
upon co-expression of NCS-1 with the
1,
2, or
4 subunit was not because of a
deleterious effect on the expression of any cloned protein, we analyzed
the level of expression of the CaV2.1 protein in these
conditions by Western blot. Fig. 2C demonstrates that a
clear band of an approximate molecular mass of 250 kDa, matching of the
theoretical molecular mass of the CaV2.1 subunit (251 kDa),
could be specifically detected only in oocytes injected with the
CaV2.1 subunit cDNA
(+
2-
+
2 subunits; see Fig.
2C, left lane). In oocytes co-injected with NCS-1
cDNA and the same combinations of Ca2+ channel
subunits, this CaV2.1 immunoreactive band was also present at similar intensity (Fig. 2C, middle lane, each
lane approximately loaded with three oocytes), whereas it
was absent in non-injected oocytes (Fig. 2C, right
lane). These results therefore revealed a true,
subunit-specific effect of NCS-1 on the Ba2+ current
amplitude that cannot be attributed to nonspecific down-regulation of
protein expression.
2-
alone or
with the
1,
2, or
3
subunits, in the presence or absence of NCS-1. These recordings were
performed using either Ba2+ or Ca2+ ions as
extracellular permeant cations to track any Ca2+-specific modulation.
subunit (
1A+
2-
subunits), no
modifications in the activation and inactivation parameters were
observed upon co-expression of NCS-1. This lack of effect was also
noted upon co-expression of the
1 or
3
subunit (see Fig. 3 and Table I). Interestingly, when co-expressed with CaV2.1 and the
2a subunit, NCS-1 significantly depolarized the
current-voltage curve (Vact =
5.0 and
0.8 mV
without and with NCS-1 respectively, p < 0.05) and
reduced inactivation (Rin = 69 and 56%
respectively, p < 0.05). Similar effects were also
found in the presence of extracellular Ba2+ (Table
II) and thus were not
Ca2+-dependent.
Effects of NCS-1 on Cav2.1 Ca2+ currents
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Fig. 3.
Effect of NCS-1 on the functional properties
of CaV2.1. Normalized current-voltage and inactivation
curves recorded in oocytes injected with the CaV2.1
1A+
2
+
1 (A)
or
1A+
2
+
2
(B) Ca2+ channel subunits with (open
circle) or without (open square) NCS-1 are shown.
Recordings were made using 10 mM extracellular
Ca2+. NCS-1 induced a significant depolarization of the
voltage for activation (marked by an asterisk on the
bottom of the current-voltage curve; see
"Experimental Procedures" and Table I) and decreased the residual
current (marked by an asterisk on the inactivation
curve). This effect was only seen with the
2
subunit.
Effects of NCS-1 on Cav2.1 Ba2+ currents
1) and a slow (
2) component. None of
these components appeared to be significantly affected by expression of
the Ca2+-binding protein NCS-1 (Fig.
4). This lack of effect was found for
channels co-expressed with the
1 or the
2
subunit and in the presence of either extracellular Ba2+ or
Ca2+. Neither the time constants (
1 and
2) nor their respective amplitude (%
2)
were changed at all voltages examined (see Fig. 4). Moreover, no effect
on channel activation and reactivation were observed upon co-expression
of NCS-1, whether the CaV2.1 subunit was expressed with the
2-
alone or with any of the four
subunits (not
shown). Therefore, although both channel expression and channel
properties seemed to be regulated by NCS-1 in a
subunit-specific
manner, they appear to require different subunit arrangements,
i.e. expression was modified when
1,
2, or
4 subunits were expressed, whereas
modifications in channel properties were only recorded in the presence
of the
2 subunit.
View larger version (18K):
[in a new window]
Fig. 4.
Inactivation kinetics of CaV2.1
are marginally modified by NCS-1. Voltage dependence of the fast
( 1) and slow (
2) time constants of
inactivation and proportion of the slow time constant
(%
2) of CaV2.1 channels co-expressed with
2-
and
1 (A) or
2 (B) subunits with (open circle)
or without NCS-1 (open square) is shown. Currents were
recorded in Ba2+, during depolarizations of 2.5 s.
NCS-1 did not statistically modify (p > 0.05)
Ba2+ current kinetics at all voltages tested. Current
traces are shown on top.
2a subunit, were conducted using two mutants of NCS-1.
NCS-1E120Q, with its third EF-hand disrupted, showed
impaired Ca2+-dependent conformational changes
(19) but was still able to bind cellular proteins.
NCS-1G2A, a myristoylation-deficient mutant of NCS-1,
relocalized NCS-1 from the perinuclear region to the cytosol (29).
2-
+
2
Ca2+ channel (i.e. a small but significant
positive shift of ~5 mV; see Fig. 5B
and Table III). They differed, however,
in their effects on the inactivation curve. Although the
NCS-1E120Q completely suppressed the effect of NCS-1 on the
residual current (Rin; see Fig. 5 and Table
III), co-expression of NCS-1G2A left this parameter unchanged but suppressed the shift in Vin
induced by wild-type NCS-1. These two mutants also had different
actions on current amplitudes. Indeed, whereas NCS-1E120Q
reduced the Ba2+ current amplitude when compared with
control currents, recorded the same day in oocytes not injected with
NCS-1, NCS-1G2A had no consistent effect, although a small,
albeit not significant, reduction could be noted (see Fig.
5A). In the case of NCS-1E120Q, however, this
effect was less marked than when recorded in oocytes co-injected with
the wild-type NCS-1, suggesting that both
Ca2+-dependent conformational changes, and
possibly myristoylation, participated to the observed effects. It
should be noted that none of these mutants had any effect on the
functional properties of the CaV2.1+
2-
Ca2+ channels, expressed alone or with the
1
or
3 subunits, whereas the effects on current amplitude
were reproduced with the
1 subunit (data not shown).
View larger version (23K):
[in a new window]
Fig. 5.
Effects of NCS-1E120Q and
NCS-1G2A on CaV2.1 expression and properties.
A, effects of wild-type NCS-1, NCS-1E120Q, and
NCS-1G2A mutants on CaV2.1 channel expression.
Oocytes were injected with
CaV2.1+ 2-
+
2a subunit
cDNAs with or without the appropriate NCS-1 mutants, and currents
were measured in 10 mM Ba2+ during a test
depolarization to +10 mV, as described for Fig. 2. Note that NCS-1 and
NCS-1E120Q both decreased current amplitude significantly
(p < 0.05). B, oocytes were injected with
CaV2.1+
2-
+
2a subunit
cDNAs with (open circle) or without (open
square; Control) the appropriate NCS-1 mutants,
NCS-1E120Q in the left panel and
NCS-1G2A in the right panel. Currents
were measured in 10 mM extracellular Ba2+ as
described for Fig. 3. Both mutations slightly shifted the
current-voltage curve in the positive direction, but only
NCS-1E120Q seemed to be able to reverse the decrease in the
residual current (Rin; see Table III for
Vact, Vin, and
Rin values).
Effects of mutated NCS-1 on Cav2.1 Ba2+ currents
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit-specific but
observed with CaV1.2, CaV2.1, and
CaV2.2 Ca2+ channels; and (2) a small
modification in the activation and inactivation parameters of the
CaV2.1, only seen when the
1 subunit is
expressed with the
2a subunit.
1 subunit (37). This sequence, located close
to the
subunit binding site (AID), acts as a retention signal in
the endoplasmic reticulum and thus reduces trafficking of the
1 subunit to the membrane, just like a surface
expression brake. This brake is usually removed by the
1/
subunit association, which occurs in the
reticulum. The fact that the decrease in current amplitude induced by
NCS-1 was only observed when
1,
2, or
4 subunits were expressed suggests that NCS-1 may
interfere with this mechanism. A similar effect on channel expression
has been recently reported (38) for the small GTPase Kir/gem, which
binds directly to the
subunit, thus preventing the
1/
association and restoring the expression brake
imposed by the retention signal. Direct specific binding of NCS-1 to
this site and/or to the
subunit is thus an attractive hypothesis to
explain the reduction of the current amplitude but needs to be further
explored by additional experiments designed to directly test the
biochemical interactions between these subunits. Such a mechanism,
however, may not be exclusive, and other pathways, acting directly or
indirectly on channel activity, such as Src-dependent
inhibition (28), or direct modulation of the G-protein-coupled receptor
pathways (19), may also exist. Although in our recording conditions,
the Src-dependent and G-protein pathways could be discarded
(the Src kinase inhibitor, PP1, had no effect; data not shown), it is
worth noting that G-protein
subunits and the Ca2+
channel
subunit possess very close binding sites on the main
1 Ca2+ channel subunit (39-41), leaving
open the possibility that interactions between the
1
subunit on one hand, and G-protein, Ca2+ channel
subunit, and NCS-1 on the other hand, could be mutually exclusive
and/or under the control of tissue-specific conditions. The I-II loop
would therefore acts as a cross-road for cell-specific regulations.
3
subunit, the
subunit that displays the lowest affinity for the AID
(42), and which also shows the weakest potency to increase current
amplitude in expression systems (43). In neurons, where the N-type
Ca2+ channel is predominantly associated with the
3 subunit (44) the increase in vesicular transport and
membrane trafficking induced by NCS-1, acting through the activation of
the phosphatidylinositol 4-kinase
(45), may overcome the effect on
the
1 retention signal, poorly masked/removed by the
3 subunit, and lead to the observed overexpression of
N-type Ca2+ channels (20). In this scenario, the tissue
specificity of the effects of NCS-1 on Ca2+ channels should
thus be critically dependent on the subunit composition of the channel.
Whether NCS-1 acts directly or indirectly on the Ca2+
channel
1 or
subunit requires further investigation.
2a subunit. Increased inactivation and a positive shift
of the current-voltage curve were the most significant changes. These
effects were seen in the presence of extracellular Ba2+ or
Ca2+ and therefore, in our conditions, did not seem to be
Ca2+-dependent. The fact that the decrease in
current amplitude and the modifications in the channel properties did
not have the same
subunit specificity suggested different
underlying mechanisms. Direct interactions between Ca2+
channels and NCS-1 at the plasma membrane have not been reported so
far, but the presence of NCS-1 in synaptic-like microvesicles and its
co-localization in presynaptic terminals with the proteins of the
soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex, VAMP (45) and syntaxin (18), also known to
interact with P/Q Ca2+ channels (1), suggest that NCS-1 is
appropriately located for these potential functional interactions.
Indeed, a previously published work (18) reported an enhancement of the
facilitation of the P/Q-type Ca2+ channel in the Calyx of
Held, occurring via an acceleration of current activation. A similar
frequency-dependent facilitation was also apparent in
oocytes (data not shown), but the large membrane capacitance of the
oocytes did not allow a precise analysis of the modifications in the
activation kinetics. Interestingly, in the Calyx of Held, these effects
were recorded during acute perfusion of NCS-1 and could be occluded by
perfusion of a C-terminal peptide of NCS-1, suggesting direct
interactions with the channel (18). A large hydrophobic crevice in this
region has been proposed as a potential site for target recognition
(15).
2a subunit,
and we have no information, for the moment, on its
subunit specificity.
subunit of the Ca2+ channel. Our results, together with
previously published work on NCS-1, CABP1, calmodulin, and
Ca2+ channels, suggest that these effects could take place
at two sites on the
1 subunit known for their regulatory
role, the I-II loop and the C-terminal tail of the channel. These sites
also interact with the Ca2+ channel
subunit and the
G
subunits of the G-protein suggesting that regulation via
G-protein-coupled receptors may also be affected. These results provide
a possible molecular explanation for the tissue and Ca2+
channel type specificity of NCS-1, which can be tested biochemically and pharmacologically. They also suggest that down-regulation of
Ca2+ channel activity may not be restricted to neuronal or
neuroendocrine cells but may also take place in cardiac myocytes, where
NCS-1 and the L-type Ca2+ channels are expressed (26).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. T. P. Snutch, Y. Mori, and E. Perez-Reyes for Ca2+ channel cDNAs, Dr. I. Lefevre, M. Franco, E. Mandart, S. Estrach, and T. Lorca for helpful discussion during the course of these experiments, and J. M. Donnay and A. Bernet for help with the preparation of oocytes.
![]() |
FOOTNOTES |
---|
* This work was supported by Association pour la Recherche contre le Cancer, the Association Française contre les Myopathies, and Ligue Nationale contre le Cancer (Comité Pyrénées Orientales).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.
§ Recipient of a doctoral fellowship from the French Ministry for Research and Education.
To whom correspondence should be addressed. Tel.:
33-467-613-352; Fax: 33-467-521-559; E-mail:
charnet@crbm.cnrs-mop.fr.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M209537200
2 A. Jeromin, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviation used is: NCS-1, neuronal Ca2+ sensor protein-1.
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