Voltage-gated Mobility of the Ca2+ Channel
Cytoplasmic Tails and Its Regulatory Role*
Evgeny
Kobrinsky,
Elena
Schwartz,
Darrell R.
Abernethy, and
Nikolai
M.
Soldatov
From the National Institute on Aging, National Institutes of
Health, Baltimore, Maryland 21224
Received for publication, November 4, 2002, and in revised form, November 26, 2002
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ABSTRACT |
Transient increase in intracellular free
Ca2+ concentration generated by the voltage-gated
Cav1.2 channels acts as an important intracellular signal.
By using fluorescence resonance energy transfer combined with patch
clamp in living cells, we present evidence for voltage-gated mobility
of the cytoplasmic tails of the Cav1.2 channel and for its
regulatory role in intracellular signaling. Anchoring of the C-terminal
tail to the plasma membrane caused an inhibition of its
state-dependent mobility, channel inactivation, and
CREB-dependent transcription. Release of the tail restored these functions suggesting a direct role for voltage-gated mobility of
the C-terminal tail in Ca2+ signaling.
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INTRODUCTION |
The ion conductance of voltage-dependent ion channels
is tightly regulated by the gating mechanism encoded in
membrane voltage sensors and cytoplasmic gates of the pore-forming
1 subunit (1). This voltage gating is due to the
displacement of membrane charges (2) that has been documented as gating
current (3) and further characterized as conformational rearrangements
detected by spectroscopy of the attached fluorescent groups (4-6).
However, little is known about a functional role for
state-dependent mobility of the channel cytoplasmic tails
(7). Such motion of the L-type (Cav1.2) Ca2+
channel C-terminal tail that binds calmodulin
(CaM)1 may serve to transfer
a regulatory signal (8, 9). Thus, investigation of the mobility of
cytoplasmic regions of Ca2+ channels associated with their
gating is important for understanding of the molecular correlates of
channel regulation and mechanisms of Ca2+ signaling.
Spectral properties of the enhanced cyan (ECFP) and yellow fluorescent
proteins (EYFP) (10) are well suited for measurements of molecular
rearrangements by FRET (11). Here we genetically fused the cytoplasmic
N and C termini of the human Cav1.2 channel
1C pore-forming subunits with EYFP and ECFP,
respectively. The labeled channels were then functionally expressed in
COS1 cells. The rearrangements of the tails due to transition into the
distinct functional states of the channel were monitored using FRET
(12) in living cells under voltage clamp conditions. A regulatory role for the voltage-gated mobility of the C-terminal tail was then characterized.
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EXPERIMENTAL PROCEDURES |
Molecular Biology--
(EYFP)N-
1C,77
and (EYFP)N-
1C,IS-IV expression plasmids
were prepared in pcDNA3 vector essentially as described earlier
(13, 14) using pEYFP vector (Clontech). To prepare
the PH-EYFP and PH-ECFP expression plasmids, the 518-bp
EcoRI/BamHI fragment of pPLCPH-enhanced green
fluorescent protein (15) was ligated into the pECFP-1 and pEYFP-1
plasmids, respectively, at EcoRI/BamHI sites. To
prepare the
1C,77-(ECFP)C expression
plasmid, the 1029-bp AatII/NotI fragment of
77pcDNA3 (13) was replaced by the coding sequence of the pECFP
vector amplified by PCR using 5'-terminal AatII linker and
3'-terminal NotI linker containing stop codon. (EYFP)N-
1C,77F-(ECFP)C and
(EYFP)N-
1C,IS-IV-(ECFP)C
expression plasmids were prepared in a similar way using
(EYFP)N-
1C,77 and (EYFP)N-
1C,IS-IV expression plasmids. To
prepare (PH-EYFP)N-
1C,77 expression plasmid,
BsrGI (
13), MfeI (412) PCR segment of pHLCC77 (16) containing 5'-terminal linker (5'-TGTACAAGGCCACC-3') was ligated
with the 6115-bp MfeI/NotI fragment of
77pcDNA3 into the NotI and BsrGI-cut PH-EYFP
plasmid. To prepare
(PH-EYFP)N-
1C,77-(ECFP)C expression plasmid, the 3474-bp NotI/PpuMI
fragment of 77CFPpcDNA3 was replaced into the respective sites of
(PH-EYFP)N-
1C,77. To prepare the
1C,77-(PH-ECFP)C expression plasmid, a
927-nucleotide 3'-terminal AatII/XhoI PCR
fragment with stop codon replaced by the XhoI linker was
ligated with the XhoI/NotI fragment of open reading frame of the PH-CFP plasmid into the AatII,
NotI-cut pHLCC77 plasmid. Its 5096-nucleotide
PpuMI/NotI fragment was then subcloned into
77pcDNA3 or (EYFP)N-
1C,77pcDNA3 to
give the
1C,77-(PH-ECFP)C and
(EYFP)N-
1C,77-(PH-ECFP)C
expression plasmids, respectively.
Electrophysiology--
COS1 cells were grown on
poly-D-lysine-coated coverslips (MatTek) in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum. Cells
were transfected with pcDNA3 vectors coding for
1C,
1 (17) (or
2a (18)), and
2
(19) subunits using the Effectene kit (Qiagen).
Effectene was used for transfection to minimize autofluorescence added
by many other transfection reagents. EGF receptor was co-expressed in
tail-anchoring FRET experiments. To release pleckstrin homology (PH)
domain, serum-deprived (24 h) cells were exposed to EGF (100 ng/ml).
Ion currents were recorded using the Axopatch 200B amplifier (Axon
Instruments) at 20-22 °C by the whole-cell patch clamp method
48-72 h after transfection of COS1 cells. Voltage protocols were
generated, and data were digitized, recorded, and analyzed using pClamp
8.1 software (Axon). The extracellular bath solution contained (in mM) NaCl, 100; BaCl2, 20; MgCl2, 1;
glucose, 10, HEPES, 10, pH 7.4. The electrodes had resistance 3-6
megohms and were filled with pipette solution containing (in
mM) CsCl, 110; MgATP, 5, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate,
10; tetraethylammonium, 20; cAMP, 0.2; HEPES, 20, pH 7.4. Currents were
sampled at 2.5-5 kHz and filtered at 1 kHz.
FRET Imaging--
Images were recorded with the Hamamatsu
digital camera C4742-95 mounted on a Nikon epifluorescent microscope
TE200 equipped with multiple filter sets (Chroma Technology). C-Imaging
(Compix) and MetaMorph (Universal Imaging) software were used to obtain and analyze images. The photobleaching experiments were conducted with
the 100-watt mercury lamp. For all other experiments, a 75-watt xenon
lamp was used. FRET was quantified with three filter sets as follows:
for EYFP cube, excitation filter 500/20 nm, dichroic beam splitter 515 nm, and emission filter 535/30 nm; for FRET (ECFP/EYFP) cube,
excitation 436/20 nm, dichroic beam splitter 455 nm, and emission
filter 540/30 nm; for ECFP cube, excitation filer 436/20 nm, dichroic
beam splitter 455 nm, and emission filter 480/40 nm. Regions of
interest were selected using the C-Imaging software where intensity (I)
from three filter sets was determined after background subtraction.
Corrected intensity of FRET (IFRETc) was
calculated (20) as (IFRET
IECFP × 58.5%
IEYFP × 11.5%). In the patch clamp experiments, the
acquisitions of fluorescence ranging from 50 to 300 ms were obtained
with simultaneous recording of the current at the indicated
conditioning voltages under steady state. In some cases, the images
were adjusted pixel-by-pixel using the reference channel of regions of
interest. With the acceptor photobleaching, we used an excitation
filter 436/10 nm and an emission filter 470/30 nm for ECFP and an
excitation filter 500/10 nm and emission filter 535/20 nm for EYFP. The
apparent efficiency of FRET was calculated as (IECFP*
IECFP)/IECFP*, where IECFP and
IECFP*
intensities of ECFP fluorescence before and after acceptor (EYFP) photobleaching, respectively. Normalization of the
corrected FRET ratios against EYFP or ECFP fluorescence showed very
similar values (Table I). Corrected FRET
and bleed-through values were obtained by standard approach (20, 21)
according to Equation 1,
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(Eq. 1)
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where a and b are, respectively,
the norm of the percentage of ECFP and EYFP bleed-through under the
FRET filter set, and IFRET, IECFP and
IEYFP are intensities in each region of interest under
FRET, ECFP, and EYFP filter sets, respectively. For the double-labeled
channels, we have experimentally determined that a = 58.5% and b = 11.5%. Background subtraction was
carried out using the standard approach (e.g. Ref. 22). In
each image, we determined the average fluorescence from the cell-free
regions. Under our experimental conditions, the average fluorescence
measured from these specified regions was not different from the
fluorescence measured with unused substrate covered with the bath
solution only. Variability of the background fluorescence ranged from 9 to 14, whereas those of the experimental cell fluorescence varied from
50 to 250 units of the same arbitrary scale. To obtain ratio of images,
10 was added to each pixel of the image corrected by the background
subtraction to avoid division by zero.
Plotting of corrected FRET images was carried out according to standard
and well established procedures (for example, see Refs. 20 and 23).
Because our images were obtained from the same cells with the same
level of ECFP expression, there was no need to additionally normalize
FRET images against ECFP. Under these conditions, we focused only on
the relative changes of fluorescence at different holding potentials.
Comparison of the ratios of corrected FRET (+40 mV/
90 mV) with those
additionally normalized against EYFP, ECFP, and square root of their
product according to Xia and Liu (24) (see Table I) clearly
demonstrates that additional normalization did not significantly change
the results of corrected FRET ratio. This result was obtained probably
because the ratio of ECFP to EYFP in experiments with the
double-labeled channels was always equal to 1.
CREB-dependent Transcription Activation--
The
plasmids YKIDN and KIXCN coding for KID and KIX domains (25),
respectively, were co-expressed (1:1) in COS1 cells with other
cDNAs at 2.5:1 ratio to cDNA coding for an
1C
subunit. To monitor transcriptional activation under voltage clamp
conditions, we used the perforated patch clamp technique (26).
-Escin (20 µM, Sigma) was added to the pipette
solution containing potassium gluconate 120, NaCl 10, MgATP 2.5, HEPES
5, and KCl, 20; pH 7.2. External solution contained (in mM)
NaCl, 140; KCl, 5.4, MgCl2 1, HEPES 5, CaCl2
2.0, and glucose 5.5, pH 7.4. Depolarization to +20 mV from the holding
potential of
90 mV was applied to elicit maximum Ca2+
current. To monitor changes of free Ca2+ concentration,
cells were loaded with Fluo-4 by incubation for 20 min at 37 °C in 5 µM Fluo-4 AM (Molecular Probes) added to the external
solution. For de-esterification of the probe, cells were incubated for
another 20 min before experiment. Ca2+ measurements were
performed at 20-22 °C using confocal microscope PCM2000 (Nikon,
Inc.) with excitation by an argon laser at 488 nm and recording of the
fluorescence emission at >515 nm.
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RESULTS |
Acceptor Photobleaching Assay--
Confirming earlier findings
(13, 27), fusion of the green fluorescent protein variants to the
cytoplasmic N and/or C termini did not compromise channel function. The
electrophysiological properties of the
(EYFP)N-
1C,77-(ECFP)C channel
were similar to those of the wild-type channel (13) except of the
acceleration by ~15% of the Ba2+ current inactivation
(Table II). A direct FRET acceptor
photobleaching assay provided strong evidence of FRET in the
(EYFP)N-
1C,77-(ECFP)C channel (Fig. 1). Illumination of the
transfected COS1 cells for 15 min with a mercury lamp caused on average
>90% photobleaching of EYFP. The resulting increase in donor
fluorescence (shown as yellow-green and
yellow-red signals, Fig. 1A, c and
e, respectively) is a direct demonstration of FRET (11). As
a positive control, under the same conditions, FRET was observed (Fig.
1A, d and f) with the co-expressed
mixture of the EYFP-PH and ECFP-PH domains of phospholipase C
1 (28).
In both cases FRET was confined to the plasma membrane region where the
functional channel molecules reside. We found that the mixture of
co-expressed (1:1) single N- and C-tail-labeled channel isoforms did
not show substantial intermolecular FRET (Fig. 1B)
indicating that with the double-labeled
1C channels we
recorded predominantly intramolecular FRET.
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Table II
Comparison of electrophysiological properties of the wild-type
1C,77 (A) and fluorescent labeled
(EYFP)N- 1C,77-(ECFP)C (B),
and
(EYFP)N- 1C,IS-IV-(ECFP)C (C)
Ca2+ channels
The 1C subunits were co-expressed with 1 and
2 accessory subunits. Ba2+ currents were elicited
by 600-ms test pulses to +10 mV from a holding potential of 90 mV.
The bath medium contained 20 mM Ba2+. The time
constants of the Ba2+ current inactivation fast and
slow were determined by two-exponential fitting. (The
approximated slow values are presented solely to reflect the
fact that slow inactivation is completely inhibited in the labeled
1C,IS-IV channel.) Vmax, voltage for the
peak current, and V0.5, voltage at 50% of the
Ba2+ current activation were determined from the
current-voltage relationship. V0.5in, the voltage at
half-maximum of inactivation, and Isust, fraction of
non-inactivating current, were determined from the fitting of
steady-state inactivation curves by Boltzmann function. Steady-state
inactivation curves were measured using a two-step voltage clamp
protocol. A 1-s conditioning pulse was applied at 30-s intervals with
10-mV increments from the holding potential Vh = 90 mV followed by a 250-ms test pulse to +10 mV. Peak current
amplitudes were normalized to maximum value. All fittings were
performed according to Ref. 33. Number of tested cells is shown in
parentheses.
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Fig. 1.
Direct FRET measurements in the
(EYFP)N- 1C-(ECFP)C
channels. A, FRET with acceptor photobleaching in the
(EYFP)N- 1C,77-(ECFP)C channel
containing the 1 accessory subunit (left
panels; scale bar, 7 µm) and in the equimolar mixture
of EYFP-PH and ECFP-PH domains co-expressed as positive control
(right panels; scale bar 14 µm). EYFP was
attached to the 124-amino acid N-terminal tail of 1C,77.
306 amino acids of the 662-amino acid C-terminal tail of the
1C subunit were replaced by ECFP so that the overall
length of the C-terminal tail of the fusion protein was 652 amino
acids. a and b, phase contrast images of the
expressing COS1 cells. c and d, ratios of ECFP
fluorescence intensity before and after photobleaching. e
and f, corresponding three-dimensional images. B,
increase of apparent efficiency of FRET due to acceptor photobleaching
in cells expressing the indicated double-labeled channels or
EYFP-PH/ECFP-PH mixture but not the mixtures of single-labeled channels
(n, number of cells; *, p < 0.0001, unpaired t test). In the schematics, Y and
C stand for EYFP and ECFP, respectively. Co-expressed
-subunits are indicated at the lower panel. Error
bars, S.E.
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Voltage-dependent FRET in the Inactivated
State--
FRET is the result of long range dipole-dipole interactions
between fluorophores. FRET depends on the distance
(r6) between the donor and acceptor fluorophore
and, to a lesser extent, the relative orientation (
2) of
the dipoles (12). To determine the differences in relative proximity
and/or angular orientation of the tagged
1C tails in the
functionally distinct resting, conducting, and inactivated states of
the channel, we combined FRET imaging with voltage clamp. We used
quantitative analysis of reversibly changing FRET in place of acceptor
photobleaching as the fluorophores became irreversibly damaged by
photobleaching. Simultaneous monitoring of the Ba2+ current
provided a direct assessment of the Cav1.2 channel's transition into the resting, conducting, or inactivated state prior to
FRET imaging. The images were acquired with a three-filter set system,
successfully used elsewhere (20, 29), for sensitized acceptor emission.
The recombinant channel, composed of the wild-type
1C,77
and accessory
1 and
2
subunits, was
stabilized in either the resting or inactivated state by whole-cell
voltage clamp at
90 or +40 mV, respectively. Depolarization to +40 mV
was selected to evoke maximum activation of the channels under
conditions when the state of inactivation could be monitored. Thus, the
amplitudes of the Ba2+ currents (Fig.
2d) were less than 50% of the
maximum ones. The analysis of FRET images of a cell expressing the
(EYFP)N-
1C,77-(ECFP)C channel
(Fig. 2A) showed that the sequential transitions between the
resting (
90 mV, Fig. 2A, c) and the inactivated states of the channel (+40 mV, Fig. 2A, b) produced a fully reversible
increase in FRET:
(IFRETc)+40 mV/(IFRETc)
90 mV = 2.9 ± 0.7 (n = 24). The steady state
inactivation analysis indicated that ~7% of the
Ba2+-conducting
(EYFP)N-
1C,77-(ECFP)C channels
remain available (Table II), suggesting that
93% of the channels are
inactivated and
7% remain in the resting closed state at the end of
the depolarization pulse shown in Fig. 2A. Since the resting
state is characterized by low FRET, this fraction has only slightly
contributed to the measurement of FRET. Taken together, these data
indicate that the cytoplasmic tails of the channel in the inactivated
state are rearranged into a conformation that gives greater FRET as compared with the closed resting state.

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Fig. 2.
Reversible changes of FRET associated with
voltage gating of Ca2+ channels. Right
panel, phase-contrast images of expressing cells with the shadow
of patch pipette (a) and schematic diagrams depicting the
arrangement of fluorophores (below). A, shown are
the ratios of corrected FRET images (b and c)
sequentially recorded at the indicated voltages with FRET cube and
their three-dimensional presentations (below) for
(EYFP)N- 1C,77-(ECFP)C channel in
b inactivated (+40 mV) and c resting ( 90 mV)
states; B, shown are the ratios when inactivation of the
same channel was slowed down by the replacement of 1
with 2a subunit, in b predominantly
conducting (+40 mV) and c resting states; and C,
shown are the ratios for the non-inactivating
(EYFP)N- 1C,IS-IV-(ECFP)C channel
in b conducting (+40 mV) and c resting states.
Scale bars, 8 µm. Image areas indicated by
arrows were digitally magnified to demonstrate confinement
of FRET to the plasma membrane. Note that in every case the
Ba2+ current recordings (d) provide evidence of
the channel state achieved prior to FRET image acquisitions (marked by
red bars). FRET at holding potential Vh = 90 mV was recorded for the same duration of time before and after the
depolarization pulse.
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The electrophysiological properties of the
1C
channel are not significantly changed by the deletion of the N-terminal
tail (30). In contrast, the proximal half of the C-terminal tail is
essential for channel function (13, 30, 31). A fusion of ECFP to the
full size, 662-amino acid C-terminal tail of
1C,77 did
not alter the electrophysiological properties of the
(EYFP)N-
1C,77F-(ECFP)C channel
(data not shown), but reduced efficiency of FRET determined in the
acceptor photobleaching assay to 0.19 ± 0.02 (n = 29), thus indicating that apparent separation of the fluorophores has increased. However, the intensity of FRET in the inactivated state (+40
mV) of this full-length
(EYFP)N-
1C,77F-(ECFP)C channel
was not significantly different as compared with the truncated channel.
Voltage-dependent FRET in the Conducting State of the
Cav1.2 Channel--
Rapid spontaneous channel inactivation
complicates direct FRET imaging of the channel in the transient
conducting state. Replacement of the cytoplasmic accessory
1 subunit with
2a (32) significantly slowed inactivation (compare Ba2+ current traces on
d in Fig. 2, A and B) and thus allowed
the channel to be maintained in a predominantly conducting state. Voltage-dependent FRET was measured with the
(EYFP)N-
1C,77-(ECFP)C/
2a channel at the end of a 3-s depolarization at +40 mV. Under these conditions, the Ba2+ current activation was
almost maximal, whereas inactivation was minimal with respect to the
time of FRET acquisition (marked by the red bar above the
current traces). The voltage-dependent increase of FRET
(3.8 ± 0.8, n = 15, see Fig. 2B)
resulting from the transition of the channels from the resting (
90
mV) to a predominantly conducting state (+40 mV) was found not to be
significantly different from that determined for the inactivated state
(2.9 ± 0.7, Fig. 2A). Thus, the folding of the channel
tails characterized by FRET in the ensemble of predominantly conducting
channels was on average closer to those determined for the inactivated
than those determined for the resting state of the same channel.
This finding was independently confirmed by the study of the
1C,IS-IV channel that is deprived of slow inactivation
by mutations introduced in the pore region (14). The
(EYFP)N-
1C,IS-IV-(ECFP)C channel
expressed in COS1 cells showed the characteristic sustained Ba2+ current (Fig. 2C, d). The amplitude of the
Ba2+ current through the
1C,IS-IV channel
was smaller than those through the
1C,77 channel.
Similar changes were observed earlier for other isoforms of the
Cav1.2 channel with impaired Ca2+-induced
inactivation (33). This was found to be due to a lower open probability
and a 10-15% reduction in single channel conductance (13). In the
stable conducting (+40 mV) state of the
1C,IS-IV channel
(Fig. 2C, b), the corrected FRET increased 7.4 ± 0.9-fold (n = 19; p < 0.0005, compared
with Fig. 2A, b) suggesting further reduction in the
apparent distance between the labeled tails and/or dipole reorientation
of the fluorophores (12) leading to an increase of FRET as compared
with the resting state (
90 mV, see Fig. 2C, c). Taken
together, these data point to distinct rearrangements of the
1C tails associated with voltage gating.
Role of the Voltage-gated Mobility of the C-terminal Tail in the
Cav1.2 Channel Inactivation--
To investigate a
regulatory role of the voltage-gated rearrangements of the
1C C-terminal tail, the tail was anchored to the plasma
membrane via the PH domain of phospholipase C
1 (Fig. 3). This domain binds specifically to
phosphatidylinositol bisphosphate in the inner leaflet of the plasma
membrane. Hydrolysis of phosphatidylinositol bisphosphate by activation
of phospholipase C induces redistribution of the PH domain to the
cytoplasm (15, 34).

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Fig. 3.
Effect of immobilization and release of the
C-terminal tail of Ca2+ channel on
state-dependent FRET. A, direct FRET
imaging. Phase contrast (a) and FRET images of COS1 cell
expressing the
(EYFP)N- 1C,77-(PH-ECFP)C channel
before (b) and after (c) release of the
membrane-anchoring PH domain. Upper panels, digitally
magnified image areas pointed by arrows. d, ratio
of images (c and b) showing increased FRET in the
membrane region in response to the C-tail release. Upper
panel, three-dimensional image. B, inhibition of
inactivation and state-dependent FRET in cell expressing
the (EYFP)N- 1C,77-(PH-ECFP)C
channel with the membrane-trapped C-terminal tail. a,
phase-contrast image of the expressing cell and schematic diagram of
the 1C channel construct (below).
b, the Ba2+ current trace elicited by
depolarization to +40 mV from Vh = 90 mV.
c and d, ratio of corrected FRET images recorded
at the indicated membrane potentials. C, restoration of
inactivation and state-dependent FRET after release of the
C-tail. a, schematic diagram of the 1C
channel construct. b, Ba2+ current trace
elicited by depolarization to +40 mV from Vh = 90
mV. c and d, ratio of corrected FRET images
recorded at the indicated potentials. Left panels, digital
magnifications of the membrane region. Low panels,
corresponding three-dimensional images. Scale bars, 8 µm.
Voltage protocol was similar to Fig. 2.
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The (EYFP)N-
1C,77-(PH-ECFP)C
channel with anchored C-tail was co-expressed in COS1 cells with
epidermal growth factor (EGF) receptor to permit a release of the
PH-tagged C-terminal tail by activating phospholipase
and
phosphatidylinositol bisphosphate hydrolysis in response to exposure of
the cell to EGF (Fig. 3A). The Ba2+ current
through the channel with the anchored C-terminal tail was activated in
the characteristic range of membrane potentials but exhibited a very
slowly inactivating component of the Ba2+ current (Fig.
3B, b). Membrane trapping of the N-terminal tail did not alter the inactivation properties of the channel (data not shown).
Very little, if any, FRET was observed with the anchored C-terminal
tail in both the conducting (Fig. 3B, c) and the
resting (d) states of the channel. The release of the tail
by EGF treatment irreversibly restored the ability of the channel to
inactivate fully as can be seen from the complete decay of the current
(Fig. 3C, b) and increased FRET in the inactivated
state (Fig. 3C, c). The ratio
(IFRETc)+40
mV/(IFRETc)
90 mV = 1.9 ± 0.9 (n = 5, p < 0.05) was not different to that observed with the
(EYFP)N-
1C,77-(ECFP)C
(Fig. 2A) and
(EYFP)N-
1C,77F-(ECFP)C (not
shown) channels. Thus, limitations imposed on free movement of the
C-terminal tail of the
1C channel affect inactivation properties as well as the associated FRET. When released, the C-tail
appears to assume a functional conformation as determined by both the
return of the normal inactivation properties of the channel and
voltage-dependent FRET. This precludes a re-insertion of
the PH domain into the membrane. Even a 30-min washout period, the
longest that we were able to achieve without losing the voltage clamp,
did not restore the membrane association of the PH domain distinguished
by the properties of the channel with the trapped tail.
Role of the Voltage-gated Mobility of the C-terminal Tail in
Regulation of CREB-dependent Transcription--
The
amplitude of the voltage-gated mobility of the Cav1.2
channel C-terminal tail may be sufficient to have a role in
Ca2+ signal transduction. It has been shown previously (8)
that Cav1.2 channels are important for
Ca2+-induced activation of cAMP-responsive element-binding
protein (CREB)-dependent transcription. In this work, to
study the role of the voltage-gated mobility of the Ca2+
channel C-terminal tail for transcriptional activation, we investigated the interaction between KID and KIX domains of CREB and co-activator CREB-binding protein under voltage clamp conditions by monitoring FRET
between (EYFP)-KID and (ECFP)-KIX, both containing nuclear localization
sequences (25). Use of perforated patch clamp technique (26) allowed us
to preserve the integrity of the cytoplasmic content of the cell,
crucial in retaining components of signaling cascade involved in
CREB-dependent transcription. We found that when the
C-terminal tail of the
1C,77-(PH)C channel
was anchored via the PH domain to the plasma membrane (Fig.
4A,
a), activation of the channel by depolarization
to +20 mV increased intracellular free Ca2+ concentration
([Ca2+]i) detected by the free Ca2+
indicator Fluo-4 (Fig. 4A, a, see image on the lower
panel). However, stimulation of the sustained Ca2+
conductance of the anchored channel by depolarization (Fig.
4A, d) did not cause strong activation of
CREB-dependent transcription despite the presence of the
CaM-binding IQ motif of the C-tail 1624-1635 (35), previously
identified as important for CREB activation (8). Release of the
C-terminal tail of the channel, induced at
90 mV by ACh stimulation
of the co-expressed type 1 muscarinic acetylcholine receptor (M1AChR),
was accompanied by activation of the inositol
1,4,5-trisphosphate-dependent Ca2+ release but did
not induce substantial activation of CREB-dependent transcription (Fig. 4A, b). It was the release of
the C-terminal tail of the channel combined with the activation of
Ca2+ conductance by depolarization that was essential to
re-establish the voltage-gated signaling sufficient for
CREB-dependent transcription activation (Fig.
4A, c). The fact that this response was inhibited by co-expression of CaM1234 (Fig. 4B), a
Ca2+-insensitive analogue of CaM retaining the affinity to
CaM-binding sites (36), points to a critical role of CaM in signal
transduction by the voltage-gated mobile C-terminal tail of the
1C channel. Selective disruption of the apo-CaM-binding
site 1572-1598 (37-39) on the C-terminal tail of the
1C channel (33) (Fig. 4C) abolished activation of CREB-dependent transcription despite
unrestricted mobility of the C-terminal tail and the presence of CaM
and IQ motif (8). These results, for the first time, demonstrated the
role of the Ca2+ channel C-tail as a voltage-gated mobile
carrier of the signal for CREB-dependent transcriptional
activation (Fig. 4D). This signal appears to be associated
with apo-CaM binding to the C-tail and may involve its Ca2+
loading (9) and translocation to the IQ motif (8, 36), a
Ca2+-filled CaM-binding site on the C-tail.

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|
Fig. 4.
CREB-dependent activation of
transcription. CREB activation was examined under perforated patch
conditions by FRET between the EYFP-labeled KID domain of CREB and
ECFP-labeled KIX domain of CREB-binding protein as interaction
partners. A, CREB activation in COS1 cells expressing M1AChR
and the (EYFP)N- 1C,77-(PH-ECFP)C
channel with the membrane-trapped C-tail, before (a) and
after (b and c) its release by ACh activation of
M1AChR. d, representative trace of the Ca2+
current (20 mM Ca2+ in bath medium) showing the
sustained component of Ca2+ conductance due to the C-tail
anchoring, which was completely eliminated by the C-tail release (not
shown). The CREB activation (A, c), shown by
intensive FRET signal in the nucleus, was abolished (B) by
the co-expression of the Ca2+-insensitive analogue of CaM
(36) (CaM1234) or (C) by disruption of the apoCaM-binding site in the
free C-tail of the 1C,77L channel (33), which lost
Ca2+-dependent inactivation property as the
result of this mutation. Upper panels show voltage and ACh
application protocol. Twelve steps of depolarization from 90 mV to
+20 mV, 10-mV increment for 1-s pulse with 10-s intervals were applied,
and a 100-ms FRET images a-c were recorded at the end of
the last depolarization before (a) or after
(c) 5-min application of 5 µM ACh.
b shows effect of 5 µM ACh applied at 90 mV.
Left panels, phase-contrast images of expressing cells with
the shadow of patch pipette; scale bars, 4 µm. Free
Ca2+ measurements with Fluo-4 (lower panels)
were carried out under the same conditions and show that the activation
of CREB-induced transcription did not depend strongly on free
intracellular Ca2+ unless the C-tail was released.
Schematic diagrams depict positions of the 1C C-tail
vis-à-vis the plasma membrane. D, histogram of FRET
intensity ratio of images c and b
(n = 3, p < 0.01).
|
|
 |
DISCUSSION |
In this report, we provide strong evidence that the C-terminal
cytoplasmic tail is a functionally important moving part of the
voltage-gated Ca2+ channel. Previously implicated as
cytoplasmic channel elements that respond to voltage gating were the
cytoplasmic domain between repeat II-III of the skeletal muscle
1S subunit (40), inactivation gates of Na+
channel (41), and the ball-and-chain (N-type) inactivation determinant
of K+ channel (7, 42). The state dependence of FRET in the
1C channel is associated with conformational refolding
of the cytoplasmic parts in the resting, conducting, and inactivated
states. FRET increased upon transition from the resting to the
inactivated or conducting states of the Ca2+ channel
suggesting gating-dependent conformational mobility of the
channel tails. The anchoring of the C-terminal tail to the plasma
membrane impaired both the channel inactivation and FRET until fully
recovered with its release. Thus, the state-dependent mobility of the cytoplasmic C-terminal tail is essential for regulation of the Ca2+ channel.
In addition to relative proximity of the tagged
1C
tails, their angular orientation may contribute to FRET because the
environment of the fluorophores in the
(EYFP)N-
1C-(ECFP)C channels may
be structured and their segmental motions may not independently
randomize the orientations (
2
2/3) (12). The
2 factor adds to the uncertainties complicating
interpretation of the FRET measurements in terms of translational
distances and is of particular concern when dipoles become oriented
perpendicular to one another (
2 = 0). Because FRET was
consistently observed, such perpendicular orientation seems unlikely.
In fact the corresponding voltage-dependent movement of the
1C cytoplasmic C-tail is sufficient to reach other
targets and be involved in signal transduction. An important regulatory
signal associated with the C-terminal tail is CaM, which supports
Ca2+-induced inactivation of the Ca2+ channel
(35, 37, 38). It is possible that the C-terminal tail mobility may, in
a state-dependent manner, provide a coordinated transfer of
CaM between the channel pore inner mouth, where it is loaded by
Ca2+ when the channel opens, and protein targets like
CaM-activated protein kinase (8), where Ca2+/CaM exerts
signaling. This hypothesis may be generalized to the role of
CaM-carrying mobile tails in other Ca2+ channels (29,
43).
 |
ACKNOWLEDGEMENTS |
The discussions with C. Romanin, M. Morad and
H. Reuter motivated much of the early work of this study. The help of
A. Blatt and O. Carlson in molecular biology and A. Yu in image
analysis and preparation of figures is greatly appreciated. We thank T. Balla for PH domain coding vector; F. Hofmann for plasmids encoding the
channel accessory subunits; A. Sorkin for the EGF receptor-coding plasmid; M. R. Montminy for YKIDN and KIXCN constructs; and S. Sollott for valuable suggestions.
 |
FOOTNOTES |
*
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: NIA, National
Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.:
410-558-8343; Fax: 410-558-8318; E-mail:
soldatovn@grc.nia.nih.gov.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M211254200
 |
ABBREVIATIONS |
The abbreviations used are:
CaM, calmodulin;
FRET, fluorescence resonance energy transfer;
CREB, cAMP-responsive
element-binding protein;
ECFP, enhanced cyan fluorescent proteins;
EYFP, enhanced yellow fluorescent protein;
nt, nucleotide;
PH, pleckstrin homology;
EGF, epidermal growth factor;
ACh, acetylcholine;
M1AChR, muscarinic acetylcholine receptor..
 |
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