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The Journal of Neuroscience, April 15, 2003, 23(8):3446
Interactions with PDZ Proteins Are Required for L-Type Calcium
Channels to Activate cAMP Response Element-Binding
Protein-Dependent Gene Expression
Jason P.
Weick,
Rachel D.
Groth,
Ann L.
Isaksen, and
Paul G.
Mermelstein
Department of Neuroscience, University of Minnesota, Minneapolis,
Minnesota 55455
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ABSTRACT |
After brief periods of heightened stimulation, calcium
entry through L-type calcium channels leads to activation of the
transcription factor cAMP response element-binding protein (CREB) and
CRE-dependent transcription. Many of the details surrounding the
mechanism by which L-type calcium channels are privileged in signaling
to CREB, to the exclusion of other calcium entry pathways, has remained unclear. We hypothesized that the PDZ interaction sequence
contained within the last four amino acids of the calcium channel
1C (CaV1.2) subunit [Val-Ser-Asn-Leu
(VSNL)] is critical for L-type calcium channels (LTCs) to interact
with the signaling machinery that triggers activity-dependent gene
expression. To disrupt this interaction, hippocampal CA3-CA1 pyramidal
neurons were transfected with DNA encoding for enhanced green
fluorescent protein tethered to VSNL (EGFP-VSNL). EGFP-VSNL
significantly attenuated L-type calcium channel-induced CREB
phosphorylation and CRE-dependent transcription, although somatic
calcium concentrations after stimulation remained unchanged. The effect
of EGFP-VSNL was specific to the actions of L-type calcium channels,
because CREB signaling after NMDA receptor stimulation remained intact.
The importance of the PDZ interaction sequence was verified using
dihydropyridine (DHP)-insensitive 1C subunits.
Neurons transfected with 1C lacking the terminal five
amino acids (DHP-LTCnoPDZ) exhibited attenuated CREB responses in
comparison with cells expressing the full-length subunit (DHP-LTC). Collectively, these data suggest that localized calcium responses, regulated by interactions with PDZ domain proteins, are necessary for
L-type calcium channels to effectively activate CREB and CRE-mediated gene expression.
Key words:
NIL-16; CREB; NFAT; L-type calcium channel; 1C; CaV1.2; CIPP; NMDA
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Introduction |
Activity-dependent gene expression
is critical for the changes that occur during development, learning and
memory, induction of chronic pain, and the use of abusive drugs (Morgan
and Curran, 1991
; Bonni and Greenberg, 1997; Nestler and Aghajanian,
1997; Lanahan and Worley, 1998; Melzack et al., 2001). The
transcription factor cAMP response element-binding protein (CREB) plays
an important role in each of these processes (Finkbeiner et al., 1997;
Ji and Rupp, 1997; Carlezon et al., 1998; Milner et al., 1998; Silva et
al., 1998; Anderson and Seybold, 2000). Within the hippocampus and
other brain regions, CREB activation after brief stimulation (
3 min)
is primarily mediated by calcium entry via L-type calcium channels
(with a smaller component attributable to NMDA receptors) (Sheng et
al., 1991; Bading et al., 1993; Deisseroth et al., 1996; Mermelstein et
al., 2000). Calcium influx through L-type channels activates calmodulin
(CaM), leading to phosphorylation of CREB on
Ser133 by CaM-dependent protein kinases
(Enslen et al., 1995; Tokumitsu et al., 1995; Bito et al., 1996; Ahn et
al., 1999; Ho et al., 2000; Ribar et al., 2000; Kang et al.,
2001). Phosphorylation of CREB promotes its interaction with CREB
binding protein (CBP) or a similar coactivator, leading to
CRE-dependent transcription (Parker et al., 1996). Yet after various
forms of stimulation, L-type channels contribute only a small
percentage to the overall rise in cytoplasmic calcium (Deisseroth et
al., 1998; Dolmetsch et al., 2001). Thus the mechanism by which calcium
entry specifically through L-type channels mediates CREB activation is
not completely understood.
Several lines of evidence suggest that the localization of L-type
channels is an important determinant in their ability to signal to
CREB. Using various calcium chelators, a recent study proposed that
subcellular microdomains adjacent to the plasma membrane contain the
calcium-sensitive signaling machinery necessary for CREB
phosphorylation (Deisseroth et al., 1996). To play a privileged role in
activity-dependent gene expression, L-type calcium channels would be
localized to these regions, to the exclusion of other calcium entry
routes. 1C (CaV1.2), the
principal subunit of a large proportion of L-type calcium channels
expressed in brain, ends with the PDZ interaction sequence
Val-Ser-Asn-Leu (VSNL) (Koch et al., 1990; Snutch et al., 1991). VSNL
promotes 1C interactions with at least two PDZ
domain proteins, channel-interacting PDZ domain protein and neuronal
interleukin-16 (Kurschner et al., 1998; Kurschner and Yuzaki,
1999). Because PDZ domain proteins are critical for linking channels
and receptors to specific second messenger systems (Craven and Bredt,
1998; Sheng and Pak, 2000), we hypothesized that the interactions
between 1C and PDZ domain proteins are
required for L-type calcium channels to efficiently activate CREB.
Two separate lines of experiments were pursued to test this hypothesis.
First, VSNL was expressed in hippocampal pyramidal neurons. In theory,
VSNL would compete with endogenous L-type calcium channels for the PDZ
domain proteins with which 1C typically interacts, resulting in the displacement of these channels from the
calcium-sensing microdomains normally involved in CREB activation. To
visualize VSNL, the peptide was added to the C terminus of enhanced
green fluorescent protein (EGFP-VSNL), generating a fluorescent protein
that in many respects would mimic 1C
localization. Thus, the effect of EGFP-VSNL on L-type calcium
channel-dependent CREB signaling was first determined. In the second
set of experiments, neurons were transfected with DNA encoding for
either full-length 1C subunits that generate
dihydropyridine (DHP)-insensitive L-type calcium channels (DHP-LTC)
(Dolmetsch et al., 2001) or a dihydropyridine-insensitive 1C subunit containing a premature stop codon,
resulting in the deletion of the PDZ interaction sequence
(DHP-LTCnoPDZ). The results from both studies suggest that the VSNL
sequence on 1C plays a critical role in L-type
calcium channel-mediated CREB phosphorylation and CRE-dependent transcription.
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Materials and Methods |
Cell culture and transfection. Hippocampal pyramidal
neurons from 1- to 2-d-old rats were cultured as described previously (Mermelstein et al., 2000). Briefly, after decapitation and brain removal, the CA3-CA1 region of the hippocampus was isolated in an
ice-cold modified HBSS solution containing 20% fetal bovine serum
(FBS) (Hyclone, Logan, UT) and (in
mM): 4.2 NaHCO3, 1 HEPES, pH 7.35, 300 mOsm. All chemicals were obtained from Sigma
(St. Louis, MO) unless stated otherwise. After dissection, the
hippocampi were washed and digested for 5 min with 10 mg/ml Trypsin
(type XI) in a solution that contained (in mM):
137 NaCl, 5 KCl, 7 Na2HPO4, 25 HEPES, and DNase (1500 U), pH 7.2, 300 mOsm. The tissue was washed
and then dissociated using a series of Pasteur pipettes of decreasing
diameter. The cell suspension was pelleted twice to remove
contaminants, plated on 10 mm coverslips, and allowed to adhere for 15 min before addition of 2 ml MEM (Invitrogen, Grand Island,
NY) containing (in mM): 28 glucose, 2.4 NaHCO3, 0.0013 transferrin
(Calbiochem, La Jolla, CA), 2 glutamine, 0.0042 insulin,
and 10% FBS, pH 7.35, 300 mOsm. Twenty-four hours after plating, 1 ml
of media was replaced with a similar solution containing 4 µM cytosine
1-
-D-arabinofuranoside and 5% FBS. Three days
later, 1 ml of media was replaced with modified MEM containing 5% FBS. Media solutions contained 2 µg/ml gentamicin
(Invitrogen, Carlsbad, CA) to prevent bacterial growth.
Cultured neurons were transfected 7-9 d in vitro (DIV)
using a calcium phosphate-based technique (Graef et al., 1999). African Green Monkey kidney (COS-7) cells and human embryonic kidney
(HEK)-293 cells were maintained in a DMEM-F12 solution (Life
Technologies) containing 2 µg/ml gentamicin. Transfections
were performed at 70-80% confluency using Lipofectamine 2000 following the manufacturer's instructions (Invitrogen).
DNA subcloning and immunocytochemistry. pEGFP-C1 was
obtained from Clontech (Palo Alto, CA). The sequences
encoding for VSNL and Ile-Thr-Thr-Leu (ITTL) were inserted into the
multiple cloning site using standard procedures and verified by
direct sequencing. NIL-16 was a gift from C. Kurschner (Myriad
Proteomics); the DHP-LTC construct was a gift from M. Greenberg
(Harvard University) and R. Dolmetsch (Stanford University).
Generation of the DHP-LTCnoPDZ construct was the result of a single
round of QuickChange mutagenesis following the manufacturer's
instructions (Stratagene, La Jolla, CA), replacing the DNA
sequence that encodes for the final tyrosine immediately before VSNL in
1C with a premature stop codon. The mutagenesis was verified by direct sequencing.
For experiments measuring CREB phosphorylation, ~20 hr after
transfection, cells were preincubated for 3 hr in a standard Tyrode
solution containing 1 µM TTX. When applicable, 5 µM nifedipine and 25 µM D-AP5
(Tocris, Ellisville, MO) were used to block L-type calcium channels and
NMDA receptors. KN-93 (2 µM;
Calbiochem) was used to block CaM-dependent protein
kinases, and the inactive analog KN-92 (10 µM) was used
because a control. Diltiazem (100 µM) was used to block
the dihydropyridine-insensitive calcium channels. AP5, nifedipine,
KN-93, KN-92, and diltiazem were present 30 min before and throughout
the stimulation. Three minute application of 50 µM NMDA
was used to initiate NMDA receptor-mediated CREB phosphorylation.
After stimulation, cells were fixed for 20 min with ice-cold 4%
paraformaldehyde (Electron Microscopy Sciences, Ft.
Washington, PA) in PBS containing 4 mM EGTA. Cells were
washed three times with PBS and then permeabilized for 5 min with 0.1%
Triton X-100 (VWR, West Chester, PA). After PBS wash, cells were
incubated for 0.5 hr at 37°C in a PBS-based block solution containing
1% BSA and 2% goat serum (Jackson ImmunoResearch, West
Grove, PA). Afterward, coverslips were exposed to the primary antibody
[1:1000 dilution of the polyclonal anti-pCREB antibody (Upstate
Biotechnology, Lake Placid, NY)] for 1 hr at 37°C. For
detecting 1C, neurons were fixed for ~12 hr
after transfection with EGFP, EGFP-VSNL, or EGFP-ITTL. In these
experiments, the preincubation and stimulation steps were omitted, and
the primary antibody (1:100; Alomone Labs, Jerusalem,
Israel) incubation was performed overnight at 37°C. After exposure to
the primary antibody, cells were washed with PBS and incubated for 1 hr
with a Cy3-conjugated secondary antibody (Jackson
ImmunoResearch). Cells were washed again and mounted using the
anti-quenching reagent Citifluor (Ted Pella, Redding, CA).
Acquisition and quantification of fluorescent intensities (n = ~15 per group) were determined using a
Bio-Rad confocal workstation (MRC 1024) and Metamorph
software (version 4.6). Transfected neurons were compared with
untransfected cells on the same coverslip, often in the same image. To
verify consistency across cover slides, multiple coverslips (two to
three) were prepared following the same experimental conditions.
Experiments were also replicated multiple times to verify results.
Immunocytochemistry on COS-7 cells followed similar protocols. NIL-16
was detected with an antibody (1:100) from PharMingen (San
Diego, CA) (Kurschner and Yuzaki, 1999). Expression of DHP-LTC and
DHP-LTCnoPDZ was determined with an Xpress antibody (1:1000) from Invitrogen.
For quantification of 1C and EGFP-VSNL
staining, confocal images were taken under equivalent magnification and
laser intensity. In addition, the confocal section that included
the primary dendrite was used for analysis. For the analysis of
1C clustering, a single fluorescence threshold
was set and applied equally across groups to minimize the inclusion of
background staining. On the basis of a Gaussian distribution,
individual puncta were defined as fluorescent regions of two to six
adjacent pixels (~0.5 µm), all above the threshold value.
Fluorescent regions larger than six pixels were considered to be
multiples. The total number of fluorescent aggregations within a 7 µm
radius from the center of the nucleus was included in the analysis.
Because of the inherent differences in transfection level and EGFP
expression, no single background threshold could be applied for the
quantification of EGFP-VSNL puncta. Thus, individual thresholds were
set for each cell, and puncta were counted according to the methods
described above. Multiple observers (n = 4) performed
analyses of each cell without knowledge of experimental hypotheses, and
the average counts were used for the final analysis.
Coimmunoprecipitation. HEK-293 or COS-7 cells transfected
with NIL-16 and either EGFP or EGFP-VSNL were washed once with 4°C PBS and then lysed in ice-cold modified radioimmunoprecipitation (RIPA) buffer without SDS (Harlow and Lane, 1988) that contained (in mM): 1 EDTA, 200 PMSF, 200 Na3VO4, and 200 NaF plus 1 µg/ml of aprotinin, leupeptin, and pepstatin. Cells were collected in 1.5 ml Eppendorf tubes and spun in a microcentrifuge at 14,000 rpm for
10 min. The supernatant was transferred to a separate 1.5 ml tube
containing 30 µl of protein-G beads and agitated for 1 hr at 4°C.
After centrifugation at 2000 rpm for 2 min, the supernatant was removed
and added to a fresh 1.5 ml tube containing 30 µl of protein-G beads
and 5 µg of the anti-NIL-16 antibody. After agitation for 1 hr at
4°C, the solution was centrifuged at 2000 rpm for 2 min. The pellet
was washed three times with RIPA buffer and prepared for Western
blotting using commercially available reagents
(Invitrogen). After blocking in a Tris-buffered saline solution containing 10% milk and 1% BSA, the blot was incubated with
a chicken polyclonal anti-EGFP antibody (1:1000; Upstate Biotechnology) overnight at 4°C. Both the primary and
secondary antibodies were diluted in Tris-buffered saline containing
1% milk, 1% BSA, and 0.1% Tween 20. The membrane was washed in 0.1% Tween-Tris-buffered saline and probed with an anti-chicken-HRP antibody (1:25,000) (Pierce, Rockford, IL) for 1 hr at
25°C and then exposed to X-Omat XB-1 film (Kodak,
Rochester, NY). Direct loading of the cell lysis solution was used as a
positive control for transfection efficiency.
Electrophysiology. Whole-cell patch-clamp recordings of
cultured hippocampal neurons (~10 DIV, 24 hr after transfection) were performed at room temperature using standard techniques. Warner GC120T-10 borosilicate glass electrodes (Warner Instrument
Corp., Hamden, CT) were pulled on a Flaming/Brown p-97 puller
(Sutter Instrument Co., Novato, CA) and fire polished with
an MF-830 microforge (Narishige, Hempstead, NY). The
intracellular recording solution contained (in
mM): 190 N-methyl-D-glucamine, 40 HEPES, 5 BAPTA, 4 MgCl2, 12 phosphocreatine, 3 Na2ATP, and 0.2 Na3GTP, pH
7.2, 275 mOsm. The external recording solution contained (in
mM): 135 NaCl, 20 CsCl, 1 MgCl2, 10 HEPES, 0.0001 TTX, and 5 mM BaCl2. Bath solution
contained (in mM): 140 NaCl, 2 KCl, 23 glucose, 15 HEPES, 1 CaCl2, 2 MgCl2,
and 0.01 glycine. All reagents were obtained from Sigma
except ATP and GTP (Boehringer Mannheim, Indianapolis, IN)
and BAPTA (Calbiochem). The junction potential (<2 mV)
was not compensated. Recordings were obtained using an Axopatch 200B amplifier (Axon Instruments, Union City, CA) controlled by
a PC running pCLAMP software (version 8.0) using a 125 kHz interface. Electrode resistances were ~3 M
. The series resistance was
compensated >70%.
Gene expression assay. Cultured neurons (~7-9 DIV) were
transfected with a luciferase-based reporter for CRE- or nuclear factor of activated T-cells (NFAT)-dependent transcription and EGFP or EGFP-VSNL. After transfection, 2 µM TTX was
added to the media. The next day, cells were washed with DMEM, and half
were stimulated for 3 min in the high K+
solutions indicated. For the CRE-luciferase assays, all cells were
pretreated for 30 min with 25 µM AP5, which was
also present during stimulation. Afterward, the original media was
supplemented with 2 µM TTX and reapplied to the
coverslips. Sixteen hours after stimulation, cells were lysed and
cellular protein was isolated. Luciferase expression was measured using
standard procedures. When measuring the L-type calcium channel
component of synaptically mediated NFAT-dependent transcription, half
of the neurons were treated with 5 µM
nifedipine after transfection. As a control for cellular viability, a
constitutively active reporter (PBJ5-luciferase) was transfected
with either EGFP or EGFP-VSNL. In separate experiments, the
CRE-luciferase reporter was transfected with either DHP-LTC or
DHP-LTCnoPDZ. The following day, 25 µM AP5 and
5 µM nifedipine were added to the cell media.
Half of the coverslips were also exposed to 100 µM diltiazem. Thirty minutes later, coverslips were stimulated with a modified cell media solution containing 20 mM K+ and either AP5
and nifedipine or AP5, nifedipine, and diltiazem. Three hours later,
cells were lysed and luciferase expression was measured. Luciferase
expression was not significantly different across groups in
unstimulated neurons. All experiments were replicated to verify results.
Calcium photometry. Internal calcium concentrations were
determined using the calcium indicator Indo-1 (Grynkiewicz et al., 1985), using methods published previously (Werth et al., 1996). Briefly, ~24 hr after transfection, cells were loaded with 2 µM Indo-1 AM ester at room temperature for 40 min. During recording, EGFP- and EGFP-VSNL-transfected cells were
superfused with a Tyrode solution containing 1 µM TTX and 25 µM AP5 at
a rate of 1-2 ml/min. In separate experiments, 5 µM nifedipine was also added to the Tyrode
solution. After acquisition of a stable baseline, the perfusate was
switched for 3 min to a 20 mM
K+ solution containing the appropriate
channel blockers. Complete solution exchanges occurred within 10 sec.
To determine which cells were transfected with the DHP-LTC or
DHP-LTCnoPDZ, neurons were cotransfected with EGFP (5:1 ratio of
channel to EGFP). Similar to the findings reported by Dolmetsch et al.
(2001), under these conditions, >90% of the neurons positive for EGFP
also exhibited Xpress-tagged calcium channels (n > 50). For neurons transfected with dihydropyridine-insensitive L-type calcium channels, recording solutions contained TTX, AP5 and
nifedipine. To determine the contribution of DHP-LTCs and
DHP-LTCnoPDZs to the overall increase in
[Ca2+]i after 20 mM K+
depolarization, diltiazem (100 µM) was added to
the recording solutions in a subset of experiments (n = 8). Differences in the residual increase in
[Ca2+]i after
diltiazem exposure was not significantly different between groups (0.4 nM; p > 0.05). After completion of each experiment, background light levels
were determined. Records were later corrected for background and
converted to
[Ca2+]i by the
equation [Ca2+]i = Kd (R 
Rmin)/(Rmax R), in which R is the 405/490 nm fluorescence ratio. The dissociation constant (Kd)
used for Indo-1 was 250 nM.
Rmin,
Rmax, and were determined in
ionomycin-permeabilized cells in calcium-free (1 mM EGTA) and 5 mM
Ca2+ buffers. Values of
Rmin,
Rmax, and were 0.19, 2.3, and 3.5, respectively.
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Results |
Interactions between EGFP-VSNL and PDZ domain proteins
Previous reports have shown that 1C is
expressed in a punctate pattern, heavily concentrated in the cell soma
and proximal dendrites of neurons (Westenbroek et al., 1990; Hell et
al., 1993). Using immunocytochemistry and confocal microscopy
techniques, we found that expression of EGFP alone did not affect the
normal distribution of 1C (Fig.
1A). Three-dimensional
reconstructions of immunolabeled neurons indicated that the vast
majority of 1C expression was localized to the
membrane surface. We hypothesized that the clustering of L-type calcium
channels was based on 1C binding to PDZ domain
proteins. Because the last four amino acids of
1C are believed to promote its interaction
with PDZ domain proteins, we overexpressed EGFP-VSNL in an attempt to
out-compete endogenous 1C protein for PDZ
binding sites. In neurons transfected with EGFP-VSNL, the distribution
of endogenous 1C was markedly altered (Fig.
1B). The number of intense clusters of fluorescence generated by immunolabeled 1C was
significantly decreased (Fig. 1D) (n = ~15 per group; p < 0.001). Furthermore, the
1C immunofluorescence in EGFP-VSNL-expressing
neurons was reminiscent of the staining observed in glial cells (Fig.
1B, arrowheads) (Agrawal et al., 2000; Chung et al.,
2001; Latour et al., 2001).
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Figure 1.
Disruption of endogenous 1C
clustering in neurons transfected with EGFP-VSNL. A,
Paired confocal images of a neuron transfected with EGFP (green) and
stained using an antibody for the calcium channel 1C
subunit (red). The distribution of endogenous 1C protein
in EGFP-transfected neurons was indistinguishable from untransfected
neurons (data not shown). B, EGFP-VSNL exhibited a
punctate expression pattern similar to the distribution of
1C in control cells. In neurons transfected with
EGFP-VSNL, 1C labeling tended to be more diffuse,
similar to staining in glial cells (arrowheads). C,
EGFP-ITTL exhibited uniform fluorescence similar to EGFP, with no
effect on 1C localization. D, EGFP-VSNL
expression resulted in a significant (p < 0.001) reduction in the fluorescent clustering of 1C.
The number of EGFP-VSNL puncta, when added to the number of residual
1C aggregations, was not significantly different
(p > 0.05) from the total number of
fluorescent clusters of 1C observed in control
conditions.
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Although EGFP was uniformly distributed throughout the cell, expression
of EGFP-VSNL fluorescence was concentrated into puncta within the cell
soma and dendrites (Fig. 1A,B). This expression pattern was similar to the localization of 1C
protein under control conditions, suggesting that EGFP-VSNL competed
with 1C for localization at PDZ binding sites.
In keeping with this idea, the number of EGFP-VSNL puncta, when added
to the number of residual 1C aggregations, was
not significantly different (p > 0.05) from the
total number of fluorescent 1C clusters
observed under control conditions (Fig. 1D). The
punctate distribution of EGFP-VSNL peaked ~10-15 hr after
transfection, becoming more difficult to resolve at 24 hr
(see Fig. 4C), consistent with the idea that the PDZ binding sites normally taken up by L-type calcium channels were being fully
occupied by EGFP-VSNL.
To verify that mislocalization of 1C by
EGFP-VSNL was specifically dependent on the VSNL sequence, EGFP was
tethered to ITTL. ITTL are the last four amino acids of one
splice variant of the calcium channel 1D
(CaV1.3) subunit (Ihara et al., 1995; Safa et
al., 2001) and were not predicted to interact with PDZ binding domains.
As with EGFP, EGFP-ITTL exhibited a homogenous distribution throughout
the cell, without altering 1C labeling (Fig.
1C,D). Because most of the L-type calcium channels in brain
are composed of either an 1C or an
1D subunit, the data suggest that
these two populations of L-type calcium channels likely have different intracellular distributions and thus may interact with distinct second
messenger systems (see Discussion).
Because the last four amino acids of receptors and ion channels promote
specific interactions with particular PDZ domain proteins, and VSNL
expression appeared to alter the subcellular distribution of endogenous
1C protein, we wanted to verify that EGFP-VSNL did indeed interact with PDZ domain proteins. To test this, COS-7 cells
were transfected with cDNA encoding for NIL-16 (a PDZ domain protein
hypothesized to interact with 1C) and either
EGFP or EGFP-VSNL. In control experiments, EGFP expression was diffuse within the cytosol and nucleus (Fig.
2A) and did not
accumulate at the plasma membrane with NIL-16 (Fig.
2B,C). Interestingly, when EGFP-VSNL was expressed
without NIL-16, its cellular distribution was similar to EGFP (Fig.
2D, bottom cell). However, when coexpressed with
NIL-16, EGFP-VSNL was also observed as aggregations at the plasma
membrane (Fig. 2D, top cell), colocalizing with
NIL-16 (Fig. 2E,F). Coimmunoprecipitation
experiments verified the interaction between EGFP-VSNL and NIL-16 (Fig.
2G, lane 6). The interaction between these two proteins was
dependent on VSNL, because EGFP did not coimmunoprecipitate with NIL-16
in control experiments (Fig. 2G, lane 3).
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Figure 2.
EGFP-VSNL interacts with the PDZ domain protein
NIL-16. A-C, Confocal image of a COS cell expressing
EGFP (green) and NIL-16 (red). The two proteins do not appreciably
colocalize. D, Two cells transfected with EGFP-VSNL.
E, The top cell also expressed NIL-16. F,
By colocalizing with the fluorophore, NIL-16 promoted EGFP-VSNL
expression at the plasma membrane (yellow). G, EGFP-VSNL
(lane 6), but not EGFP (lane 3), coimmunoprecipitated with NIL-16,
demonstrating that the interaction between the two proteins is
dependent on the PDZ interaction sequence. EGFP and EGFP-VSNL are not
detected when the antibody for NIL-16 is omitted from the
immunoprecipitation step (lanes 1, 4). Direct probing of the cell
lysate indicated comparable concentrations of EGFP and EGFP-VSNL
protein (lanes 2, 5).
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To verify that the alteration in neuronal 1C
immunolabeling after EGFP-VSNL expression was not caused by a change in
protein expression, L-type calcium channel currents were isolated using patch-clamp methods. Application of the L-type calcium channel blocker
nifedipine (5 µM) resulted in a 34.1 ± 2.6%
(mean ± SEM) reduction of the whole-cell calcium current in
EGFP-VSNL-expressing neurons (n = 14). This percentage
of L-type current was not significantly different from untransfected
(35.6 ± 2.9%; n = 14) or EGFP transfected (37.8 ± 3.2%; n = 11) neurons (Fig.
3). Furthermore, whole-cell calcium
current densities remained unchanged, eliminating the possibility that
EGFP-VSNL produced a general decrease in calcium channel expression
(not transfected: 7.79 ± 0.98 pA/pF; EGFP: 9.62 ± 1.60 pA/pF; EGFP-VSNL: 9.32 ± 1.12 pA/pF).
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Figure 3.
EGFP-VSNL did not alter L-type calcium channel
expression. A, A representative whole-cell patch-clamp
recording taken from a neuron transfected with EGFP-VSNL (5 mM Ba2+ was used as the charge carrier).
Application of nifedipine (5 µM) isolated the L-type
current. The voltage waveform is shown above the current traces.
B, The L-type calcium channel component of the
whole-cell current was not significantly different between
untransfected neurons and those transfected with either EGFP or
EGFP-VSNL.
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Although the above data demonstrate that the quantity of L-type calcium
current remained unchanged in EGFP-VSNL-expressing neurons, we
considered the possibility that the retention of current was caused by
the replacement of 1C subunits with
1D. Because L-type calcium channels composed
of 1C are more sensitive to DHP than those
containing 1D (Xu and Lipscombe, 2001), the
dose dependence of nifedipine block was determined. For each
concentration of nifedipine examined (1 nM TO 10 µM), block of L-type calcium current in
EGFP-VSNL-expressing neurons (n = 7) did not differ significantly from control (n = 9) (supplemental
Fig. 1; available at www.jneurosci.org). Furthermore, in both
groups, a high-affinity (~30 nM) and
low-affinity (~1 µM) DHP binding site was
found, consistent with previous reports regarding the concentrations required to block 1C and
1D L-type calcium channels.
Global calcium dynamics remain unchanged after
EGFP-VSNL expression
The next series of experiments determined that EGFP-VSNL did not
affect global calcium influx. Neurons transfected with either EGFP or
EGFP-VSNL were loaded with the calcium indicator Indo-1. Somatic
calcium concentrations were measured during a 20 mM
K+ (+25 µM AP5) stimulus
protocol that mimicked the conditions used to activate CREB via calcium
entry through L-type calcium channels (see below). Intracellular
calcium concentrations in EGFP-VSNL-transfected neurons
(n = 7) increased by 106.2 ± 20.5 nM, from a basal concentration of 46.7 ± 6.0 nM, which was not significantly different
from their EGFP-transfected (n = 8) counterparts
(110.3 ± 10.3 nM, 46.4 ± 4.4 nM) (Fig.
4A,B). Furthermore, in
the presence of 5 µM nifedipine, intracellular
calcium concentrations increased by 40.2 ± 3.8 nM in EGFP-VSNL-transfected (n = 9) and 39.4 ± 5.6 nM in EGFP-transfected (n = 8) neurons (Fig. 4C,D). The approximate
60% block by nifedipine after 20 mM
K+ depolarization was consistent with
previous biophysical and imaging work suggesting that L-type calcium
channels activate at more negative potentials than other voltage-gated
calcium channels (Mermelstein et al., 2000, 2001).
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Figure 4.
EGFP-VSNL does not affect global calcium
signaling. A, Representative recordings of
[Ca2+]i measurements during
application of 20 mM K+ in EGFP- and
EGFP-VSNL-expressing neurons. AP5 (25 µM) was present
throughout the recording to block NMDA receptors. B, The
stimulus-induced increase in somatic calcium concentrations did not
differ between groups. C, D, Similar
calcium photometry experiments in the presence of nifedipine. The rise
in somatic intracellular calcium attributable to L-type and non-L-type
calcium channels after depolarization is not different between EGFP-
and EGFP-VSNL-expressing neurons.
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EGFP-VSNL specifically disrupts L-type calcium channels from
signaling to CREB
The effect of EGFP-VSNL on CREB activation was determined using a
phospho-Ser133-specific CREB antibody
(Ginty et al., 1993) with nuclear immunofluorescence quantified from
confocal sections. To activate CREB via calcium entry through L-type
calcium channels, neurons were depolarized for 3 min with 20 mM K+ (Mermelstein et al.,
2001). To eliminate NMDA receptor-mediated CREB phosphorylation, AP5
was applied both 30 min before and during depolarization. In neurons
expressing EGFP, 20 mM K+
produced a significant increase (p < 0.001) in
CREB phosphorylation, equal to the effect observed in neighboring
untransfected cells (Fig. 5A).
The 20 mM K+-induced
increase in CREB phosphorylation was blocked by both nifedipine and
KN-93 (2 µM) (p < 0.001) but not by the control drug KN-92 (10 µM) (Fig. 5B), demonstrating that
under mild stimulation conditions, L-type channels mediate CREB
phosphorylation via activation of a CaM-dependent protein kinase (Bito
et al., 1996; Wu et al., 2001).
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Figure 5.
EGFP-VSNL specifically attenuated L-type calcium
channel-mediated CREB phosphorylation. A, Immunolabeling
studies demonstrated that expression of EGFP did not alter 20 mM K+- (+AP5) induced CREB
phosphorylation. B, Nifedipine and the CaM kinase
inhibitor KN-93 (2 µM) significantly
(p < 0.001) attenuated CREB phosphorylation
after depolarization by 20 mM K+. The
negative control KN-92 (10 µM) had no effect.
C, D, Expression of EGFP-VSNL
significantly (p < 0.001) attenuated L-type
calcium channel-mediated CREB phosphorylation. The arrow indicates a
neuron expressing EGFP-VSNL. The arrowhead points to an untransfected
neuron. The bar graph depicts summarized data. EGFP-VSNL attenuation of
CREB phosphorylation was observed only following parameters designed to
activate L-type calcium channels (i.e., 20 mM
K+ + AP5). Conversely, NMDA receptor-mediated CREB
phosphorylation (50 µM NMDA + nifedipine) was unaffected
by EGFP-VSNL expression.
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Although EGFP-transfected neurons exhibited a stimulation-induced
increase in CREB phosphorylation identical to untransfected neurons,
CREB phosphorylation was significantly reduced
(p < 0.001) in neurons expressing EGFP-VSNL
(Fig. 5C,D). The reduction in CREB phosphorylation occurred
although the amount of calcium entering the cell through L-type calcium
channels remained the same (Fig. 4). Intriguingly, the reduction in
CREB phosphorylation by EGFP-VSNL was observed only with stimulation
parameters designed to activate CREB specifically via calcium entry
through L-type channels. EGFP-VSNL-transfected neurons treated for 3 min with NMDA (50 µM) in the presence of nifedipine did not exhibit decreased levels of CREB phosphorylation (Fig. 5D). Under these conditions, AP5 completely
blocked stimulus-induced CREB phosphorylation, indicating an NMDA
receptor-mediated event (data not shown). Furthermore, similar to
L-type calcium channels, a significant component of CREB
phosphorylation after brief NMDA receptor stimulation is dependent on
activation of CaM-dependent protein kinases (Bito et al., 1996; Wu et
al., 2001; Impey et al., 2002). Consistent with this finding, KN-93
significantly decreased CREB phosphorylation after depolarization in
the presence of nifedipine (supplemental Fig. 2; available at
www.jneurosci.org). The effect of KN-93 was identical in both
untransfected and EGFP-VSNL-transfected neurons, demonstrating that the
influence of EGFP-VSNL on L-type calcium channel-mediated CREB
signaling could not be attributed to a nonspecific effect on calcium,
CaM, or CaM-dependent protein kinases.
We next sought to determine whether expression of EGFP-VSNL
would impact CREB-dependent gene expression. In addition to
either EGFP or EGFP-VSNL, neurons were transfected with a
luciferase-based reporter of CRE-dependent transcription. A
3 min 20 mM K+ stimulus (+AP5)
significantly (p < 0.05) increased
CRE-dependent transcription [22,370 ± 9416 reflective light
units (RLU)] in EGFP-transfected neurons (n = 9) in
comparison with unstimulated cells (n = 10).
Conversely, the increase in CRE-dependent transcription observed in
EGFP-VSNL-transfected neurons (3213 ± 1697 RLU; n = 9) was not significantly different from their unstimulated controls (n = 10) (Fig.
6A). Furthermore, when
transfected with a constitutively active reporter, expression between
groups was not significantly different (EGFP: 599,875 ± 59,744 RLU; EGFP-VSNL: 529,254 ± 25,171 RLU; n = 10 per
group), eliminating the possibility that the reduction in CRE-dependent
transcription observed in EGFP-VSNL transfected neurons was caused by a
nonspecific decrease in gene expression.
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Figure 6.
EGFP-VSNL inhibited CRE-dependent transcription,
whereas NFAT-dependent transcription was unaffected. A,
A 3 min 20 mM K+ stimulus activated
CRE-dependent transcription in EGFP (p < 0.05) but not in EGFP-VSNL-expressing neurons. Transcription was
measured using a luciferase-based reporter construct. Similarly, after
a 3 min depolarization with 90 mM K+,
CRE-dependent transcription in EGFP-VSNL-transfected neurons was
significantly attenuated (p < 0.02). AP5
was used to block NMDA receptors. B, After a 90 mM K+ stimulus, NFAT-dependent
transcription was unaltered by EGFP-VSNL expression. The L-type calcium
channel component of NFAT-dependent transcription triggered by
endogenous synaptic activity was also unaffected by EGFP-VSNL.
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K+ (90 mM) has also been used
as a stimulus to activate L-type calcium channel-mediated gene
expression (Bito et al., 1996). This stimulus will activate not only
CREB, but other transcription factors as well, including NFATc4
(Graef et al., 1999). For CREB, the stronger depolarization
results in increased calcium entry through L-type channels and greater
CREB activation. Thus we tested whether expression of
EGFP-VSNLwould again result in a diminution of CREB signaling. In
EGFP-transfected neurons (n = 4), 90 mM K+ (+AP5)
increased CRE-luciferase expression by 56,842 ± 18,978 RLU
relative to unstimulated controls (n = 4). In contrast,
EGFP-VSNL neurons (n = 5) exhibited an increase of only
6346 ± 1222 RLU when compared with unstimulated EGFP-VSNL
neurons (n = 5). CRE-dependent transcription triggered
by the brief stimulus was significantly less (p < 0.02) in EGFP-VSNL neurons. Consistent with the idea that
attenuation of CRE-dependent transcription is through inhibition of
CREB phosphorylation, EGFP-VSNL-expressing neurons also exhibited a
significant (p < 0.05) decrease in 90 mM K+-induced CREB
phosphorylation (data not shown).
Because NFAT-dependent transcription has been shown to rely on calcium
entry through L-type channels after a 90 mM
K+ depolarization, we tested whether
displacement of 1C L-type channels would also
affect the activation of this transcription factor. Neurons were
transfected with EGFP or EGFP-VSNL, along with a luciferase-based
reporter of NFAT transcription. After a 3 min 90 mM
K+ stimulus, EGFP-VSNL-transfected neurons
(n = 10) exhibited an increase of 7217 ± 2409 RLU
in comparison with unstimulated controls (n = 10),
which was not significantly different from the stimulus-induced effect
in EGFP neurons (8459 ± 1742 RLU; n = 10 per
group) (Fig. 6B). AP5 was omitted from these
experiments because NMDA receptors do not directly activate
NFAT-dependent transcription under these conditions (Graef et al.,
1999). In another test, nifedipine was used to block NFAT-dependent
transcription mediated by endogenous synaptic activity. Again,
EGFP-VSNL failed to block L-type channel-mediated NFAT-dependent
transcription (EGFP: 21,437 ± 3982; EGFP-VSNL: 21,426 ± 2429 RLU; n = 10 per group), suggesting that a
mechanism independent of 1C localization is
responsible for L-type channel regulation of NFAT activity (see Discussion).
1C subunits lacking the PDZ interaction sequence
exhibit a significant impairment in the ability to signal to CREB
Recently, Dolmetsch et al. (2001) constructed an epitope-tagged
1C subunit containing a threonine to tyrosine
point mutation at position 1039, resulting in the generation of a
DHP-insensitive L-type calcium channel (DHP-LTC).
Depolarizations in the presence of DHPs isolate CREB phosphorylation
and CRE-dependent transcription arising from calcium entry specifically
through these channels. Furthermore, DHP-LTCs are still sensitive to
diltiazem (100 µM), a non-DHP blocker of L-type calcium
channels. As such, we used this construct to examine the role of the
PDZ interaction sequence on L-type calcium channel CREB responses.
Hippocampal neurons were transfected with DNA encoding for either the
full-length DHP-LTC or a truncated version in which the PDZ
interaction sequence was absent because of the insertion of a premature
stop codon (DHP-LTCnoPDZ). Both constructs localized predominantly to
the soma and proximal dendrites of neurons (Fig. 7B,C),
suggesting that removal of the terminal five amino acids did not have a
dramatic effect on insertion of the subunit into the plasma membrane
(see Discussion). Furthermore, after a 3 min stimulation with 20 mM K+ (+AP5 and
nifedipine), increases in intracellular calcium attributable to both
channels were not significantly different (DHP-LTC: 40.9 ± 6.4 nM, n = 7; DHP-LTCnoPDZ:
50.1 ± 5.4 nM, n = 6) (Fig.
7A). Under these same stimulation conditions, neurons
expressing DHP-LTCs exhibited a significant increase in CREB
phosphorylation that could be blocked by diltiazem (Fig.
7B). Conversely, neurons transfected with DHP-LTCnoPDZ did
not exhibit the same stimulus-induced increase in CREB phosphorylation
(Fig. 7C).
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Figure 7.
The PDZ interaction sequence on 1C
is required for L-type calcium channels to effectively signal to CREB.
A, Diltiazem-sensitive (100 µM) increases
in [Ca2+]i after a 3 min 20 mM K+ (+AP5 and nifedipine)
depolarization are not significantly different between neurons
transfected with the full-length, dihydropyridine-insensitive L-type
calcium channel (DHP-LTC) and those expressing DHP-LTCnoPDZ.
B, Neurons expressing DHP-LTCs (arrow) exhibit
significant (p < 0.001) increases in CREB
phosphorylation after the same stimulation conditions outlined above;
untransfected neurons do not (arrowheads). The stimulus-induced
increase in CREB phosphorylation was blocked by diltiazem.
C, Unlike full-length channels,
dihydropyridine-insensitive L-type calcium channels lacking the PDZ
interaction sequence do not trigger CREB phosphorylation after a 3 min
depolarization. D, In comparison with neurons
transfected with DHP-LTC, neurons transfected with DHP-LTCnoPDZ
exhibit significantly (p < 0.001) less
diltiazem-sensitive CRE-dependent transcription after a 3 hr 20 mM K+ stimulus in the presence of AP5
and nifedipine.
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In the final experiment, neurons were transfected with the
CRE-luciferase reporter and either DHP-LTC or DHP-LTCnoPDZ. As reported originally by Dolmetsch et al. (2001), whereas DHP-LTCs are
capable of activating CREB, they are not as proficient as endogenous
L-type calcium channels. Thus, we were not surprised to find that
neurons expressing DHP-LTCs did not consistently exhibit increases in
luciferase expression after a 3 min 20 mM K+ stimulus (in the presence of AP5 and
nifedipine). Consequently, neurons were stimulated with 20 mM K+ (+AP5 and nifedipine)
for 3 hr in the presence or absence of diltiazem (Fig. 7D).
Under these conditions, DHP-LTCs generated an increase in luciferase
activity by 102,569 ± 9,899 RLU (n = 10 per
group), significantly greater than CRE-dependent transcription attributable to DHP-LTCnoPDZs (42,752 ± 9077 RLU;
n = 10 per group). Intriguingly, the results suggest
that although DHP-LTCnoPDZs are capable of activating CREB, they are
less efficient than those channels containing the PDZ interaction
sequence (see Discussion).
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Discussion |
The subcellular localization of
1C-comprised L-type calcium channels
appears critical for CREB-dependent gene expression. EGFP-VSNL, which
can interact with the PDZ domain protein NIL-16, reduced L-type calcium
channel aggregations when expressed in neurons. After expression of
EGFP-VSNL, CREB phosphorylation and CRE-dependent transcription
mediated by calcium entry through L-type calcium channels was
significantly attenuated, although increases in somatic calcium
concentrations after stimulation remained unchanged. Furthermore, the
ability of L-type calcium channels to activate CREB and CRE-dependent
transcription after the removal of the PDZ interaction sequence was
significantly compromised. Collectively, our results suggest that local
calcium signaling plays a critical role in L-type calcium
channel-mediated activation of CREB-dependent gene expression.
Over the last several years, PDZ domain-containing scaffold
proteins have been shown to link ion channels with both cytoskeletal and signaling proteins. Because the highly polarized nature of neurons
requires different cellular locales to support distinct functions, the
active assembly of signal transduction pathways within particular
regions of the plasma membrane provides a mechanism by which efficient
signaling can occur. The most frequently studied of these structures
are the postsynaptic densities, in which PDZ domain protein
interactions regulate glutamate receptor activity and glutamate-induced
plasticity. Here we provide evidence that L-type calcium channels may
also be targeted to distinct intracellular scaffolding and signaling
proteins to activate gene expression. Considering that different
classes of voltage-gated calcium channels support unique cellular
functions, the utilization of linking proteins is not surprising. In
fact, compartmentalization of calcium channels may be a common theme,
because recent data have shown that the N-type calcium channel subunit
1B-1 (CaV2.2a) contains targeting sequences to promote its interaction with the adaptor proteins Mint/x11-like protein 1 (Mint1) and
calcium/calmodulin-dependent serine protein kinase
(CASK) (Maximov et al., 1999; Kaneko et al., 2002; Maximov and
Bezprozvanny, 2002). The resulting interactions target N-type calcium
channels to the presynaptic terminal where they play a critical role in
neurotransmitter release.
By manipulating various regions of the 1C
subunit, a clearer understanding of how L-type calcium channels are
preferentially linked to CREB and CRE-dependent
transcription is emerging. Dolmetsch et al. (2001) found that
removal of the CaM-binding "IQ motif" from
1C produces L-type calcium channels incapable
of signaling to CREB, demonstrating an inherent prerequisite within the
channel for activation of CRE-dependent transcription. Yet various
calcium channel 1 subunits, not just
1C, contain similar (or identical) IQ motifs
and interact with CaM (Lee et al., 1999; Peterson et al., 1999). Thus,
another mechanism must provide L-type calcium channels with a
"private line" to the nucleus, allowing only these voltage-gated
calcium channels to trigger CREB phosphorylation. We believe that this
second requirement is fulfilled by interactions between
1C and PDZ domain proteins. Anchoring
1C to regions where CREB signaling is
initiated would not only promote the ability of these particular L-type
calcium channels to activate gene expression but also help to exclude
other calcium channels from localizing to these same regions and thus
signaling themselves.
The studies with DHP-LTC and DHP-LTCnoPDZ provide support for this
hypothesis. After a brief and mild stimulation (3 min with 20 mM K+), activation of
DHP-LTCs, but not DHP-LTCnoPDZs, results in the phosphorylation of
CREB (Fig. 7). Interestingly, with longer stimulation conditions (e.g.,
3 hr), L-type calcium channels lacking the PDZ interaction sequence can
partially overcome their disadvantage in signaling to CREB,
perhaps by overwhelming endogenous calcium buffering. Consistent
with this idea, after a prolonged and robust depolarization (resulting
in micromolar increases in intracellular calcium), DHP-LTCs
containing the IQ motif but lacking the final 502 amino acids from its
C terminus (including VSNL) exhibit CREB phosphorylation and
CRE-dependent transcription to a lesser extent than the full-length
channel (Dolmetsch et al., 2001).
In our experiments, we used two distinct strategies in examining
the importance of the 1C PDZ interaction
sequence on L-type calcium channel-mediated CREB responses. With
EGFP-VSNL, we took a freely diffusible protein (EGFP) and promoted its
targeting to the membrane surface (Fig. 1B). As a
result, 10-15 hr after transfection, a clear difference in the
distribution between EGFP and EGFP-VSNL was observed. At later time
points (Fig. 5C), differences between the two fluorophores
became more difficult to resolve. We attribute this phenomenon to
EGFP-VSNL saturation of the PDZ protein binding sites, with excess
EGFP-VSNL distributed similarly to EGFP. When comparing the
localization of DHP-LTC with DHP-LTCnoPDZ, no striking
differences were observed. Both proteins contain 24 transmembrane-spanning regions and the intracellular domains that promote interactions with the calcium channel accessory subunits necessary for channel insertion into the plasma membrane. As such, both
DHP-LTC and DHP-LTCnoPDZ were targeted to the cell soma and dendrites
(Fig. 7B,C). Presumably, at the
subcellular level, differences in their localization exist.
Currently, only two PDZ domain proteins are known to interact with
VSNL: CIPP and NIL-16 (Kurschner et al., 1998; Kurschner and Yuzaki,
1999). CIPP is expressed within the cerebellum; NIL-16 is expressed
within the cerebellum and hippocampus. Thus, our working hypothesis is
that in hippocampal neurons, interactions between L-type calcium
channels and NIL-16 are necessary for efficient signaling to CREB.
However, coimmunoprecipitation experiments examining potential
interactions between 1C and NIL-16 in neurons have thus far been unsuccessful (Kurschner and Yuzaki, 1999; J. P. Weick and P. G. Mermelstein, unpublished observations). This is
most likely attributable to various factors, including the relative low
abundance of NIL-16 in neurons and the poor sensitivity of the NIL-16
antibody (Kurschner and Yuzaki, 1999). Future experiments will need to
overcome these technical limitations to definitively reveal which PDZ
domain protein(s) interacts with L-type calcium channels.
Future research will also need to identify other proteins localized to
these signaling complexes. Because of its multiple roles in L-type
calcium channel function, CaM may be concentrated within these
subcellular regions. Interactions between
1C-comprised L-type calcium channels and CaM
are required for channel inactivation and facilitation (Peterson et
al., 1999; Zühlke et al., 1999, 2000; Erickson et al., 2001; Pitt
et al., 2001). Therefore, by modulating calcium entry through L-type
calcium channels, CaM may indirectly influence CREB phosphorylation.
Furthermore, after brief stimulation, L-type calcium channel-induced
CREB phosphorylation occurs via CaM-dependent protein kinase kinase and
CaM-dependent protein kinase IV (CaMKIV), indicating that CaM plays a
more direct role in CREB activation (Enslen et al., 1995; Tokumitsu et
al., 1995; Bito et al., 1996; Ahn et al., 1999; Mermelstein et al., 2001). A necessary step in this process is the translocation of CaM
from the cytosol into the nucleus (Deisseroth et al., 1998; Mermelstein
et al., 2001; Wei et al., 2002). With longer depolarizations, L-type
calcium channels mediate CREB phosphorylation through MAPK (mitogen-activated protein kinase) (Xing et al., 1996; Impey et al., 1998), a process also requiring CaM (Dolmetsch et al., 2001). Consequently, interactions within a clustered signaling complex, in
which PDZ domain proteins play a critical role, may ensure that L-type
calcium channels have access to sufficient concentrations of CaM to
carry out multiple cellular functions.
Outside the hippocampus, the privileged role of L-type calcium channels
in CREB signaling has been observed in the cerebral cortex, cerebellum,
neostriatum, olfactory bulb, and retina (Murphy et al., 1991; Yoshida
et al., 1995; Liu and Graybiel, 1996; Cigola et al., 1998). Calcium
entry through NMDA receptors also activates CREB via a mechanism that
uses local calcium microdomains (Hardingham et al., 2001, 2002).
Cooperation between NMDA receptors and L-type calcium channels can
occur to enhance CREB signaling (Nakazawa and Murphy, 1999;
Rajadhyaksha et al., 1999), but depending on the stimulus conditions,
activation of a single pathway can also take place (Hardingham et al.,
2002). Our work has focused on studying how mild depolarizations lead
to CREB phosphorylation and CRE-dependent transcription via CaM/CaMKIV
activation, because this is the signaling pathway responsible for CREB
regulation after synaptic activity at multiple stimulus frequencies
(Deisseroth et al., 1996) and is a critical mediator of plasticity
in vivo (Ho et al., 2000; Ribar et al., 2000).
With the expression of EGFP-VSNL, we were able to differentiate CREB
phosphorylation caused by calcium entry through L-type calcium channels
from NMDA receptors. But what is the purpose of having both L-type
calcium channels and NMDA receptors signal to CREB? One possibility is
that along with CREB phosphorylation, each pathway also activates other
signaling molecules specific to that particular cascade, resulting in
differences between L-type calcium channel- and NMDA receptor-mediated
plasticity. For example, calcium entry through L-type calcium channels
activates NFATc4, whereas calcium through NMDA receptors will not
(Graef et al., 1999). Recently, L-type calcium channel-mediated, but
not NMDA receptor-mediated, long-term potentiation and long-term
depression were altered after disruption of the gene encoding
extracellular matrix glycoprotein tenascin-C (Evers et al., 2002).
These data further suggest that activation of L-type calcium channels
and NMDA receptors lead to distinct aspects of plasticity.
In addition to CREB and NFATc4, L-type calcium channels have been shown
to activate the transcription factors serum response factor and myocyte
enhancer factor-2 (Misra et al., 1994; Mao et al., 1999). Because
NFAT-dependent transcription was not affected by EGFP-VSNL,
interactions between PDZ domain proteins and
1C are apparently not required for linking
L-type calcium channels to the second messenger systems that trigger
NFATc4 activation. Thus, multiple mechanisms seem to exist by which
L-type calcium channels signal changes in gene expression. This adds
another layer of cellular complexity to stimulus-induced signaling,
because these separate gene activation pathways could be differentially regulated.
Notably, other discrepancies exist between activation of CREB and
NFATc4 after the opening of L-type calcium channels. For example,
NFATc4 requires dephosphorylation by calcineurin (Rao et al., 1997),
previously shown to limit CREB phosphorylation (Bito et al., 1996).
CREB and NFATc4 also exhibit differential sensitivities to the patterns
of synaptic stimulation used to trigger their responses (P. G. Mermelstein and R. W. Tsien, unpublished observations).
Furthermore, calcium entry through NMDA receptors, but not release from
intracellular stores, will lead to rapid CREB activation, whereas the
opposite is true for NFATc4. What can account for these differences
between CREB and NFATc4 activation? One possibility is that
NFAT-dependent transcription is being regulated by calcium entry
through 1D-comprised L-type calcium channels.
Experiments are under way to address this issue, as well as to
determine whether interactions with PDZ domain proteins are required
for L-type calcium channels to signal to transcription factors other
than CREB.
Understanding how L-type calcium channels regulate different
transcription factors is of significant interest, because their coordinated activities guide the expression of immediate-early genes,
growth factors, signaling, and structural proteins. Furthermore, through changes in gene expression, L-type calcium channels affect cell
fate and axonal and dendritic guidance and influence long-term potentiation and depression (for review, see Mermelstein et al., 2000).
The concept of localized calcium signaling within subcellular microdomains has been suggested as a mechanism of signaling specificity for years. Here we provide some of the first evidence that L-type calcium channels signal to the transcription factor CREB using such a
process. Further examination of this and other signaling pathways
leading to variations in gene expression after L-type calcium channel
activation will play an important role in understanding how cellular
activity leads to long-term changes in cell structure and function.
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FOOTNOTES |
Received Sept. 4, 2002; revised Dec. 31, 2002; accepted Jan. 29, 2003.
This work was supported by National Institutes of Health (NIH) Grant
NS41302 and a Whitehall Foundation grant (P.G.M.). R.D.G. is supported
by NIH Training Grant DA07234. We thank Drs. Linda Boland, Eric
Newman, Harry Orr, and Kevin Wickman for their scientific input; Drs.
Stan Thayer and Yuriy Usachev for their assistance with the photometry
studies; Dr. Chris Gomez, Robert Raike, and Holly Kordasiewicz for
providing COS cells; and Bryan Becklund, Marissa Iden, and Kathryn
Klammer for their technical support. We thank Drs. Michael Greenberg
and Ricardo Dolmetsch for the DHP-LTC construct and Dr. Cornelia
Kurschner for the NIL-16 plasmid.
Correspondence should be addressed to Paul G. Mermelstein, Department
of Neuroscience, University of Minnesota, 6 145 Jackson Hall, 321 Church Street Southeast, Minneapolis, MN 55455. E-mail: pmerm{at}umn.edu.
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TOP
ABSTRACT
Introduction
