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University of Alabama at Birmingham, Department of Physiology and Biophysics, Birmingham, Alabama 35294, USA
2Correspondence: University of Alabama at Birmingham, Department of Physiology and Biophysics, McCallum Building, Rm. 898, 1918 University Blvd., Birmingham, Alabama 35294, USA. E-mail: blalock{at}uab.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: calmodulin • CALP • Fura-2 • cation channel • complementary peptide • antisense peptide
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4)
. Thus,
agents that block Ca2+ entry often inhibit
apoptosis (5
6
7
8
9)
. Although such Ca2+
channel blockers often act on the channel’s pore region or ligand
binding site, a new regulatory site representing a potential target for
a new generation of Ca2+ channel blockers has
recently been described. Specifically, certain
Ca2+ binding motifs, termed EF hands, or binding
sites for proteins with EF hands such as calmodulin (CaM) are
responsible for inactivating Ca2+ channels
(10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29)
. EF hand motifs consist of a helix-loop-helix
with the coordination site for Ca2+ encompassed
by the loop. Consequently, these sites might be exploited to control
apoptosis, which is dependent on Ca2+ conduction
through such channels.
As an approach toward rationally designing molecules that specifically
bind the aforementioned Ca2+ sensors, we have
used the technique of inverted hydropathy (30
, 31)
to
produce a series of peptides, termed calcium-like peptides or CALP
(32
, 33)
. These peptides are thought to interact with
Ca2+ binding motifs as a result of having a
complementary surface contour to a stretch of eight amino acids of the
loop sequence of EF hands (33)
. This study presents the
biological activity of two such peptides, CALP1 and CALP3. In a recent
report we have demonstrated that both of these octameric peptides share
similar biochemical activity in that they are able to bind the EF hands
of CaM. The peptide/CaM complex in turn was able to activate
phosphodiesterase in the absence of Ca2+. These
results demonstrated a specific and productive interaction of CALP with
the desired target protein. This also suggested the possibility that
these peptides might block Ca2+- and
CaM-regulated Ca2+ channels and consequently
inhibit apoptosis, which is dependent on Ca2+
entry.
In this report, we describe the ability of CALP to enter cells and directly as well as indirectly via CaM block glutamate receptor channels and a store-operated nonselective cation channel (NSCC). This inactivation is apparently due to a peptide/EF hand interaction involved in regulating such channels. As a consequence, CALP inhibited apoptosis mediated by glutamate as well as HIV-1 gp120. Collectively, these results demonstrate a new type of Ca2+ channel blocker that may have utility in controlling apoptosis in diseases such as AIDS or neuronal loss due to ischemia.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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for sequences) were synthesized in our laboratory using solid-phase
peptide synthesis with Fmoc chemistry and
Opfp(O-pentafluorophenyl ester) amino acids activated with
HOAt (1-hydroxy-7-azabenzotriazole) on a PerSeptive 9050 Peptide
synthesizer. All reagents for peptide synthesis were purchased from
PerSeptive Biosystems (Framingham, Mass.) unless otherwise mentioned.
Preloaded PEG-PS (polyethylene glycol graft polystyrene) resin was
used, and all the peptides were purified by reverse phase-high
performance liquid chromatography (RP-HPLC) on a Delta Pack C18 300A
30 x 3 id cm column. The purity of the product (always >90%)
was checked by RP-HPLC on a Dynamax C18 (300x4.8 id) column with a
gradient from 0 to 90% acetonitrile and 0.1% trifluoacetic acid in 40
min. The identities of the peptides were confirmed by TOF-MALDI mass
spectrometry (UAB core facility).
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Cyclic-CALP3 (Table 1)
was synthesized starting from
Fmoc-Asp(PEG-PS)-OAl. After amino-terminal Fmoc removal and allyl side
chain deprotection with
Pd[(C6H5)3P]4
(Fluka, Ronkonkoma, N.Y.), the peptide was cyclized on the support
using PyAOP ([7-azabenztriazol-1-yloxytris(pyrrolidino)
phosphonium-hexafluorophosphate) and DIPEA (diisopropylethylamine). The
cyclization was confirmed by mass spectroscopy.
For F-CALP1 (Table 1)
the sequence was extended in the
carboxyl-terminal direction with a cysteine. The side chain sulfur was
selectively functionalized using fluorescein-5-malemide (Pierce,
Rockford, Ill.) at pH 6.5. The final product was purified by RP-HPLC
and confirmed by mass spectroscopy.
Peptides were stored as lyophilized 50 µl aliquots of a 5 mM stock at -20°C, reconstituted in MilliQ water before being diluted in buffer or media at final concentrations shown.
Cell culture
Neocortical cultures were made from the frontal cortex of
Sprague-Dawley rats (Harlan, Indianapolis, Ind.) taken at embryonic day
18. Briefly, after removal of all surrounding tissue and vasculature,
the cortical layer was peeled off and placed in 0.25% trypsin (Gibco
BRL, Gaithersburg, Md.) for 10 min at room temperature. This was
followed by mechanical dissociation using a graded series of
fire-polished Pasteur pipettes. Cells were plated in 96-well culture
plates or on 22 mm glass coverslips that had been coated with 1 mg/ml
poly-L-lysine (Sigma). The media consists of basal medium
Eagle (Gibco-BRL) supplemented with 100 µg/ml
penicillin-streptomycin, 2 mM L-glutamine (Cell-Gro), 100
µg/ml Gentamicin sulfate (Sigma), 5% iron-supplemented bovine
calf serum (Atlanta Biologicals, Atlanta, Ga.), 5% CPSR-3 (Sigma),
and 5 g/l glucose. The media was always warmed to 37°C before adding
to the cells. Cells were fed every 5 days by replacing half of the
supernatant from each culture well with fresh media. Experiments were
conducted between days 8 and 10 in culture.
T cells were isolated from human peripheral blood (American Red Cross, Birmingham, Ala.) first by centrifuging the buffy coat over a layer of Ficoll-Hypaque (using Histopaque 1.077g, Sigma) to remove red blood cells; then white blood cells were passed over a nylon wool column to remove adherent B cells. This procedure yielded a culture that was ~80% T cells as determined by flow cytometry (data not shown).
Both Jurkat E6–1 T cells (American Type Culture Collection, Norcross, Ga.) and T cell-enriched human peripheral blood cells were cultured at 37°C with 5% CO2 in RPMI media with 10% fetal bovine serum (Atlanta Biologicals), 100 µg/ml Penicillin/streptomycin, (1x) nonessential amino acid solution, 2 mM L-glutamine, 1 mM sodium pyruvate (Cell-Gro, Herndon, Va.), 23.8 mM sodium bicarbonate, 10 mM HEPES (Sigma, St. Louis, Mo.). Jurkat cells were diluted 1:10 in fresh media every 2–3 days.
Fluorescent microscopy of F-CALP1-loaded cells
Approximately 50 µM F-CALP1 was added to the culture media and
the cells were incubated at room temperature in the dark for 15 min.
Then Hoechst 33258 (7.5 µM, Sigma) was added and the cells were
incubated for an additional 5 min. This was followed by washing the
cells with normal Ringer’s solution and fixing for 5 min at room
temperature with Bouin’s solution (Sigma). The cells were washed again
twice with Ringer’s solution and mounted on glass slides. Digital
images were acquired using an Olympus IX70 digital confocal microscope
(Lake Success, N.Y.) equipped with a Sensys 1400 CCD camera (Photonics,
Tucson, Ariz.). Image analysis was done using IPLab Spectrum software.
Ca2+ imaging using Fura-2
Neuronal cultures made on 22 mm glass coverslips grown in 6-well
plates were loaded with 2 µM fura 2-acetoxymethlyester (Molecular
Probes, Eugene, Oreg.) in the cell’s media at room temperature for
1 h in the dark. This loading was stopped by washing the cells
three times in normal Ringer’s solution. Cells were allowed to
de-esterify for 15 min at room temperature before they were used.
Coverslips containing loaded cells were placed in a chamber on the
stage of a Nikon Diaphot 200 inverted epifluorescence microscope and
visualized using a 40x oil immersion objective. The dye was
alternately excited at 340 and 380 nm with a single wavelength
monochrometer (Photon Technologies International, South Brunswick,
N.J.) and a ratio was obtained every 4 s. The emitted fluorescence
above 520 nm was captured by video camera (Hamatsu, Hamal Su
City, Japan), then digitized and saved on a personal computer, and
analyzed using Image Master software (Photon Technologies
International). The ratio of the two images (340/380 nm) was
calculated, which is reflective of
[Ca2+]i. Glutamate and
CALP were added as a bolus to the 1 ml chamber from a 10 mM and 5 mM
stock solution, respectively. Ionomycin (10 µM final concentration)
was added at the end of each experiment to check for variability and to
determine whether the limits of saturation of the Fura-2 had been
reached. For each data point, three experiments were done, recording 20
cells from each coverslip. The data are represented as the mean
increase in [Ca2+]i ±
SE.
Electrophysiological studies by patch clamping
For recordings on Jurkat cell, we used a conventional whole-cell
technique. Briefly, Jurkat E6–1 cells were washed twice with the
external solution (see below). The cells were resuspended and allowed
to settle on a glass coverslip for 10 min; the unsettled cells were
removed by washing the coverslip twice with the external solution.
Recording was conducted by using a List EPC-7 patch clamp amplifier
(LIST Medical, Darmstadt-Eberstadt, Germany). Pipettes were
pulled from KG12 capillaries (World Precision Instrument, Inc.,
Sarasota, Fla.), Q-Dope coated and fire-polished to produce a tip
resistance of 3–5 M
in the external solution. Internal solution
contained (in mM): 140 CsGlut, 2 MgCl2, 1
CaCl2, 10 EGTA-Cs, 5 HEPES, pH 7.2. The external
solution contained (in mM): 131 NMDGCl, 10 CaCl2,
2.2 MgCl2, 5.6 D-glucose, 39 HEPES, pH 7.4. A tip
junction potential of +10 mV was not corrected. The capacitative
current was compensated. After whole-cell configuration was achieved,
the cells were held at 0 mV, and a series of voltage steps from -80 to
+80 mV with a 20 mV increment were applied to the cell each minute to
measure the current. Currents were sampled at 2 kHz and filtered at 1
kHz by an 8 pole low-pass Bessel filter (Model 900, Frequency device).
Drugs were added directly into the chamber.
For cell-attached and inside-out recording of NSCC in Jurkat cells, the pipette contained (in mM): 150 NaCl, 11 MgCl2, 10 HEPES, 5 glucose, pH 7.3 (with NaOH). Bath solution contained (in mM): 150 KCl, 1 CaCl2, 2 MgCl2, 10 EGTA·K, 10 HEPES, pH 7.2 (with KOH). Gp120 (8.3 nM) and CALP1 (120 µM) were added to the bath solution as specified. Holding potential was -80 mV, and the signal was digitized at 20 kHz and recorded by a Sony VCR on a magnetic tape. For analysis and display, the data were resampled at 10 kHz and filtered at 300 Hz by an 8 pole low-pass Bessel filter.
We used a perforated whole-cell technique to record from neurons. The
external solution contained (in mM):162 NaCl, 2.4 KCl, 1.3
CaCl2, 10 HEPES, 10 D-glucose, 0.01 glycine (pH
7.4) adjusted with NaOH. Tetrodotoxin (0.5 µM), picrotoxin(50 µM),
and strychnine(2 µM) were added to block voltage-gated
Na+ channels, GABA receptor, and glycine
receptors, respectively. The pipette solution contained (in mM): 55
CsCl, 70 Cs2SO4, 7
MgCl2, 1 CaCl2, 5
D-glucose, 10 HEPES, pH 7.2, adjusted with CsOH. A final concentration
of 300 µM amphotericin-B from freshly prepared stock (60 mg/ml in
DMSO) was added to the pipette solution. Whole-cell configuration could
be achieved within 10 min. Cells were used only if they had series
resistance of less than 20M. After whole-cell configuration was
achieved, the capacitative currents were compensated and neurons were
held at -80 mV. Series resistance compensation was not used. The drugs
were delivered to the chamber by a gravity-feeding perfusion system.
The solution change was achieved within 1 s as measured by
blockage of glutamate current by 200 µM CNQX and 40 µM AP-5. The
signals were digitized at 20 kHz and recorded by a VCR (Sony, Tokyo,
Japan) on a magnetic tape. For presentation, the signal was resampled
at 50 Hz and analyzed using PCLAMP software (Axon Co., Foster
City, Calif.).
For cell-attached recordings of the neurons, the pipettes were
fabricated the same way as in the whole-cell recording, but with a tip
resistance of 1–3 M in the same solution. Neurons were bathed in
the same external solution as in perforated whole-cell recording. The
pipette contained the same external solution with 25 µM glutamate
added. Holding potential was 0mv. The other recording conditions were
the same as in perforated whole-cell recording. For display and
analysis, the signal was resampled at 5 kHz and filtered at 100 Hz by
an 8 pole low-pass Bessel filter.
Neurotoxicity studies
Apoptosis was induced in neuronal cultures by stimulating the
cells either with glutamate (75 µM) or a combination of gp120 (100
pM) and glutamate (25 µM). This involved washing the cells twice with
a salt solution consisting of (in mM): 120 NaCl, 5.4 KCl, 10
CaCl2, 25 Tris·HCl, 15 glucose, adjusted to pH
7.4 with NaOH, followed by a 15 min incubation of the cells with the
gp120 and glutamate with or without CALP or control compounds at the
specific concentrations. After stimulation, the cells were washed twice
more in the control salt solution; fresh media warmed to 37°C was
replaced and the cells were returned to the 37°C incubator for 20–24
h.
Cell death was determined in these experiments by using fluorescein
diacetate and propidium iodide staining (34
35
36)
. Cultures
were washed twice with Hanks’ balanced salt solution (HBSS), then
incubated with fluorescein diacetate (4 µM) and propidium iodide (36
µM) in HBSS for 3 min at room temperature. This was followed by a
quick wash with HBSS and the cells were then viewed using a Nikon
Diaphot 200 inverted epifluorescence microscope (Japan) with a
20x objective. An average of five microscope fields containing ~75
cells per field were counted from each well. Each condition was
repeated in at least three different wells; thus, the total cells
counted for each data point is ~1100 cells. The graph shows the mean
of these triplicates ± SE.
To specifically measure apoptosis in these cells, the TUNEL method was
used (37)
. First the cells were briefly fixed with
Bouin’s solution (Sigma) and permeabilized with phosphate-buffered
saline (PBS)/1% Tween 20. The TUNEL method involves labeling the
nicked DNA (a characteristic of apoptotic cells) with biotin-16-dUTP
and subsequently with alkaline phosphatase (Boehringer Mannheim,
Indianapolis, Ind.), and developed using Fast Red (Sigma). The cells
were then viewed on a light microscope (Zeiss) using a 20x objective
and counted as for the FDA/PI experiments. Similar numbers of cells
were counted as for the FDA/PI experiment and are represented as
mean ± SE.
Lymphocytotoxicity studies
Apoptosis was induced in ~3 x 105
peripheral blood mononuclear cells by the addition of 5 µg/ml native
gp120 (Advanced Biotechnologies Inc., Columbia, Md.), 3 µg/ml
superantigen mixture (consisting of 1 µg/ml of each Staphylococcal
enterotoxin A and B, and toxic shock syndrome toxin-1; Sigma) or 10
µM dexamethasone in the RPMI media. CALP (100 µM) was added in the
specified conditions. After 24 h of incubation at 37°C, the
cells were washed with PBSAz (PBS containing 2% fetal calf serum and
0.01% sodium azide). Cells were then pelleted and resuspended in 20
µg/ml of 7-amino actinomycin D (7AAD; Sigma) in PBSAz and incubated
at 4°C for 20 min. The cells analyzed by flow cytometry on a Becton
Dickinson FACSCalibur (Mountain View, Calif.). Each condition was done
in triplicate and graphed as mean ± SE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was conjugated to fluorescein malemide so that it
could be visualized by fluorescent microscopy. F-CALP rapidly entered
rat neocortical neuronal cultures (Fig. 1A, B
) or Jurkat T cells (Fig. 1C
), resulting in
staining throughout the cytoplasm. A hydrophilic control peptide of the
same size and conjugated to fluorescein malemide (see F-MBP in Table 1
)
did not enter the cells (Fig. 1D
). This demonstrates that
the fluorescein malemide is not responsible for uptake of the CALP
peptide by the cells.
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Further evidence that the CALP peptides can enter the cells comes from several electrophysiological studies discussed below. The most striking of these come from recordings done in the cell-attached configuration, in which the channels being recorded are separated from the bath by the giga ohm seal of the pipette. Addition of CALP to the extracellular bath results in these channels being blocked, which demonstrates the ability of the peptide to enter the cell and diffuse to the site of action.
CALP is a Ca2+ channel blocker
The first approach we took to examine whether CALP could
inhibit Ca2+ entry used the ratiometric
Ca2+ indicator dye, Fura-2. Cultured rat
neocortical neurons loaded with this dye were exposed to glutamate
(75 µm) either in the presence or absence of CALP or controls
(Fig. 2
), and the resulting increase in the cytoplasmic free
Ca2+ concentration
([Ca2+]i) was measured.
Given as a bolus, glutamate caused a large sustained increase in
[Ca2+]i, (Fig. 2A
). This was inhibited by CALP1 and CALP3 in a
dose-dependent manner (Fig. 2C
;
IC50=54.43±2.767 µM and 37.25±2.585 µM,
respectively). Cyclizing CALP3 abolishes its ability to interact with
EF hands of CaM (33)
. Consequently, cyclic CALP3 was
unable to inhibit Ca2+ influx, and this peptide
served as a negative control. Since the majority of
Ca2+ channels activated by glutamate in these
cells are of the N-methyl-D-aspartate (NMDA) type, the specific
antagonist to these channels, MK801, was an effective positive control
(IC50=51.26±6.368 µM) (38)
. The
ability of the CaM antagonist
N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) to abolish
the inhibitory effect of CALP and restore a large measure of the
response of these cells to glutamate(Fig. 2D
) demonstrates
that the mode of action of these peptides is different from MK801 and
involves CaM as an intermediary.
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To study the actual channel activity involved in this process, we used
patch clamping techniques. Figure 3
shows a recording done in the perforated whole-cell configuration of
rat neocortical neurons. When glutamate (25 µM) is applied to the
bath, a large inward conductance is activated. Subsequent application
of 100 µM CALP1 to the chamber almost completely blocked the current.
However, CALP1 had little effect on glutamate-induced current in
conventional whole-cell configuration (data not shown). This suggests
that the inhibition of glutamate-induced current by CALP1 is dependent
on a diffusible factor, such as CaM, which was washed out rapidly
during conventional whole-cell recording. To confirm that CALP1 acted
on glutamate-induced currents by interacting with a binding site in the
cytoplasm, we recorded from these cells in the cell-attached mode. In
this instance, glutamate (25 µM) was placed in the pipette solution,
activating multiple channels in this patch (Fig. 4
). Each of these opened channels had a single-channel conductance of
50–70 pS, suggesting it was predominantly carried by the NMDA
receptor. Application of 100 µM CALP1 to the bath reversibly blocked
the NMDA currents (Fig. 4)
. CALP1 blockage of the NMDA current in the
cell-attached mode demonstrates that the peptide was able to gain
access to the interior of the cell. Furthermore, the reversibility of
the glutamate-induced current after the CALP was washed from the bath
solution demonstrates that the peptide did not accumulate in the
pipette. Therefore, we concluded that CALP1 blocked the NMDA receptor
by acting on the cytoplasmic side. Single-channel analysis of the
inhibition caused by CALP1 revealed that the effect was partially due
to a decrease in the single-channel conductance (Fig. 4C
) as
well as a decrease in the open probability (Fig. 4D
).
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It has been demonstrated that the HIV-1 envelope glycoprotein, gp120,
can cause a Ca2+ influx in
CD4+ T cells due to an interaction with the
chemokine receptor, CCR5 (39)
. We found that gp120 can
activate a nonselective cation channel (INSCC) in
Jurkat cells, which is permeable to Ca2+. We
tested the effects of CALP1 on this channel. As shown in Fig. 5
, addition of 8.3 nM gp120 activated a large, outwardly rectified
conductance with a reversal potential of around 30 mV, indicating it
was carried by a nonselective cation channel. Addition of 40 µM CALP1
blocked 60% of the current (Fig. 5C
). This blockage is also
demonstrated on isolated patch recordings in the inside-out
configuration, demonstrating a direct effect of the peptide on the
channel (Fig. 6
). In contrast to the effect of the peptide on glutamate receptor
channels, single-channel analysis reveals that the inhibitory effect of
CALP1 on INSCC is due to the decrease of the open
probability, but not the single-channel conductance (Fig. 6C
). Furthermore, at equivalent concentrations of CALP, the
blockage of these channels was greater on recordings done in the
inside-out configuration (Fig. 6)
than outside-out (data not shown).
This provides further evidence that CALP is interacting with a target
on the cytoplasmic face of the channel.
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CALP inhibits apoptosis
Glutamate is the major excitatory amino acid used in the central
nervous system, but if the concentration becomes too high (for example,
in ischemic conditions) it can result in significant cytotoxicity
(5
, 6
, 40)
. This provided an excellent model for studying
the possible anti-apoptotic effects of CALP, since this process of cell
death is dependent on the sustained increase in
[Ca2+ ]i due to the
opening of glutamate receptor channels. Cultured rat neocortical
neurons were exposed to glutamate (75 µM) either in the presence or
absence of CALP or control substances for 15 min, then washed with
fresh media and incubated overnight. Cytotoxicity (Fig. 7A
) was measured by staining the cells with fluorescein
diacetate, which live cells take up and cleave resulting in green
fluorescence, and propidium iodide, which dead cells take up resulting
in red fluorescence (34
35
36)
. CALP1 and CALP3 were able to
inhibit this cytotoxicity in a dose-dependent manner
(IC50= 52.48±1.651 µM,
IC50 =50.97±1.583 µM, respectively), as was
MK801. Apoptosis (Fig. 7B
) was also measured in this system
by using the TUNEL method (37)
to stain nuclei of the
cells with cleaved DNA, which is characteristic of apoptosis. CALP’s
dose-dependent inhibition of apoptosis in this system (CALP1
IC50=44.78±0 µM, CALP3
IC50 =33.41±1.099 µM) was similar not only to
the cytotoxicity measurements, but also to the Fura-2 imaging studies.
This suggests that the inhibition of apoptosis is mainly due to the
inhibition of the influx of Ca2+. This ability of
Ca2+ channel blockers to inhibit cytotoxicity has
been shown previously and is further supported by the action of MK801
in this system. Once again, cyclic CALP3 did not inhibit the effect of
glutamate.
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In another system, we studied the anti-apoptotic effects of CALP in a
well-characterized model of HIV neurotoxicity in cultured rat
neocortical cells. The HIV-1 envelope glycoprotein gp120 (100 pM), in
addition to a relatively small amount of glutamate (25 µM), induces a
rapid apoptotic effect that is known to be dependent on the influx of
Ca2+ through glutamate receptor channels
(7
8
9
, 41
42
43
44
45)
. In contrast to the glutamate
excitotoxicity model described above, the combination of these two
compounds results in cytotoxicity that is mostly due to apoptosis. As
shown in Table 2
, CALP1 (30 µM) was as effective at inhibiting apoptosis in this
system as the combination of NMDA-R and non-NMDA-R blockers (10 µM
MK801 and 100 µM DNQX, respectively) or even the omission of
Ca2+ from the extracellular media during
stimulation. Other positive controls known to inhibit this type of
apoptosis are the L-type Ca2+ channel blocker
nifedipine (100 µM), as well as an agent, dantrolene (30 µM), which
is known to inhibit the release of Ca2+ from
internal stores (40)
.
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HIV causes apoptosis in uninfected CD4+ T cells
by an as yet unknown mechanism. It is known, however, that the
engagement of the CD4 receptor by gp120, followed by stimulation of the
T cell receptor (TCR) complex, can lead to apoptosis of these cells
(46
47
48
49
50)
. Therefore, we induced apoptosis in T
cell-enriched human peripheral blood lymphocytes using a system whereby
the cells are exposed to gp120 prior to superantigen stimulation of the
TCR. After 24 h incubation, the cells were stained with 7AAD and
analyzed by flow cytometry. Loss of membrane integrity leading to the
uptake of this dye, together with a decrease in cell size, are the two
characteristics of apoptosis measured by this procedure
(51)
. As Fig. 8
shows, gp120 and a superantigen mixture have a synergistic effect on
these cells, resulting in a much larger apoptotic signal than the
combined effect of each reagent separately. However, if either CALP1 or
CALP3 (100 µM) is present beforehand, then this signal and the
resulting apoptosis can be inhibited. One again, cyclic CALP3 did not
inhibit this cytotoxic process at all. Not all apoptosis is dependent
on an initial influx of Ca2+. One example of this
is induced by dexamethasone. This compound causes a signal leading to
apoptosis by direct interaction with the glucocorticoid receptor
leading to DNase I susceptibility (52
53
54)
. The fact that
CALP did not inhibit apoptosis induced by dexamethasone demonstrates
that the peptide is acting as an anti-apoptotic agent through its
channel blocking activity, and not some downstream effector.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20
21
22
23
24
25)
. Although the NSCC channel mentioned above
has not been cloned, we have observed a negative feedback regulated by
Ca2+ (unpublished observations); however, more
work needs to be done to elucidate the exact mechanism by which this
occurs.
We have previously demonstrated that CALP1 and CALP3 mimic the effect
of Ca2+ through their specific interaction with
EF hands (32
, 33)
. We have shown that they bind to CaM in
the carboxyl-terminal Ca2+ binding sites, and
this interaction results in a conformational change whereby the
CALP/CaM complex can activate phosphodiesterase in the absence of
Ca2+. In this study we have extended our
investigations to show the biological relevance of designing such
peptides. Specifically, these peptides represent a new class of
Ca2+ channel blocker that exert their action not
on the pore regions of the channel or the ligand binding sites of
certain receptor channels, but rather on the Ca2+
sensing mechanisms. Depending on the channel, this inhibitory effect
can be mediated through a direct interaction of the peptide with the
cytoplasmic face of the channel, as demonstrated with the NSCC, or it
can be indirect—mediated through CaM, as demonstrated with the
glutamate receptor channels. The different mechanisms of interaction of
the peptide with the channels were also demonstrated by the distinct
changes produced in the single-channel properties.
As a consequence of the channel blocking ability of these peptides, apoptosis mediated by an influx of Ca2+ through these channels was also inhibited. This inhibition was demonstrated to be specific, since neither CALP1 nor CALP3 were able to inhibit dexamethasone-induced apoptosis. Given the importance of apoptosis mediated by a Ca2+ influx in many disease processes, some of which have been modeled here, these peptides could be useful lead products for the design of therapeutic agents to interfere with such cytotoxicity.
Furthermore, since Ca2+ plays a role in other
signaling processes apart from the induction of apoptosis, CALP may
also be useful in their modulation. We have observed the effect of CALP
on the contraction of urinary smooth muscle (32)
and
canine heart muscle (our unpublished observation). Another
Ca2+-mediated process is airway
hyperresponsiveness, which can be modeled as the constriction of
isolated guinea pig trachea perfused with acetylcholine or histamine.
Nitric oxide (NO) is normally produced by the epithelia surrounding the
lumen of the trachea in a Ca2+-dependent manner,
which acts as an important feedback mechanism against
bronchoconstriction induced by histamine or cholinergic receptor
agonists. We have observed the modulation of the histamine or
acetylcholine-induced constriction of isolated guinea pig trachea by
CALP (unpublished results). This demonstrates the ability of these
peptides to modulate Ca2+-mediated signaling
pathways apart from those involved in a cytotoxic response.
In a more general sense, by using the design principles for CALP, it
may be possible to produce peptides to interact with other ion channels
in a more specific and targeted manner. With the continuing elucidation
of channel structure and function, from pore regions to gating
mechanisms and other regulatory domains, come further opportunities for
such design. We have also demonstrated with the CALP series the ability
to design peptides to interact with the same region on CaM with
opposing action (33)
. This suggests that it may be
possible not only to regulate distinct types of ion channels, but to
regulate them in opposing ways. Certainly this could lead to many new
and specific channel agonists and antagonists that would have great
benefit in a vast number of disease processes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication November 18, 1999. Accepted for publication February 4, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1C calcium channels. Biophys. J. 76,A340abstr.
-actinin with the NR1 subunit modulate Ca2+-dependent inactivation of NMDA receptors. J. Neurosci. 19,1165-1178-actinin and calmodulin to the NMDA receptor. Nature (London) 385,439-442[Medline]
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), and MK801 (
), but
cyclic CALP3 (
) did not inhibit this response at all.
D) Exposure to W-7 (5 µM) prior to CALP3 (100 µM)
restores the responsiveness of these cells to glutamate (75 µM).
)
inhibited the toxic effects of glutamate in these cultures as did MK801
(gray filled triangle), whereas cyclic CALP3 (gray filled inverted
triangle) had no effect. A) Cell death was measured by
staining cells with fluorescein diacetate, which is taken up by live
cells and cleaved, resulting in green fluorescence, and propidium
iodide, which is taken up by dead cells and makes them fluorescent red.
B) Apoptosis was specifically measured by staining the
cells using the TUNEL method.