From the Departments of Pharmacology and Toxicology and
§ Medical Physics and Biophysics, Karl-Franzens-University
Graz, A-8010 Graz and the Department of Biophysics,
Johannes-Kepler-University Linz, A-4040 Linz, Austria
Received for publication, September 8, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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Modulation of smooth muscle, L-type
Ca2+ channels (class C, CaV1.2b) by
thionitrite S-nitrosoglutathione (GSNO) was investigated in
the human embryonic kidney 293 expression system at the level of
whole-cell and single-channel currents. Extracellular administration of
GSNO (2 mM) rapidly reduced whole-cell Ba2+
currents through channels derived either by expression of The identification of nitric oxide
(NO)1 as the major
endothelium-derived relaxing factor led to the discovery of a variety of NO-mediated signal transduction mechanisms (1-4). NO-mediated control of vascular functions involves the modulation of various ion
transport systems including the high voltage-activated L-type Ca2+ channels (5-7). Both increases (5) and decreases (6,
7) of L-type Ca2+ current in response to NO donors have
been reported. Cellular regulation of Ca2+ current was
suggested to depend at least in part on NO-induced activation of
guanylyl cyclase and subsequent modification of the phosphorylated
state of channel proteins (8-11). However, S-nitrosothiols
(thionitrites) were recently demonstrated to exert a potent inhibitory
effect on class C, L-type Ca2+ channels, which was
suggested to be independent of intracellular cGMP accumulation (12).
Thus, a more direct modulation of L-type Ca2+ channels by
NO donors appears likely. So far it is unclear whether the inhibition
of Ca2+ channels by thionitrites requires the release of NO
and how single-channel properties are affected by this mechanism of
regulation. Modulation of Ca2+ channels by the
S-nitrosothiol nitrosoglutathione (GSNO) appears to be of
particular physiological interest, because GSNO is the most abundant
endogenous thionitrite and has been suggested as a potential NO storage
site or transport species (11, 13, 14). GSNO decomposes slowly
to generate NO, a reaction which is catalyzed by traces of metal ions
(14-17). Alternatively GSNO is able to modify protein thiol residues
via nitrosation or glutathionylation reactions. In the present study we
investigated whether Ca2+ channel inhibition by GSNO
involves (i) decomposition of the thionitrite to yield NO, (ii)
transnitrosation reactions, or (iii) the formation of mixed disulfides.
Moreover, the changes in single Ca2+ channel function
associated with GSNO-induced inhibition were characterized. Our results
strongly suggest intracellular S-nitrosation as a mechanism
involved in physiological control of Ca2+ channel function
and demonstrate a unique modification of Ca2+ channel
function by GSNO, involving joint alteration of gating and permeability
of the pore-forming Cells--
Electrophysiology--
Whole-cell membrane currents were
recorded with a bath solution containing (in mM) 40 BaCl2, 60 NaCl, 30 tetraethylammoniumchloride, 15 HEPES,
0.5 EDTA, pH adjusted to 7.4 with methane sulfonic acid. The pipette
solution contained (in mM) 115 cesium methanesulfonate, 20 CsCl, 15 HEPES, 10 EGTA, pH 7.4 adjusted with
N-methyl-D-glucamine.
For single-channel cell-attached recordings, the whole-cell bath
solution was used as pipette solution. In experiments with single
Analysis of channel gating (open probability
(Po) and availability (Ps)) was
performed with custom-made software (20). Statistical analysis was
performed using Student's t test considering a level of
p < 0.05 as significant. For analysis of effects on
whole-cell currents, the slow run-down of Ba2+ current was
accounted for by averaging the values immediately before administration
and after washout of drugs to obtain the reference (control) value.
Measurement of cGMP Accumulation--
HEK293 stably expressing
Solutions and Compounds--
GSNO, as well as 8-Br-cGMP, was
dissolved freshly in the bath solution. For administration of SIN-1 and
DEA/NO, 100 mM stock solutions were prepared in water or 10 mM NaOH, respectively. Neocuproine (2, 9-dimethyl-1,10-phenanthroline) was directly dissolved in the pipette
solution. Diethylenetriamine-pentaacetic acid (DTPA) was
dissolved in 10 mM NaOH and further diluted in pipette
solution. All NO donors were obtained from Alexis (San Diego, CA), and
all other compounds were from Sigma.
Inhibition of
Fig. 3 summarizes the effects of GSNO,
DEA/NO, and SIN-1 on Ba2+ currents through Inhibition of Inhibitory Modulation by GSNO of Class C, L-type Ca2+
Channels Is Associated with a Reduction of Open Probability and the
Occurrence of a Reduced Conductance State--
Inhibition by GSNO of
The results of the present study suggest that nitrosothiols such
as GSNO affect central functions of the pore-forming Molecular Basis of L-type Ca2+ Channel Modification by
GSNO--
Three potential NO donors (GSNO, DEA/NO, and SIN-1) were
compared for their effects on whole-cell Ba2+ currents in
HEK293 cells stably transfected with the smooth muscle Ca2+
channel
It is tempting to speculate about the modification of a critical
cysteine residue of the Ca2+ channel Protein Thiol Nitrosation as a Unique Regulatory Mechanism That
Affects both Gating and Conductance of L-type Ca2+
Channels--
Our analysis of the inhibitory effect of GSNO on
single-channel parameters revealed that the reduction of whole-cell
currents was mainly because of suppression of single-channel open
probability. In addition we observed a slightly reduced channel
availability and number of active channels in the patches already
within 5-10 min of exposure to GSNO, and channel activity was
completely lost during prolonged (>10 min) exposure. A detailed
inspection of the single-channel behavior during the development of
GSNO inhibition uncovered a striking phenomenon. A defined and
reproducible subconductance level of about 50% of the full unitary
conductance was recorded in about 30% of the experiments. This
conductance state was observed only transiently during the development
of the GSNO-induced inhibition of channel activity. Thus we report here
on a subconductance state of
In summary, we provide evidence for inhibitory modulation by GSNO of
the principle pore-forming subunit of smooth muscle L-type Ca2+ channels by intracellular S-nitrosation.
GSNO-mediated regulation of Ca2+ channels involves unique
changes in the functional properties of the pore-forming 1C-b or by coexpression of
1C-b plus
2a and
2-
. The
non-thiol nitric oxide (NO) donors
2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which
elevated cellular cGMP levels to a similar extent as GSNO,
failed to affect Ba2+ currents significantly. Intracellular
administration of copper ions, which promote decomposition of the
thionitrite, antagonized its inhibitory effect, and loading of cells
with high concentrations of dithiothreitol (2 mM) prevented
the effect of GSNO on
1C-b channels. Intracellular loading of cells
with oxidized glutathione (2 mM) affected neither
1C-b
channel function nor their modulation by GSNO. Analysis of
single-channel behavior revealed that GSNO inhibited Ca2+
channels mainly by reducing open probability. The development of
GSNO-induced inhibition was associated with the transient occurrence of
a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the
1 subunit of smooth muscle L-type
Ca2+ channels by an intracellular mechanism that is
independent of NO release and stimulation of guanylyl cyclase. We
suggest S-nitrosation of intracellularly located sulfhydryl
groups as an important determinant of Ca2+ channel gating
and conductance.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1C subunit.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1C-b
channels2 were stably
expressed in HEK293 cells (18). In some sets of experiments, cells
were transfected to express
2a and
2-
subunits3,4,
in addition to the pore-forming subunit. Positively transfected cells
were identified because of the expression of the human T-cytotoxic cell
cluster of differentiation (CD8) as a marker gene (19). The cells were
cultured at 37 °C in Dulbecco's modified Eagle's medium + 10%
fetal calf serum and 0.25 mg/ml G418 (Sigma).
1C-b channels, the pipette solution was supplemented with S-Bay-K
8644 (1 µM) to increase open times to a level that allows reasonable analysis of conductance and gating. The bath solution in
these experiments contained (in mM) 110 potassium
aspartate, 50 Tris, 10 KCl, 3 EGTA, and 2 MgCl2 at a pH of
7.4 adjusted with N-methyl-D-glucamine. Pipettes
(2-5 megohm) were pulled from borosilicate glass (Clark Electromedical
Instruments, Pangbourne, United Kingdom) and were coated with
Sigmacoat (Sigma) for single-channel recordings. Experiments were performed in a 200 µl bath chamber with a
gravity-driven perfusion system. Currents were recorded at room
temperature using an EPC-7 patch clamp amplifier (List, Darmstadt,
Germany). Signals were low pass-filtered at 1 kHz and digitized with 5 kHz. Voltage clamp protocols (depolarizing pulses from
70 to +30 mV
or voltage ramps from
100 to +80 mV/0.6 V/s; 0.2 Hz) were controlled
by pClamp software using a Digidata 1200 computer interface (Axon Instruments, Foster City, CA).
1C-b channel protein were subcultured on 24-well plates and
incubated for 15 min at 37 °C in a buffer containing (in
mM) 110 potassium aspartate, 50 Tris, 10 KCl, 3 EGTA, and 2 MgCl2 at a pH of 7.4 adjusted with
N-methyl-D-glucamine. Cells were incubated in
the presence of 1 mM 3-isobutyl-1-methylxanthine with NO
donors (GSNO, SIN-1, or DEA/NO) for 4 min, and the incubation was
stopped by removal of medium and addition of 0.01 M HCl.
cGMP was measured in the samples by radioimmunoassay (3).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1C-b Currents by GSNO Is Independent of NO Release
and Involves an Intracellular Site of Action--
Fig.
1 compares the effects of GSNO, DEA/NO,
and SIN-1 on whole-cell Ba2+ currents through
1C-b
channels. Extracellular administration of GSNO (2 mM)
rapidly inhibited the inward Ba2+ currents as illustrated
in Fig. 1, A and B. The current to voltage relation was not affected by GSNO (not shown). GSNO was tested at
concentrations between 50 µM and 2 mM, which
inhibited
1C-b currents in the range of 13 ± 5% (50 µM; n = 3) to 38 ± 6% (2 mM; n = 17). Neither DEA/NO nor SIN-1
mimicked the action of GSNO. DEA/NO (2 mM) moderately
promoted the Ba2+ current run-down (Fig. 1C),
and SIN-1 (2 mM) failed to affect
1C-b currents (Fig.
1D). Similarly, channels comprised of
1C-b,
2a, and
2-
, corresponding to channel complexes expressed in cardiovascular tissues (21, 22), were rapidly inhibited by GSNO
(n = 5; see Fig. 2) but
barely affected by DEA/NO or SIN-1 (data not shown).
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Fig. 1.
GSNO inhibits Ba2+ currents
through 1C-b channels whereas the NO-donors
DEA/NO and SIN-1 are without effect. A, C,
and D, time courses of peak Ba2+ inward currents
recorded at 30 mV. Exposure to the NO donors GSNO (2 mM;
A), DEA/NO (2 mM; C), and SIN-1 (2 mM; D) is indicated. B, individual
current traces taken from the experiment shown in A.
Ba2+ inward currents were recorded during depolarizing
voltage steps from
70 to 30 mV before (1) and after (2) extracellular
administration of GSNO.
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Fig. 2.
GSNO inhibits L-type Ca2+
channels comprised of 1C-b and the auxiliary
subunits
2a and
2-
. Left,
time course of peak Ba2+ inward current recorded at
30 mV. GSNO induces a partial inhibition of L-type current in
cells expressing
1C-b*
2a*
2-
. Right, individual
current traces taken from the experiment shown on the left.
Ba2+ inward currents were recorded during
depolarizing voltage steps from
70 to 30 mV before (1) and after (2)
extracellular administration of GSNO.
1C-b channels
and shows corresponding cellular cGMP levels. Only GSNO exerted a
significant inhibitory action on Ba2+ currents (Fig.
3A) through
1C-b channels, whereas all three compounds
elevated cellular cGMP levels to a similar extent (about 40-fold; see
Fig. 2B). These measurements of cGMP levels were performed
in the presence of the phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine (1 mM). When
3-isobutyl-1-methylxanthine was omitted, significant rises in cellular
cGMP were detectable only with DEA/NO (1.3-fold) but not with GSNO or
SIN-1.5 Thus the effect of
the NO donors were not correlated with cGMP accumulation. To test
whether intracellular decomposition of GSNO to yield NO was required
for the observed Ca2+ channel modulation, the action of
GSNO was studied under conditions that promote decomposition of the
thionitrite within the cell. Copper ions are well known to effectively
promote homolytic cleavage of GSNO and were therefore considered as a
suitable tool to deliberately increase the decomposition rate. Because
reduced metal ions, in particular Cu+, were reported to
catalyze decomposition of GSNO (15, 23, 24), both CuSO4 (50 µM) and the reductant DTT (500 µM) were included in the pipette solution. If decomposition of GSNO within the
cell was essential to Ca2+ channel inhibition, loading of
the cells with copper ions would be expected to enhance inhibition,
otherwise this treatment should suppress the action of the thionitrite.
Fig. 4A shows that the effects
of GSNO (2 mM) on whole-cell Ba2+ currents were
completely abolished in cells that were loaded with a pipette solution
containing 50 µM CuSO4 plus DTT (500 µM). To confirm that this inhibition is dependent on
metal ions we performed experiments using the metal chelators
neocuproine (50 µM) plus DTPA (50 µM) to
remove catalytically active metal ions, in particular Cu+.
Addition of neocuproine plus DTPA recovered the inhibitory effects of
GSNO to the extent observed in presence of DTT (500 µM)
alone (Fig. 4, B and C). Thus the intracellular
presence of metal ions significantly antagonized the inhibitory action
of GSNO. Nonetheless, the inhibitory effects of GSNO observed in the
presence of DTT (500 µM) plus metal chelators or in the
presence of DTT (500 µM) alone were significantly blunted
as compared with control (see Figs. 1 and 2), indicating that DTT by
itself interferes with the action of the thionitrite.
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Fig. 3.
Inhibition of whole-cell Ba2+
currents by GSNO is not mediated by cGMP. A,
Ba2+ current inhibition by GSNO (2 mM;
n = 17), DEA/NO (2 mM; n = 7), and SIN-1 (2 mM; n = 5). The
asterisk indicates significant difference versus
control (reference value). B, intracellular cGMP levels in
the absence of NO donors (control) and in the presence of GSNO (2 mM; n = 3), DEA/NO (2 mM;
n = 3), and SIN-1 (2 mM; n = 3).
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Fig. 4.
Inhibition of whole-cell Ba2+
currents by GSNO is antagonized by intracellular administration of
copper ions. Time courses of peak Ba2+ current
recorded at 30 mV in the presence of 50 µM
CuSO4/500 µM DTT (A) and in the
presence of 50 µM CuSO4/500 µM
DTT plus the metal chelators neocuproine (1 mM) and DTPA
(50 µM) (B) in the pipette solution are shown.
Extracellular application of 2 mM GSNO is indicated. The
inset shows current recordings during single voltage steps
before and after inhibition by GSNO. Scaling bars, 200 pA/200 ms. C, mean values ± S.E. of GSNO-induced
current inhibition in the absence or presence of CuSO4 and
the metal chelators neocuproine plus DTPA (MC) in DTT
containing pipette solution are given (n = 6-9). The
asterisk indicates a statistically significant
difference.
1C-b Currents by GSNO Involves Nitrosation of
Critical Thiol Residues--
An excess of DTT is expected to compete
with cellular thiols in terms of transnitrosation or oxidation and is
therefore expected to inhibit thionitrite effects that are based on
these reactions. Indeed, GSNO-induced inhibition of whole-cell
Ba2+ currents in cells dialyzed with 500 µM
DTT plus metal chelators was clearly less pronounced (15 ± 4%;
n = 6) as compared with control conditions (38 ± 6%; n = 17). Therefore we tested whether loading of
the cells with high concentrations of DTT (2 mM) by itself
is sufficient to prevent the action of GSNO. Fig.
5 illustrates that GSNO barely inhibits
1C-b currents in cells that were dialyzed with 2 mM DTT
(n = 6). Prevention of GSNO effects by excess of DTT
was not dependent on metal ions as metal chelators did not recover
inhibitory effects of GSNO in the presence of 2 mM DTT. Inhibition of GSNO effects by intracellular DTT suggests an
intracellular transnitrosation or glutathionylation reaction as the
chemical basis of GSNO effects. To test whether oxidation of thiols
and/or the formation of mixed disulfides can account for the effects of
GSNO, we performed experiments in which the cells were loaded with 2 mM oxidized glutathione (GSSG). As shown in Fig.
5B, dialysis with GSSG failed to suppress
1C-b currents
(n = 3) and did not affect the inhibitory action of
GSNO. Thus GSNO-induced modulation of
1C-b channels is suggested to
be based on the nitrosation of critical thiol residues.
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Fig. 5.
Inhibition of whole-cell Ba2+
currents by GSNO is antagonized by intracellular administration of DTT
but not affected by GSSG. Time courses of peak Ba2+
current recorded at 30 mV in the presence of 2 mM DTT
(A) and in the presence of 2 mM GSSG
(B) in the pipette solution are shown. Extracellular
application of 2 mM GSNO is indicated.
C, mean values ± S.E. of GSNO-induced current
inhibition in the absence (n = 17) and presence of DTT
(n = 6). The asterisk indicates a
statistically significant difference. MC, metal
chelators neocuproine plus DTPA.
1C-b channels was confirmed in single-channel recordings, using the
cell-attached configuration (Fig. 6).
Note that in this recording configuration, access of bath-applied GSNO
to the channels in the patch requires transport across the cell
membrane. Overall channel activity in cell-attached patches (mean patch
current) decreased to values between 40 and 10% of control within a 2- to 10-min exposure to GSNO (2 mM), respectively. Prolonged
exposure to GSNO resulted in complete loss of channel activity in most
experiments. The inset in Fig. 6A shows that the
control activity in cell-attached patches was fairly stable during
prolonged perfusion with bath solution. Fig. 6B summarizes the inhibitory effects of GSNO observed in cell-attached experiments. A
similar inhibition was observed in cells expressing
1C-b*
2a*
2-
as illustrated in Fig.
7. Neither DEA/NO (2 mM) nor
SIN-1 (2 mM) or 8-Br-cGMP (2 mM) exerted a
significant inhibitory modulation (data not shown). In an attempt to
gain further information on Ca2+ channel modulation by
GSNO, we analyzed the changes in single-channel gating properties
induced by the thionitrite. Because most of the cell-attached patches
contained more than one channel, we analyzed single-channel
Po and Ps, as well
as the number of active channels in the patches, by use of a recently
developed method (20) that allows the analysis of multichannel data.
GSNO inhibited
1C-b, as well as
1C-b*
2a*
2-
, channels
mainly by reduction of Po (Fig.
8). In addition we observed a slight
reduction of Ps from 0.48 ± 0.02 to
0.31 ± 0.03 for
1C-b, which was statistically significant, and
from 0.39 ± 0.05 to 0.31 ± 0.06 for
1C-b*
2a*
2-
channels. It is of note that the single-channel experiments with
1C-b were performed in the presence of S-Bay-K 8644 (1 µM) to prolong open times and to enable a reasonable
analysis of the data. A detailed inspection of single-channel currents
during the onset of the inhibitory modulation revealed that GSNO
induced the transient occurrence of a reduced conductance state. This GSNO-induced subconductance level was detectable in the early phase of
GSNO-induced channel modulation in 1 of 5 patches with
1C-b*
2a*
2-
and in 3 of 9 patches with
1C-b channels. A
typical experiment is illustrated in Fig.
9. The GSNO-induced conductance state
exhibited relatively long dwell times at a current amplitude of ~0.4
pA (at 40 mM Ba2+ and 0 mV) corresponding to a
subconductance level of ~50% of the full conductance state. These
results provide evidence for a combined modulation of gating and
permeability properties of the
1C-b subunit by GSNO as the basis of
its inhibitory effects on L-type Ca2+ channels.
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Fig. 6.
GSNO inhibits unitary Ba2+
currents through 1C-b channels in the
cell-attached configuration. A, upper panel,
time course of the mean patch current of
1C-b channels in a
cell-attached patch (during depolarizing pulses from
70 to 0 mV).
Extracellular administration of 2 mM GSNO is indicated. The
inset shows a time course of the mean patch current in a
control experiment; perfusion of the cell with vehicle is indicated.
Lower panel, individual records of single-channel activity
during depolarizing pulses at the indicated time points. Scaling
bars, 1 pA/200 ms. B, mean patch current values
calculated from 8-min intervals before and after administration of 2 mM GSNO in individual experiments.
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Fig. 7.
GSNO inhibits unitary Ba2+
currents through
1C-b*
2a*
2-
channels in the cell-attached configuration. A,
upper panel, time course of the mean patch current of
1C-b*
2a*
2-
channels in a cell-attached patch (during
depolarizing pulses from
70 to 0 mV). Extracellular administration of
2 mM GSNO is indicated. Lower panel, individual
records of signal-channel activity during depolarizing pulses at the
indicated time points. Scaling bars, 1 pA/200 ms.
B, mean number of open channels calculated from 8-min
intervals before and after administration of 2 mM GSNO in
individual experiments.
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Fig. 8.
Inhibition of 1C-b
and
1C-b*
2a*
2-
currents by GSNO is because of a reduction of open probability of
the channels. Mean Po ± S.E. before
(black columns) and 2-10 min after (white
columns) extracellular administration of 2 mM GSNO are
given for
1C-b (left; n = 5) and
1C-b*
2a*
2-
(right; n = 6).
S-Bay-K 8644 (1 µM) was used in experiments with the
1C-b subunit alone to prolong open times and enable analysis. The
asterisks indicate statistically significant
differences.
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Fig. 9.
GSNO-induced modulation of
1C-b channels is associated with changes in unitary
conductance of
1C-b channels.
A, upper panel, time course of mean patch
currents recorded during depolarizing pulses from
70 to 0 mV before
and after perfusion with 2 mM GSNO. Lower panel,
individual traces of channel activity at the indicated time points.
Scaling bars, 1 pA/200 ms. The asterisks indicate
the subconductance levels. B, amplitude histograms derived
from the experiment shown in A before and after
administration of 2 mM GSNO. The asterisk
indicates the subconductance level.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 subunit of
class C L-type Ca2+ channels, i.e.
gating, as well as ion permeation, because of S-nitrosation
of a critical thiol group.
1C-b subunit. All three compounds are well known to increase
cGMP levels in various cellular systems (6, 10, 11, 25). Despite this
principle similarity, the three compounds favor distinct chemical
reactions that have been suggested to mediate (patho-) physiological
effects of NO (26, 27). DEA/NO releases NO, which is readily available
to stimulate soluble guanylyl cyclase, whereas SIN-1 decomposes to
produce both NO and superoxide (28), two species that combine rapidly
to form peroxynitrite, a well known nitrating and oxidizing species. It
is unlikely that peroxynitrite contributes to the effects of GSNO in
our whole-cell experiments, because HEPES, which has recently been
demonstrated to react with peroxynitrite to form NO donors (29), was
used as a buffer system. GSNO, by contrast, not only decomposes slowly to release NO but is, in addition, able to react with protein thiol
groups to form S-nitrosothiols or mixed disulfides (30). Consistent with the view that all three compounds release NO under certain conditions, they all elevated cGMP levels of HEK293 cells, with
DEA/NO being most effective. In clear contrast to its superior action
as a stimulator of soluble guanylyl cyclase, DEA/NO exerted rather
moderate inhibitory effects on L-type Ca2+ channels,
indicating that cGMP does not control
1C-b channels in the HEK293
expression system. The notion that the observed Ca2+
channel inhibition by GSNO does not involve cGMP was further corroborated by the observation that 8-Br-cGMP (2 mM) did
not affect
1C-b channels in cell-attached experiments. These
results are in line with a previous study demonstrating that inhibition of L-type Ca2+ channels by S-nitrosothiols is
insensitive to inhibition of soluble guanylyl cyclase (12). Thus GSNO
appears to exert a specific, cGMP-independent effect on L-type
Ca2+ channels. To test whether this effect of GSNO is based
on a direct interaction of the thionitrite with intracellular protein
thiol residues or requires the release of NO, we studied its effect in
cells loaded with copper ions to promote decomposition of GSNO (13, 14,
17, 23, 24) or excess of DTT, which competes with endogenous thiols for
S-nitrosation and oxidation (31-33). Both conditions
clearly blunted or even eliminated the action of GSNO, supporting the
concept that Ca2+ channel modulation by GSNO involves a
reaction of the thionitrite with intracellular sulfhydryls. Two types
of reaction between GSNO and protein thiol residues were considered,
transnitrosation and glutathionylation. Our observation that even
excessive loading of cells with GSSG failed to affect channel function,
which argued against glutathionylation as a mechanism of channel
modulation. This conclusion is further supported by our previous
experiments with the lipophilic oxidant
tert-butylhydroperoxide, which is known to induce oxidation
of cellular thiols and formation of mixed disulfides but failed to
affect
1C-b channels in the HEK293 expression system.5
Thus we suggest nitrosation of critical protein thiol groups as a
mechanism of L-type Ca2+ channel regulation. The modest
inhibitory effects observed with the NO donor DEA/NO may, therefore, be
because of nitrosation of thiol groups, a reaction that was shown to
occur at high concentrations of NO in the presence of oxygen
(28).
1 subunit itself as
the regulatory principle mediating the effects of GSNO. Although
modification of a yet unknown regulatory protein cannot be excluded,
our experiments demonstrate that Ca2+ channel regulation by
GSNO does not require expression of any of the known auxiliary subunits
of the channel. Thus the
1C protein appears to be the most likely
target for GSNO modulation. An intracellular localization of the
regulatory thiol residue is suggested by the following lines of
evidence: (i) the effects of GSNO were sensitive to intracellular
administration of copper ions and DTT, and (ii) GSNO inhibited L-type
Ca2+ channels not only in the whole-cell but also in the
cell-attached configuration of the patch clamp technique. A truncated
form of
1C that lacks the cytoplasmic C-terminal 438 amino acids
including 13 cysteine residues was also sensitive to inhibition by
GSNO.5 The sequence of this truncated
1C includes 11 cysteine residues that are potentially accessible from the cytoplasmic
side and may be considered as candidates for a regulation by transnitrosation.
1C-b channels that is induced by a
potential (patho-) physiologic regulator of L-type Ca2+
channels. The transient nature of the GSNO-induced subconductance state
precluded a more detailed analysis of this phenomenon. Nonetheless, our
results strongly suggest that GSNO exerts a modulatory effect on both
gating and permeation in the
1C-b protein. The observed GSNO-induced
modulation of gating and conductance was independent of the
expression of auxiliary
2a and
2-
subunits and may
well explain inhibitory effects of nitrosothiols on native
cardiovascular Ca2+ channels (5, 34, 35).
1 subunit
and may represent an important mechanism that links NO signaling to
Ca2+ channel functions in the cardiovascular system.
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ACKNOWLEDGEMENTS |
---|
We thank N. Klugbauer and F. Hofmann for
providing HEK293 cells stably transfected with 1C-b, as well as
2a and
2-
cDNA. We also thank M. Rehn for performing the
RIA and I. Hauser for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by the Fonds zur Förderung der Wissenschaftlichen Forschung (Spezialforschungsbereich Biomembranes F715 and P12667 to K. G., F708 to W. S., and P12728 to C. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 43-316-380-5570; Fax: 43-316-380-9890; E-mail: klaus.groschner@kfunigraz.ac.at.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M008244200
3 GenBankTM/EMBL accession number X64298.
4 SPTREMBL Q9NY47.
5 M. Poteser and K. Groschner, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: NO, nitric oxide; GSNO, S-nitrosoglutathione; HEK, human embryonic kidney; Po (open probability), probability of a single channel to be found in an open state; Ps (availability), probability of a single channel to open during depolarization; SIN-1, 3-morpholinosydnonimine-hydrochloride; DEA/NO, 2,2-diethyl-1-nitroso-oxhydrazin; DTPA, diethylenetriamine-pentaacetic acid; DTT, dithiothreitol.
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