Niflumic acid differentially modulates two types of
skeletal ryanodine-sensitive Ca2+-release channels
Toshiharu
Oba
Department of Physiology, Nagoya City University Medical School,
Nagoya 467, Japan
 |
ABSTRACT |
The effects of niflumic acid on ryanodine
receptors (RyRs) of frog skeletal muscle were studied by incorporating
sarcoplasmic reticulum (SR) vesicles into planar lipid bilayers. Frog
muscle had two distinct types of RyRs in the SR: one showed a
bell-shaped channel activation curve against cytoplasmic
Ca2+ or niflumic acid, and its mean open probability
(Po) was increased by perchlorate at 20-30
mM (termed "
-like" RyR); the other showed a sigmoidal
activation curve against Ca2+ or niflumic acid, with no
effect on perchlorate (termed "
-like" RyR). The unitary
conductance and reversal potential of both channel types were
unaffected after exposure to niflumic acid when clamped at 0 mV. When
clamped at more positive potentials, the
-like RyR
channel rectified this, increasing the unitary current. Treatment with
niflumic acid did not inhibit the response of both channels to
Ca2+ release channel modulators such as caffeine,
ryanodine, and ruthenium red. The different effects of niflumic acid on
Po and the unitary current amplitude in both types
of channels may be attributable to the lack or the presence of
inactivation sites and/or distinct responses to agonists.
frog skeletal muscle sarcoplasmic reticulum; single-channel
activity; activation and inactivation sites; perchlorate; ryanodine
receptor agonists
 |
INTRODUCTION |
NIFLUMIC ACID, an anti-inflammatory reagent, is
generally accepted to function as a blocker of anion transport or
Cl
channels in various cells (2, 8, 30). The inhibitory effect of niflumic acid on the Ca2+-activated
Cl
channel of smooth muscle has been studied extensively
(10, 12, 25). By comparison, there have been relatively few studies on
the action of niflumic acid on Ca2+ release from the
sarcoplasmic reticulum (SR) of smooth muscle, with these studies
providing different conclusions. For example, Hogg et al. (9) reported
that niflumic acid enhanced the evoked release, whereas Criddle et al.
(5) reported no effect. In skeletal muscle, on the other hand, our
previous paper (18) demonstrated that niflumic acid has a dual effect
on the Ca2+-release channel prepared from frog skeletal
muscle SR, which was blocked by 1 mM cis Ca2+. The
mean open probability (Po) of the channel was
increased by niflumic acid at 10 µM and blocked at 100 µM. SR in
nonmammalian skeletal muscles has been reported to express two isoforms
of the ryanodine receptor (RyR), i.e.,
- and
-isoforms (see Refs. 22, 28 for reviews). The
- and
-isoforms of frog RyR
are homologous to the mammalian skeletal muscle RyR (RyR1) and brain RyR (RyR3), respectively (16), although the
-isoform has been suggested to have characteristics similar to the cardiac RyR (RyR2) on
the basis of an immunological study (see Ref. 28 for review). In
preparations isolated from bullfrogs, we provided evidence that there
are distinct RyRs having different dependence on cis Ca2+ (19). Most recently, Murayama and Ogawa (16) reported
that RyR3 shares functional characteristics of the
Ca2+-induced Ca2+-release (CICR) channel with
bullfrog
-isoform. However, there is no report on the effect of
niflumic acid on RyR2 or RyR3. Therefore, it is of interest to
elucidate the effects of niflumic acid on each isoform existing in frog
skeletal muscle. Here we report that niflumic acid dose dependently
activates one isoform of the Ca2+-release channels (termed
"
-like" RyR, as it is likely to be the
-isoform), which
displayed a sigmoidal activation curve against cytoplasmic
Ca2+ and was not affected by perchlorate. The unitary
conductance of this channel was increased by niflumic acid at large
positive potentials. The channel was maximally activated through the
binding of niflumic acid to at least one binding site, as evidenced by the Hill plot. By contrast, niflumic acid affected the
Po of the other channel (termed "
-like"
RyR, as it is likely to be the
-isoform) in a dual manner, with no
effect on the unitary conductance of the channel, consistent with our
previous report (18). Therefore, it seems likely that the
-isoform
lacks an inactivating site or is much less responsive against agonists,
compared with the
-isoform.
 |
MATERIALS AND METHODS |
Heavy SR vesicle preparation.
Microsomal fractions enriched in terminal cisternae of the SR were
prepared from leg skeletal muscle of Rana catesbiana as described previously (11). Heavy SR membrane vesicles were suspended in
100 mM KCl, 20 mM tris(hydroxymethyl)aminomethane maleate (pH 6.8), 20 µM CaCl2, and 0.3 M sucrose and, after quick freezing in
liquid N2, stored at
50°C until use. Protein
concentration was determined by the biuret reaction using serum bovine
albumin as the standard.
Planar lipid bilayer methods and single-channel data acquisition
and analysis.
Single-channel recordings were carried out by incorporating heavy SR
vesicles into planar lipid bilayers, according to our previous method
(19). Lipid bilayers consisting of a mixture of
L-
-phosphatidylethanolamine,
L-
-phosphatidyl-L-serine, and L-
-phosphatidylcholine (5:3:2 by wt) in decane (40 mg/ml) were formed across a hole ~200 µm in diameter in a
polystyrene partition separating two compartments, the cis
(vol, 3 ml) and trans (vol, 2.2 ml) chambers. The cis
and trans solutions consisted of 250 mM (cis) or 50 mM
(trans) CsCH3SO3 and 10 mM CsOH,
adjusted with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid to
pH 7.4. SR vesicles (~2 µg/ml) were added to the cis side.
After channel fusion was confirmed by occurrence of flickering
currents, unfused SR vesicles were removed from the cis chamber
by perfusion with a new cis solution (20 ml). The cytoplasmic
surface of the RyR faced the cis side (19). K+ and
Cl
channels present in the SR membrane were blocked by
using Cs+ as the charge carrier and methanesulfonate as the
impermeant ion, respectively. All reagents were added to the
cis chamber, except for perchlorate, which was added to both
sides. The trans side was held at ground potential, and the
cis side was clamped at 0 mV using a 1.5% agar bridge in 3 M
KCl and Ag-AgCl electrodes, unless otherwise indicated. Experiments
were done at room temperature (18-22°C).
Single-channel currents, amplified by a patch-clamp amplifier
(CEZ-2300, Nihon-Kohden, Tokyo, Japan), were displayed on an oscilloscope, filtered at 0.5 kHz using a four-pole low-pass Bessel filter, and digitized at 2 kHz for analysis. Data were saved on the
hard disk of a NEC personal computer. Po and the
lifetime of open and closed events from records of duration >2 min
were calculated by 50% threshold analysis using QP-120J software
(Nihon-Kohden), as described previously (18, 19). When more than two
channels were incorporated into bilayers, data were discarded from the analysis.
The results are presented as means ± SE. Statistical analysis was
performed with Wilcoxon's U-test or paired t-test.
Values of P < 0.05 were regarded as significant. The number
of binding sites required for niflumic acid to maximally activate the
channel was estimated from the Hill slope.
Chemicals.
Stock solutions for niflumic acid (200 mM; Sigma, St. Louis, MO) and
ryanodine (1 mM; Wako Pure Chemical, Osaka, Japan) were dissolved in
pure ethanol and stored at
20°C. Ruthenium red (1 mM; Sigma) and
NaClO4 (1 M; Wako) were dissolved in ultrapure water and
stored at 0-4°C. Caffeine (0.5 M; Sigma) was prepared in
ultrapure hot water just before each experiment.
L-
-phosphatidylethanolamine (egg yolk),
L-
-phosphatidyl-L-serine (bovine brain), and
L-
-phosphatidylcholine (egg yolk) were purchased from
Sigma. Other reagents were of analytical grade.
 |
RESULTS |
RyR with distinct channel gating kinetics.
Our frog SR preparations had two distinct RyRs showing different
concentration dependence on cytoplasmic Ca2+ (19). In order
to obtain particular basal data to investigate the effects of niflumic
acid on Ca2+-release channels, we first studied the gating
kinetics of each isoform activated by exposure to different
concentrations of cis Ca2+ in detail.
As shown in Fig. 1, A and
B, the channel activity showed either the bell-shaped (
-like
RyR) or the sigmoidal (
-like RyR) curve against cis
Ca2+. The
-like RyR was slightly activated at pCa 6 (Po = 0.079 ± 0.053, n = 3) but not
at pCa 7 or 8. The maximum Po was observed at pCa 4 (0.202 ± 0.028, n = 15). A further decrease in pCa to 3 led to a decreased Po (0.024 ± 0.006,
n = 18). In contrast, the other type of channel was more
sensitive than the
-like RyR channel against cis
Ca2+. The channel was activated at pCa <7, and
Po reached a maximum at pCa 5 (Po = 0.356 ± 0.061, n = 17). High
Po was sustained over a range of pCa from 5 to 3. The slope of the Hill curve for the channel activation was 0.92 ± 0.12 (n = 4). The open (
o1,
o2) and closed (
c1,
c2) lifetime distributions in each channel were best
fit by two exponentials (Fig. 1C). Both
o1
(1.73 ms) and
o2 (6.30 ms) in the
-like RyR channel
at pCa 5 were larger than those (
o1 = 0.84 ms and
o2 = 3.16 ms) in the
-like RyR channel activated by
the same pCa. However, the unitary current amplitudes of 19.6 ± 0.8 pA (n = 10) and 20.5 ± 1.0 pA (n = 8; at holding potential of 0 mV and pCa 5), the reversal potentials of
22.4 ± 0.7 mV (n = 5) and
23.8 ± 1.2 mV (n = 6), and
the unitary conductances of 811.0 ± 21.6 pS (n = 10) and
804.9 ± 28.2 pS (n = 8) were not significantly different
between
-like and
-like RyR isoforms, respectively. Increase in
pCa to 6-6.3 from 5 resulted in a decrease in the open time
constant and an increase in the closed one in the
-like channel,
suggesting that the time constants vary as a function of pCa. When each
isoform was activated to a similar extent by using different
concentrations of cis Ca2+
(Po = 0.076 ± 0.028 in the
-like RyR
channel at pCa 5 and Po = 0.086 ± 0.017 in
the
-like channel at pCa 6-6.3), these measured parameters were
not different between the two channels (Table
1, control values).

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 1.
Occurrence of distinct Ca2+-release channels
having different dependence on cis pCa and their electrical
characteristics. A: channel activity in " -like" [open
probability (Po) = 0.035, 0.216, 0, and 0 at
pCa = 5, 4, 3, and 8, respectively] and " -like"
(Po = 0.481, 0.587, 0.368, 0.301, and 0 at
pCa = 5, 4, 3, 6, and 8, respectively) channels at various
cis Ca2+ concentrations. Holding potential was 0 mV. Dashed and solid lines, open and closed channel levels,
respectively. RyR, ryanodine receptor. B: relationship between
Po and pCa in -like ( ) and -like ( )
channels. Numbers beside symbols, numbers of preparations examined.
Vertical bars, 1 SE. C: amplitude histograms (top) and open and closed histograms (bottom) activated at 10 µM
Ca2+ (pCa = 5) in -like (left) and -like
(right) channels shown in A. Single-channel current
amplitudes were 16.9 pA (top left) and 16.2 pA (top
right), respectively. Open and closed times are shown for each
histogram (bottom).
|
|
Because perchlorate is known to activate RyR1 and
-channels but not
RyR2 or
-channels (13, 26), we used perchlorate to further
discriminate the channel types pharmacologically. Previously, we showed
that the
-like RyR channel was activated on application of 20 or 30 mM perchlorate (from Po = 0.118 in control to
Po = 0.269 or Po = 0.333,
respectively; Ref. 17). However, in the
-like RyR channel,
perchlorate between 1 and 30 mM did not affect Po (Fig. 2). Similar results were observed in
three other preparations.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of perchlorate on the -like Ca2+-release channel
activity. Top: channel activity at pCa = 4 (P0 = 0.366), 3 (Po = 0.314), and 6 (Po = 0.261). Bottom: perchlorate did not
affect channel activity (Po = 0.305, 0.264, 0.263, and 0.256 at 1, 5, 10, and 30 mM, respectively) when
cumulatively applied to both cis and trans sides.
Experiments were carried out on same channel at holding potential of 0 mV. Dashed and solid lines, open and closed channel levels,
respectively.
|
|
Effects of niflumic acid on the
-like RyR.
Figures 3A and
4 clearly demonstrate that the action of
niflumic acid on the
-like RyR is concentration dependent, as
expected from our previous results (18). Application of 3 µM niflumic acid at pCa 5 caused an increase in Po from 0.054 ± 0.030 (n = 5) in control to 0.102 ± 0.059 (Fig. 3),
although 1 µM niflumic acid did not
(Po = 0.052). An increase in drug concentration
to 10 µM increased Po to 0.126 ± 0.077. However, further increases (range, 30-1000 µM) inhibited
Po in a dose-dependent manner.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 3.
Different effects of niflumic acid on -like (A) and
-like (B) channel activity. A: control channel
activity activated at pCa 5 (Po = 0.079) and
channel activity after cumulative exposure to niflumic acid over a
range from 1 to 1,000 µM (Po = 0.115, 0.146, 0.120, 0.041, and 0.016 at 1, 3, 10, 100, and 1,000 µM, respectively). B: control channel activity activated by
cis pCa 6 (Po = 0.174) and channel
activity after cumulative exposure to niflumic acid over a range from 1 to 1,000 µM (Po = 0.116, 0.149, 0.251, 0.307, and 0.319 at 1, 3, 10, 100, and 1,000 µM, respectively).
Bottom: modification of niflumic acid-treated channel by 10 µM ryanodine. Channel was locked into long-lasting subconductance open state. Ruthenium red at 2 µM applied to cis side closed
ryanodine-modified channel. Dashed and solid lines, open and closed
channel levels, respectively. C: open (left) and
closed (right) time histograms in control (pCa 6) and after
100 µM niflumic acid treatment in -like channel. Open and closed
times are shown for each histogram.
|
|

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
Relationship between Po and niflumic acid ( and
) or ethanol ( ) concentrations in -like ( ) and -like
( ) channels activated by pCa 5 and pCa 6-6.3, respectively. The
50% effective concentration and Hill coefficient in -like channels
were 21.7 µM and 1.27, respectively. Ethanol groups are shown as
combined data from -like (n = 3) and -like
(n = 5) channels. Data are means ± SE from 5-8
experiments. Abscissa gives niflumic acid (µM) and ethanol (%, in
parentheses) concentrations on a log scale.
|
|
The possibility that this inhibition was artifactual due to high
concentrations of the vehicle (ethanol) was examined. Because niflumic
acid was applied cumulatively, the ethanol concentration resulting from
the inclusion of niflumic acid at 1000 µM was very high (1.58%).
Therefore, we tested the effect of various concentrations of ethanol on
the Ca2+-release channel activity. Eight preparations (3
-like and 5
-like RyR channels) were used. Ethanol alone over a
range from 0.1 to 1.58%, equivalent to concentrations resulting from
the inclusion of niflumic acid over a range from 1 to 1,000 µM, did not affect the Po in either type of channel (Fig.
4), similar to our previous results in bilayers (20), although
Ca2+- or caffeine-induced CICR potentiation has been
reported in the presence of ethanol (20, 23).
Exposure of the
-like RyR channel to 100 µM niflumic acid
significantly decreased the number of open events per second (from 45 s
1 in control to 22 s
1,
P < 0.05) but had no effect on the mean open time and open
time constants. An example of such an experiment is shown in Fig.
3A, and the results obtained from eight different preparations
are summarized in Table 1.
Effects of niflumic acid on the
-like RyR.
Figure 3B shows concentration-dependent increase in
Po in the
-like RyR channel after cumulative
application of niflumic acid from 1 to 1,000 µM. The control
Po of the channel was 0.133 in the presence of 1 µM cis Ca2+. Exposure to 10 µM niflumic acid
increased the Po (to 0.251), although 1-3 µM
did not affect the channel activity. On addition of 100 µM niflumic
acid, the response achieved near maximal activation (Po = 0.307). Similar results were observed in
five different channels and are depicted in Fig. 4. A relationship
between Po and niflumic acid concentration was fit
by a single sigmoidal curve with a 50% effective concentration
(EC50) of 21.7 ± 6.4 µM (n = 5). The slope
of the Hill curve was 1.27 ± 0.22 (n = 5). These data
suggest that the channel gating modification by niflumic acid may
differ between isoforms. Then, we compared effects of niflumic acid on
gating of the
-like RyR with those of the
-like RyR described
above.
The results were analyzed in 14
-like RyRs and are summarized in
Table 1. Exposure of the
-like RyR to 10 or 100 µM niflumic acid
significantly increased the number of open events per second from 48 in
control to 68 and 87, respectively. In addition, niflumic acid at 100 µM significantly decreased the closed time constants, resulting in
shortening of the mean closed time (to 12.2 ms from 35.9 ms in
controls), but open time constants and mean open time were not
affected. At 10 µM, niflumic acid had a mild effect on these
electrical parameters.
The biphasic (in
-like channel) and monophasic (in
-like channel)
activation patterns observed after treatment with niflumic acid (Fig.
4) were similar to the respective Ca2+-dependent activation
patterns of these isoforms (Fig. 1). The question arises whether the
activation by niflumic acid and that by Ca2+ are governed
by the same mechanism. To resolve this question, we investigated the
effect of niflumic acid in
-like channels that had been activated at
optimal pCa 5. No further activation was induced by application of 100 µM niflumic acid to such Ca2+-activated channels
(Po = 0.358 ± 0.126 in niflumic acid vs.
0.342 ± 0.078 in controls, n = 5).
Our previous results indicate that 100 µM niflumic acid does not
affect single-channel conductance, reversal potential, or single-current amplitude in the
-like RyR (18). Similar experiments were carried out in the
-like RyR and are depicted in Fig.
5. At holding potentials between
40 and
+10 mV, unitary conductance in the
-like RyR channel (809 ± 36
pS, n = 5) was similar to that of the
-like RyR channel
(830 ± 14 pS, n = 5), as previously observed (18).
Interestingly, exposure of the
-like RyR to 100 µM niflumic acid
increased unitary conductance at positive potentials higher than +10
mV, as shown by increased current amplitude. Current amplitudes in
control and in niflumic acid-treated channels were 27.2 ± 1.1 and
30.5 ± 1.5 pA at 10 mV holding potential, 36.0 ± 1.1 and 41.7 ± 1.9 pA at 20 mV, and 41.8 ± 1.5 and 48.4 ± 1.5 pA
( P < 0.05) at 30 mV, respectively.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of niflumic acid on unitary conductance and reversal potential
in -like channel. Top: traces on both sides show
single-channel amplitude obtained when channel was clamped at holding
potentials of 10, 0, 10, and 20 mV in control (Cont; pCa = 6) and
after 100 µM niflumic acid treatment (Nif). Experiments were done in same channel. Bottom: current-voltage relationship in this
channel. Note enhancement of unitary current at more positive holding
potentials after niflumic acid treatment. Dashed and solid lines, open
and closed channel levels, respectively.
|
|
Effects of Ca2+-release channel modulators on
channel activity of both RyRs.
Channel activity of the
-like RyR was inhibited by a shot of 100 µM niflumic acid (Fig. 6; from
Po = 0.090 in controls to Po = 0.033, n = 4). Exposure of such
channels to 2 mM caffeine increased the Po 6.8-fold
to 0.224 ± 0.117. Mean open time was prolonged twofold from 1.35 ms/event in niflumic acid to 2.69 ms/event after caffeine treatment,
and the number of open events per second also increased, from 24.3 to
76.4. The caffeine-activated channel was open-locked after exposure to
10 µM ryanodine (Fig. 6, C and D, left). Subsequent
application of 2 µM ruthenium red almost completely closed the
open-locked channel (Fig. 6E, left).

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of caffeine, ryanodine, and ruthenium red on niflumic
acid-treated -like (left) and -like (right)
channels. A: control channel activity at pCa 4 (Po = 0.269), 3 (Po = 0.008), and 5 (Po = 0.083) in -like channel and at pCa 4 (Po = 0.488), 3 (Po = 0.335), and 6.3 (Po = 0.040) in the -like channel.
B-E: channel activity in presence of 100 µM
niflumic acid (B; Po = 0.029 and 0.122 in -like and -like channels, respectively), 2 mM caffeine
(C; Po = 0.169 and 0.350 in -like and
-like channels, respectively), 10 µM ryanodine (D;
subconductive open state), and 2 µM ruthenium red (E;
closed). Chemicals were added cumulatively to cis side of each
channel. Dashed and solid lines, open and closed channel levels,
respectively.
|
|
Effects of modulators on the
-like RyR after treatment with 100 µM
niflumic acid were qualitatively the same as those on the
-like RyR.
An example is shown in Fig. 6, right. Caffeine at 2 mM elicited
a further increase in the Po of the channel
activated by exposure to niflumic acid (9.2-fold from
Po = 0.039 ± 0.015 in niflumic acid to
0.358 ± 0.128, n = 5). Mean open time was lengthened from
1.33 ± 0.22 to 4.64 ± 1.57 ms/event, and the number of open events
per second increased from 27.4 ± 9.0 to 71.5 ± 23.5 after 2 mM
caffeine application. Mean closed time was significantly shortened from
26.2 ± 3.2 ms in niflumic acid to 5.9 ± 2.7 ms after caffeine
treatment (P < 0.01). All of these channels responded to
ryanodine and ruthenium red in a manner similar to the
-like RyR.
Application of 100 µM niflumic acid to the ryanodine-modified channel
affected neither the channel activity nor unitary conductance (Fig.
7C). Such channels were closed in
response to 2 µM ruthenium red (Fig. 7D). Similar results
were observed in three other experiments.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of niflumic acid on ryanodine-modified -like channel.
A: channel activity in control
(Po = 0.438, 0.277, and 0.293 at pCa 4, 3, and 5, respectively). B: open lock of the channel by subsequent
application of 10 µM ryanodine. C: no effect of 100 µM
niflumic acid on ryanodine-modified channel activity. D: ruthenium red (2 µM) closed ryanodine-locked channel. Dashed and solid lines, open and closed channel levels, respectively.
|
|
 |
DISCUSSION |
The present study has revealed that niflumic acid can directly modulate
the RyR/Ca2+-release channel. The modulating effect of
niflumic acid on the RyR depends on its concentration and on the type
of RyR. The drug acts as an agonist in one type of RyR and as an
agonist or an antagonist depending on its concentration, in the other
channel. However, the binding sites for CICR channel modulators are
intact even after niflumic acid-induced channel modification, because all of the channels examined responded to caffeine, ryanodine, and
ruthenium red. These effects of niflumic acid were quite similar to
those elicited after exposure of the channels to various cytoplasmic concentrations of Ca2+, except for an extra effect on
single-channel conductance in one type of the channel voltage clamped
at more positive potentials.
Nonmammalian skeletal muscle SR has two isoforms (
- and
-isoforms) of the RyR, different from mammalian skeletal muscle composed of a single isoform (22, 28). In bullfrog skeletal muscle,
amino acid sequences were determined to be 5,037 amino acids for the
-isoform and homologous to RyR1, whereas the
-isoform, composed
of 4,868 amino acids, was similar to RyR3 (16, 24). This and our
previous (19) results also demonstrated the occurrence of two distinct
Ca2+-release channels/RyRs in our SR preparations, as
evidenced using Ca2+, perchlorate, and niflumic acid. One
type of channel, which we termed the
-like RyR, was inactivated by
application of 1 mM cis Ca2+ (Fig. 1 and Ref. 19)
and was activated in response to 20-30 mM perchlorate (17). The
other channel, which we termed the
-like RyR, exhibited a high
Po even in the presence of 1 mM cis Ca2+ (Fig. 1 and Ref. 19) and was not activated by
perchlorate (Fig. 2). Taking into account electrophysiological evidence
that
/RyR1 and
/RyR3 isoforms of the RyR in skeletal muscles have
distinct responses to Ca2+ and perchlorate (1, 13, 15, 21,
26), our results strongly suggest the
-like RyR to be the
/RyR1
isoform and the
-like RyR to be the
/RyR3 isoform.
Although the physiological function of
- and
-isoforms of the RyR
is poorly understood, some investigators have proposed a plausible
model that the depolarization signal of the transverse tubular
membranes sensed by the dihydropyridine receptor/voltage sensor is
transmitted directly to the
-isoform to release Ca2+
[depolarization-induced Ca2+ release (DICR)] and the
released Ca2+ elicits further release of Ca2+
from the
-isoform operating as a CICR channel (see Refs. 27, 28 for
reviews). This model is supported by the present results in that both
niflumic acid and Ca2+ at 1 mM behave as activators for the
-isoform (Figs. 1 and 4) and not for the
-isoform and in that the
-isoform is more sensitive to Ca2+ than the
-isoform,
as evaluated by the Po (Table 1). These observations also indicate either a lack of inactivation sites or a
remarkably decreased binding of the inactivation site to a high
concentration of niflumic acid and Ca2+ in the
-isoform.
These characteristic properties of the
-isoform would favor the
function of amplifying DICR through the
-isoform by subsequent CICR.
The present observations that niflumic acid and Ca2+ dose
dependently activate or inhibit the
-isoform and activate the
-isoform (Figs. 1 and 4) suggest that the former has at least two
binding sites (high and low affinities), whereas the latter has only a single high-affinity binding site. The EC50 of
-like
channel activation by niflumic acid was 21.7 µM, and the slope of the Hill curve was 1.27, although those in the
-like channel were not
determined because estimation of maximum value of the
Po was impossible using our technique, due to the
existence of the inactivation process. A similar slope of the Hill
curve (0.92) was observed in
-like channels when activated by
Ca2+. A Hill coefficient near unity supports the existence
of a single binding site for niflumic acid and Ca2+ in the
-like isoform. Interestingly, the biphasic and monophasic patterns
of
-like and
-like channel activation induced by niflumic acid
were similar to those of the pCa-dependent activation of these
isoforms, respectively (compare Fig. 1 with Fig. 4). In addition, the
present observation that niflumic acid at 100 µM elicited no further
activation of the
-like isoform, which had been activated at pCa 5, indicates that effects of niflumic acid and Ca2+ on the
channel activation are not additive, suggesting that both effects are
governed by the same mechanism. The binding sites of niflumic acid,
however, seem likely to be different from those of Ca2+, as
described below. Therefore, it is of interest to elucidate the
underlying mechanism by which niflumic acid leads to the conformational change similar to that elicited by Ca2+, but it remains
unknown. Both channels were activated by increasing the number of open
events per second and mean open time duration and by decreasing the
mean closed time duration after binding of niflumic acid to
high-affinity binding sites (Table 1). Binding of niflumic acid to
low-affinity sites in the
-like RyR led to the decrease in
Po (i.e., channel inactivation) by predominantly increasing the closed time duration. Therefore, the high-affinity binding site would be on the same amino acid sequences conserved in
both RyR isoforms. On the other hand, it seems likely that the
low-affinity binding site is in an amino acid sequence specific to the
-isoform. Both isoforms in bullfrog skeletal muscles show 69%
homologous amino acid sequences (24). However, there are two remarkable
differences in amino acid sequences between isoforms: one is in a
stretch of about 100 residues from amino acid 1,305 of
-isoform,
which is absent in the
-isoform and RyR3, and the other is in a
stretch of about 400 residues just prior to the carboxy terminal that
is highly conserved in all RyRs (24). Binding sites of niflumic acid
may be on a positively charged residue(s) at the cytoplasmic surface,
close to a hydrophobic region (i.e., transmembrane segments) of the
channel molecule (4). The RyRs of bullfrog skeletal muscle have many
positively charged amino acids such as Arg, Lys, and His (24).
Ca2+ binding sites in the RyRs have not yet been fixed but
have been deduced from experiments using an antibody against a small
peptide of rabbit skeletal muscle RyR (3) and from analysis of amino acid sequences showing EF hand structure (see Ref. 22 for
review). As discussed above, niflumic acid activated the
-like
channel via the same mechanism as Ca2+ does. However,
further biochemical experiments combined with genetic engineering or
ultrastructural methods should be carried out to elucidate the exact
binding sites to niflumic acid and Ca2+ and thus to help
understand the tertiary structure of RyRs that leads to channel
activation or inactivation.
One possibility that can not be completely excluded is that niflumic
acid modifies channel gating by acting on a secondary protein such as
triadin, calsequestrin, or FK-506-binding protein. These proteins have
been reported to be involved in the regulation of the
Ca2+-release channel (18). As shown in Fig. 5,
single-channel current amplitude in the
-like channel was increased
after exposure to 100 µM niflumic acid at more positive holding
potentials. Therefore, it seems likely the site of niflumic acid action
is not on these modulatory proteins but rather directly on the RyR.
Alternatively, the increase in the current amplitude may be explained
by niflumic acid entering the transmembrane electric field at positive
holding potentials and then increasing the channel pore size or
decreasing positive charges in the pore. There are only a few amino
acids having positively charged groups in the putative pore region
[1 amino acid in the model proposed by Takeshima et al. (29)
or 9 amino acids scatteringly in the model by Zorzato et al. (31)]. However, the fact that there are limited numbers of positively charged
amino acids in the region lining the pore makes it unlikely that the
binding site(s) of niflumic acid is in the pore.
The
-isoform or RyR1 is located mainly in skeletal muscle, and the
-isoform or RyR3 occurs not only in skeletal muscle but also in
specific brain regions representing about 2% of the total RyRs (22,
28). It has been reported that RyR3 is expressed specifically in
hippocampus, thalamus, and corpus striatum (6, 7, 16). A recently
published report has shown that the
-isoform in frog skeletal muscle
and the RyR3 in the brain are homologous amino acid sequences and may
have a similar functional role in releasing Ca2+ from
intracellular Ca2+ stores (16). However, properties of
mammalian RyR3 channel are not yet known, as the protein has not yet
been isolated. Further investigations using the
-isoform would help
elucidate the functional role of RyR3 in specific regions of the brain.
 |
ACKNOWLEDGEMENTS |
I thank Drs. D. F. Van Helden and H. Suzuki for reading the
manuscript.
 |
FOOTNOTES |
This work was supported by Grant-In-Aid for Scientific Research
086700600 from the Ministry of Education, Science, Sports, and Culture,
Japan.
Address for reprint requests: T. Oba, Dept. of Physiology, Nagoya City
University Medical School, Mizuho-ku, Nagoya 467, Japan.
Received 27 March 1997; accepted in final form 17 June 1997.
 |
REFERENCES |
1.
Bull, R.,
and
J. J. Marengo.
Sarcoplasmic reticulum release channels from frog skeletal muscle display two types of calcium dependence.
FEBS Lett.
331:
223-227,
1993[Medline].
2.
Chao, A. C.,
and
H. Mochizuki.
Niflumic and flufenamic acids are potent inhibitors of chloride secretion in mammalian airway.
Life Sci.
51:
1453-1457,
1992[Medline].
3.
Chen, S. R. W.,
L. Zhang,
and
D. H. MacLennan.
Antibodies as probes for Ca2+ activation sites in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum.
J. Biol. Chem.
268:
13414-13421,
1993[Abstract/Free Full Text].
4.
Cousin, J.-L.,
and
R. Motais.
Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds. II. Chemical properties of the flufenamate-binding site on the band 3 protein.
Biochim. Biophys. Acta
687:
156-164,
1982[Medline].
5.
Criddle, D. N.,
R. Soares de Moura,
I. A. Greenwood,
and
W. A. Large.
Effect of niflumic acid on noradrenaline-induced contractures of the rat aorta.
Br. J. Pharmacol.
118:
1065-1071,
1996[Abstract].
6.
Giannini, G.,
A. Conti,
S. Mammarella,
M. Scrobogna,
and
V. Sorrentino.
The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues.
J. Cell Biol.
128:
893-904,
1995[Abstract].
7.
Hakamata, Y.,
J. Nakai,
H. Takeshima,
T. Kita,
and
K. Imoto.
Primary structure and distribution of a novel ryanodine receptor/calcium release channel from rabbit brain.
FEBS Lett.
312:
229-235,
1992[Medline].
8.
Hals, G. D.,
and
P. T. Palade.
Different sites control voltage dependence and conductance of sarcoball anion channel.
Biophys. J.
57:
1037-1047,
1990[Abstract].
9.
Hogg, R. C.,
Q. Wang,
and
W. A. Large.
Action of niflumic acid on evoked and spontaneous calcium-activated chloride and potassium currents in smooth muscle cells from rabbit portal vein.
Br. J. Pharmacol.
112:
977-984,
1994[Abstract].
10.
Janssen, L. J.,
and
S. M. Sims.
Ca2+-dependent Cl
current in canine tracheal smooth muscle cells.
Am. J. Physiol.
269 (Cell Physiol. 38):
C163-C169,
1995[Abstract/Free Full Text].
11.
Koshita, M.,
and
T. Oba.
Caffeine treatment inhibits drug-induced calcium release from sarcoplasmic reticulum and caffeine contracture but not tetanus in frog skeletal muscle.
Can. J. Physiol. Pharmacol.
67:
890-895,
1989[Medline].
12.
Lamb, F. S.,
K. A. Volk,
and
E. F. Shibata.
Calcium-activated chloride current in rabbit coronary artery myocytes.
Circ. Res.
75:
742-750,
1994[Abstract].
13.
Ma, J.,
K. Anderson,
R. Ahirokov,
R. Levis,
A. Gonzalez,
K. Karhanek,
M. Hosey,
G. Meissner,
and
E. Rios.
Effects of perchlorate on the molecules of excitation-contraction coupling of skeletal and cardiac muscle.
J. Gen. Physiol.
102:
423-448,
1993[Abstract].
14.
Meissner, G.
Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors.
Annu. Rev. Physiol.
56:
485-508,
1994[Medline].
15.
Murayama, T.,
and
Y. Ogawa.
Purification and characterization of two ryanodine-binding protein isoforms from sarcoplasmic reticulum of bull-frog skeletal muscle.
J. Biochem. (Tokyo)
112:
514-522,
1992[Abstract].
16.
Murayama, T.,
and
Y. Ogawa.
Properties of Ryr3 ryanodine receptor isoform in mammalian brain.
J. Biol. Chem.
271:
5079-5084,
1996[Abstract/Free Full Text].
17.
Oba, T.,
M. Koshita,
T. Aoki,
and
M. Yamaguchi.
Bay K 8644 and
potentiate caffeine contracture without Ca2+ release channel activation.
Am. J. Physiol.
272 (Cell Physiol. 41):
C41-C47,
1997[Abstract/Free Full Text].
18.
Oba, T.,
M. Koshita,
and
D. F. Van Helden.
Modulation of frog skeletal muscle Ca2+ release channel gating by anion channel blockers.
Am. J. Physiol.
271 (Cell Physiol. 40):
C819-C824,
1996[Abstract/Free Full Text].
19.
Oba, T.,
M. Koshita,
and
M. Yamaguchi.
H2O2 modulates twitch tension and increases Po of Ca2+ release channel in frog skeletal muscle.
Am. J. Physiol.
271 (Cell Physiol. 40):
C810-C818,
1996[Abstract/Free Full Text].
20.
Oba, T.,
M. Koshita,
and
M. Yamaguchi.
Ethanol enhances caffeine-induced Ca2+-release channel activation in skeletal muscle sarcoplasmic reticulum.
Am. J. Physiol.
272 (Cell Physiol. 41):
C622-C627,
1997[Abstract/Free Full Text].
21.
O'Brien, J.,
H. H. Valvevia,
and
B. A. Block.
Physiological differences between the
and
ryanodine receptors of fish skeletal muscle.
Biophys. J.
68:
471-482,
1995[Abstract].
22.
Ogawa, Y.
Role of ryanodine receptors.
Crit. Rev. Biochem. Mol. Biol.
29:
229-274,
1994[Abstract].
23.
Ohnishi, S. T.,
J. L. Flick,
and
E. Rubin.
Ethanol increases calcium permeability of heavy sarcoplasmic reticulum of skeletal muscle.
Arch. Biochem. Biophys.
233:
588-594,
1984[Medline].
24.
Oyamada, H.,
T. Murayama,
T. Takagi,
M. Iino,
N. Iwabe,
T. Miyata,
Y. Ogawa,
and
M. Endo.
Primary structure and distribution of ryanodine-binding protein isoforms of bullfrog skeletal muscle.
J. Biol. Chem.
269:
17206-17214,
1994[Abstract/Free Full Text].
25.
Pacaud, P.,
G. Loirand,
J. L. Lavie,
C. Mironneau,
and
J. Mironneau.
Calcium-activated chloride current in rat vascular smooth muscle cells in short-term primary culture.
Pflügers Arch.
413:
629-636,
1989[Medline].
26.
Percival, A.,
A. Williams,
J. Kenyon,
M. Grinsell,
J. A. Airey,
and
J. L. Sutko.
Chicken skeletal muscle ryanodine receptor isoforms: ion channel properties.
Biophys. J.
67:
1834-1850,
1994[Abstract].
27.
Rios, E.,
and
G. Pizarro.
Voltage sensor of excitation-contraction coupling in skeletal muscle.
Physiol. Rev.
71:
849-908,
1991[Free Full Text].
28.
Sutko, J. L.,
and
J. A. Airey.
Ryanodine receptor Ca2+ release channels: Does diversity in form equal diversity in function?
Physiol. Rev.
76:
1027-1071,
1996[Abstract/Free Full Text].
29.
Takeshima, H.,
S. Nishimura,
T. Matsumoto,
H. Ishida,
K. Kangawa,
N. Minamino,
H. Matsuo,
M. Ueda,
M. Hanaoka,
T. Hirose,
and
S. Numa.
Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor.
Nature
339:
439-445,
1989[Medline].
30.
White, M. M.,
and
M. Aylwin.
Niflumic and flufenamic acids are potent reversible blockers of Ca2+-activated Cl
channels in Xenopus oocytes.
Mol. Pharmacol.
37:
720-724,
1990[Abstract].
31.
Zorzato, F.,
J. Fujii,
K. Otsu,
M. Phillips,
N. M. Green,
F. A. Lai,
G. Meissner,
and
D. H. MacLennan.
Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum.
J. Biol. Chem.
265:
2244-2256,
1990[Abstract/Free Full Text].
AJP Cell Physiol 273(5):C1588-C1595
0363-6143/97 $5.00
Copyright © 1997 the American Physiological Society