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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 "alpha -like" RyR); the other showed a sigmoidal activation curve against Ca2+ or niflumic acid, with no effect on perchlorate (termed "beta -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 beta -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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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., alpha - and beta -isoforms (see Refs. 22, 28 for reviews). The alpha - and beta -isoforms of frog RyR are homologous to the mammalian skeletal muscle RyR (RyR1) and brain RyR (RyR3), respectively (16), although the beta -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 beta -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 "beta -like" RyR, as it is likely to be the beta -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 "alpha -like" RyR, as it is likely to be the alpha -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 beta -isoform lacks an inactivating site or is much less responsive against agonists, compared with the alpha -isoform.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-alpha -phosphatidylethanolamine, L-alpha -phosphatidyl-L-serine, and L-alpha -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-alpha -phosphatidylethanolamine (egg yolk), L-alpha -phosphatidyl-L-serine (bovine brain), and L-alpha -phosphatidylcholine (egg yolk) were purchased from Sigma. Other reagents were of analytical grade.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (alpha -like RyR) or the sigmoidal (beta -like RyR) curve against cis Ca2+. The alpha -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 alpha -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 (tau o1, tau o2) and closed (tau c1, tau c2) lifetime distributions in each channel were best fit by two exponentials (Fig. 1C). Both tau o1 (1.73 ms) and tau o2 (6.30 ms) in the beta -like RyR channel at pCa 5 were larger than those (tau o1 = 0.84 ms and tau o2 = 3.16 ms) in the alpha -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 alpha -like and beta -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 beta -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 alpha -like RyR channel at pCa 5 and Po = 0.086 ± 0.017 in the beta -like channel at pCa 6-6.3), these measured parameters were not different between the two channels (Table 1, control values).


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Fig. 1.   Occurrence of distinct Ca2+-release channels having different dependence on cis pCa and their electrical characteristics. A: channel activity in "alpha -like" [open probability (Po) = 0.035, 0.216, 0, and 0 at pCa = 5, 4, 3, and 8, respectively] and "beta -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 alpha -like (open circle ) and beta -like (bullet ) 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 alpha -like (left) and beta -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).

                              
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Table 1.   Effects of niflumic acid on Po and lifetimes of distinct isoforms of Ca2+-release channel

Because perchlorate is known to activate RyR1 and alpha -channels but not RyR2 or beta -channels (13, 26), we used perchlorate to further discriminate the channel types pharmacologically. Previously, we showed that the alpha -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 beta -like RyR channel, perchlorate between 1 and 30 mM did not affect Po (Fig. 2). Similar results were observed in three other preparations.


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Fig. 2.   Effects of perchlorate on the beta -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 alpha -like RyR. Figures 3A and 4 clearly demonstrate that the action of niflumic acid on the alpha -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.


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Fig. 3.   Different effects of niflumic acid on alpha -like (A) and beta -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 beta -like channel. Open and closed times are shown for each histogram.


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Fig. 4.   Relationship between Po and niflumic acid (black-square and bullet ) or ethanol (open circle ) concentrations in alpha -like (black-square) and beta -like (bullet ) channels activated by pCa 5 and pCa 6-6.3, respectively. The 50% effective concentration and Hill coefficient in beta -like channels were 21.7 µM and 1.27, respectively. Ethanol groups are shown as combined data from alpha -like (n = 3) and beta -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 alpha -like and 5 beta -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 alpha -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 beta -like RyR. Figure 3B shows concentration-dependent increase in Po in the beta -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 beta -like RyR with those of the alpha -like RyR described above.

The results were analyzed in 14 beta -like RyRs and are summarized in Table 1. Exposure of the beta -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 alpha -like channel) and monophasic (in beta -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 beta -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 alpha -like RyR (18). Similar experiments were carried out in the beta -like RyR and are depicted in Fig. 5. At holding potentials between -40 and +10 mV, unitary conductance in the beta -like RyR channel (809 ± 36 pS, n = 5) was similar to that of the alpha -like RyR channel (830 ± 14 pS, n = 5), as previously observed (18). Interestingly, exposure of the beta -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.


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Fig. 5.   Effect of niflumic acid on unitary conductance and reversal potential in beta -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 alpha -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).


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Fig. 6.   Effects of caffeine, ryanodine, and ruthenium red on niflumic acid-treated alpha -like (left) and beta -like (right) channels. A: control channel activity at pCa 4 (Po = 0.269), 3 (Po = 0.008), and 5 (Po = 0.083) in alpha -like channel and at pCa 4 (Po = 0.488), 3 (Po = 0.335), and 6.3 (Po = 0.040) in the beta -like channel. B-E: channel activity in presence of 100 µM niflumic acid (B; Po = 0.029 and 0.122 in alpha -like and beta -like channels, respectively), 2 mM caffeine (C; Po  = 0.169 and 0.350 in alpha -like and beta -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 beta -like RyR after treatment with 100 µM niflumic acid were qualitatively the same as those on the alpha -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 alpha -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.


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Fig. 7.   Effects of niflumic acid on ryanodine-modified beta -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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (alpha - and beta -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 alpha -isoform and homologous to RyR1, whereas the beta -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 alpha -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 beta -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 alpha /RyR1 and beta /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 alpha -like RyR to be the alpha /RyR1 isoform and the beta -like RyR to be the beta /RyR3 isoform.

Although the physiological function of alpha - and beta -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 alpha -isoform to release Ca2+ [depolarization-induced Ca2+ release (DICR)] and the released Ca2+ elicits further release of Ca2+ from the beta -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 beta -isoform (Figs. 1 and 4) and not for the alpha -isoform and in that the beta -isoform is more sensitive to Ca2+ than the alpha -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 beta -isoform. These characteristic properties of the beta -isoform would favor the function of amplifying DICR through the alpha -isoform by subsequent CICR.

The present observations that niflumic acid and Ca2+ dose dependently activate or inhibit the alpha -isoform and activate the beta -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 beta -like channel activation by niflumic acid was 21.7 µM, and the slope of the Hill curve was 1.27, although those in the alpha -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 beta -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 beta -like isoform. Interestingly, the biphasic and monophasic patterns of alpha -like and beta -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 beta -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 alpha -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 alpha -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 alpha -isoform, which is absent in the beta -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 beta -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 beta -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 alpha -isoform or RyR1 is located mainly in skeletal muscle, and the beta -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 beta -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 beta -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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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