SciELO - Scientific Electronic Library Online

vol.33 issue2Lipid peroxidation and antioxidants in hyperlipidemia and hypertensionAmyloid-ß-peptide reduces copper(II) to copper(I) independent of its aggregation state author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand




Related links


Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.33 n.2 Santiago  2000 

SH Oxidation Stimulates Calcium Release Channels
(Ryanodine Receptors) From Excitable Cells


1Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile
2Centro de Estudios Científicos de Santiago
*Present address: Department of Anesthesia, Brigham and Women’s Hospital, 75 Francis Street, Boston MA 02115, USA.


The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors) of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal) modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub µM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed.

KEY TERMS: calcium dependence; neurons; redox state; ryanodine receptors; sarcoplasmic reticulum; skeletal and cardiac muscle.


The cytosolic concentration of free calcium ions ([Ca2+]) is precisely regulated in all cells and can rapidly increase in response to various types of stimuli (Berridge, 1997). An increase in intracellular [Ca2+] regulates a wide range of cellular events, including gene expression and cell growth, the generation of second messengers and the activity of key enzymes of cell signaling pathways. Cell specific responses such as fertilization, endocrine secretion, neuronal signaling and muscle contraction are also brought about by an increase in intracellular [Ca2+]. Yet prolonged increases in cytosolic [Ca2+] are harmful to cells and have been implicated in apoptosis and cell death. Accordingly, cells use several mechanisms to exert a precise control of cytosolic [Ca2+] to ensure only transient increases in cytoplasmic [Ca2+] in response to stimuli (Berridge, 1997; Berridge et al., 1998).

Cytosolic [Ca2+] can increase either by the entry of Ca2+ from the extracellular medium or the release of Ca22+ from internal stores (Berridge et al., 1998). Two different receptors mediate Ca2+ release: the IP3-gated calcium channels and the ryanodine receptors/calcium release channels (RyR channels) that originate calcium-induced calcium release (CICR) in cardiac muscle and neurons (Berridge, 1997; Berridge et al., 1998). It is therefore important to characterize the way in which Ca2+ affects RyR channel activity and how other ions or metabolic reactions normally occurring in cells modulate the effects of Ca2+ on the channels. Multiple cellular components and reactions regulate RyR channels. In addition to Ca2+, these include other ions such as Mg2+ and protons, adenine derivatives such as cyclic ADP-ribose, ATP and other adenine nucleotides, interaction with other proteins, and metabolic reactions including phosphorylation and oxidation (Meissner, 1994; Coronado et al. 1994; Franzini-Armstrong and Protasi, 1997; Zucchi and Ronca-Testoni, 1997).

Oxidation of SH groups induces the release of calcium from cardiac or skeletal sarcoplasmic reticulum (SR) vesicles and activates the corresponding RyR channels incorporated in lipid bilayers (Prabhu and Salama, 1990; Abramson et al., 1995; Donoso et al., 1997; Eager et al., 1997; Eager and Dulhunty, 1999). Furthermore, highly reactive sulfhydryl groups of the channel protein are involved in the interactions between subunits of the homotetrameric channel protein (Wu et al., 1997) or between the RyR channel and other proteins (Liu and Pessah, 1994; Zhang et al., 1999).

Here we present experiments showing that the oxidation of SH groups modifies the calcium dependence of RyR channels incorporated in planar bilayers, decreasing in particular channel inhibition by mM [Ca2+] (Marengo et al., 1998). Since the inhibitory sites for Ca2+ and Mg2+ seem to be the same (Meissner et al., 1986; Laver et al., 1997), we investigated whether SH oxidation decreased the Mg2+ inhibition of skeletal RyR channels. For this purpose, the time course of calcium release from native skeletal SR vesicles at different free Mg2+ concentration was determined and was compared with that of oxidized vesicles. The results obtained indicate that oxidation overrides the Mg2+ inhibition of CICR.


Isolation of vesicles. Skeletal muscle SR vesicles enriched in triads were isolated from frog (Caudiverbera caudiverbera) or rabbit (New Zealand) fast skeletal muscle in the presence of a combination of protease inhibitors, as described previously (Hidalgo et al., 1993). Brain endoplasmic reticulum vesicles were obtained from the brain cortex of the rat (Sprague-Dawley) as described in detail elsewhere (Marengo et al., 1996). Cardiac SR vesicles were isolated from rabbit hearts using a protocol described previously (Marengo et al., 1998). Small aliquots of the vesicular preparations were quickly frozen in liquid N2 and were stored at -80 °C for up to one month.

Purification of RyR channels from frog skeletalal muscle. RyR channels were purified by a modification of the method described by Zhang et al (1997). Briefly, triad enriched amphibian SR vesicles were incubated with 20 mM MOPS, pH 7.2, containing 3% CHAPS [w/v], 0.25 M sucrose, 5 mg/ml phosphatidylcholine, 1 M NaCl, 5 mM DTT, 0.2 mM CaCl2 and a mixture of protease inhibitors, and were sedimented at 100.000 x g. The resulting supernatant was loaded on top of a discontinuous sucrose gradient (10%, 13% and 15%) containing 1% CHAPS, 5 mg/ml phosphatidylcholine and protease inhibitors. Fractions containing the highest ryanodine binding were pooled and extensively dialyzed to eliminate CHAPS.

Bilayer experiments. Channel recording and analysis were performed as described in detail previously (Bull and Marengo, 1994; Marengo et al., 1996). All experiments were carried out at room temperature (22 – 24 ºC). Open probability values (Po) were calculated from single channel records lasting at least 180 s. The recording conditions were 40 mM Ca2+-HEPES, 15 mM HEPES/Tris, pH 7.4 in the trans compartment; 225 mM HEPES/Tris, pH 7.4 and variable [Ca2+] in the cis compartment. To set the desired cis [Ca2+], 0.5 mM total Ca2+ and sufficient N-(2-hydroxyethyl)-ethylenediamine-triacetic acid (HEDTA) or ethyleneglycol-bis(ß-aminoethyl ether) N, N, N’, N’-tetraacetic acid (EGTA) were added to the cis compartment. Resulting cis [Ca2+] values were routinely checked with a calcium electrode.

After channel incorporation into the bilayer and establishment of recording conditions (Bull and Marengo, 1994), oxidation was carried out by addition of 2,2'-dithiodipyridine (DTDP) or thimerosal to the cis compartment (Marengo et al., 1998). SH oxidation was stopped by the removal of the non-reacted reagent through extensive perfusion of the cis compartment (5-10 times the cis volume) with a solution containing 225 mM HEPES/Tris, pH 7.4. Native or oxidized channels were treated with SH-reducing agents following essentially the same procedure. The cis [Ca2+] used during incubation with SH reagents is specified in the text. The theoretical analysis of the three different calcium dependencies was done as described previously (Marengo et al., 1996). Due to the fact that purified RyR channels displayed four conductance levels plus rapid and complex kinetics, the normalized mean current Po* was used as an index of channel activity and was calculated as described previously (Perez et al., 1998).

Calcium release kinetics. We measured calcium release kinetics in an SX.18MV fluorescence stopped-flow spectrometer from Applied Photophysics Ltd. (Leatherhead, U.K.). Extravesicular [Ca2+] was determined by measuring the fluorescence of the intermediate affinity calcium indicators Calcium Green 5N (CG5N) or Oregon Green 488 Bapta 5N (OG488B5N). The fluorescence emission of CG5N was measured using a 515 nm cut-off long-pass filter and an excitation wavelength of 488 nm, and that of OG488B5N using a 530 nm cut-off long-pass filter and an excitation wavelength of 490 nm. The Kd of both fluorescent indicators was determined in calcium buffered solutions containing 150 mM KCl, 20 mM imidazole-MOPS, pH 7.2. All calcium buffers were calculated with the WinMaxC program (file bers.ccm, and checked with a calcium electrode using a standard commercial kit to calibrate the electrode (WPI, Sarasota, FL). The fluorescence of both calcium indicators increased swiftly upon mixing the dye with solutions containing µM [Ca2+], with a Kd of 24 µM for CG5N and of 42 µM for OG488B5N. Addition of up to 2 mM free [Mg2+] did not affect dye fluorescence, yielding in both cases the same Kd for CaV, regardless of the presence of Mg2+.

Calcium release was measured in triads isolated from rabbit skeletal muscle actively loaded with calcium at 25 ºC. Active loading was done by incubating triads (1 mg/ml) in a solution containing (mM) 0.05 CaCl2, 150 KCl, 5 MgCl2, 20 imidazole-MOPS, pH 7.2, plus or minus 0.5 thimerosal. After 5 minutes, 5 mM ATP and 5 mM phosphocreatine plus 10 U/ml creatine kinase were added and active loading was continued for several minutes in the presence or absence of thimerosal. In some experiments, thimerosal was added 5 min after initiating active loading by the addition of ATP, and oxidation was continued during active loading for up to 10 min. Calcium release was initiated by mixing 1 volume of the solution containing the vesicles with 10 volumes of releasing solution containing (mM): 0.165 CaCl2, 150 KCl, 20 imidazole-MOPS, pH 7.2, plus 1 µM CG5N or OG488B5N. Variable concentrations of ATP and MgCl2 were added to the releasing solution to obtain after mixing pCa 5.0, 1.4 mM free [ATP] and variable free [Mg2+], ranging from 25 to 990 µM. The concentrations of ATP and Mg2+ of all releasing solutions were calculated using the WinMaxC program as above.


All reagents used were of analytical grade. Lipids were obtained from Avanti Polar Lipids, Inc., Birmingham, AL. Thimerosal, dithiothreitol and protease inhibitors (Leupeptin, Pepstatin A, benzamidine and phenylmethylsulfonyl fluoride) were obtained from Sigma Chemical Co. The fluorescent calcium indicators CG5N or OG488B5N were obtained from Molecular Probes, Inc.


Calcium dependence of native RyR channels in bilayers. The activity of all channels studied in this work was modulated by cis [Ca2+]. As illustrated in Figure 1, RyR channels from brain cortex neurons, amphibian or mammalian skeletal muscle showed three different calcium dependencies, which we have named the Low Po, the MS, and the C calcium dependencies (Marengo et al., 1996). RyR channels of the Low Po type had bell-shaped calcium dependence with Po < 0.1 in the entire [Ca2+] range studied. Channels that exhibit the MS or the C calcium dependencies are activated by µM [Ca2+] to Po values > 0.2 and differ in that MS channels are inhibited by 0.5 mM [Ca2+], whereas the C channels are not. Cardiac RyR channels exhibited the MS and the C calcium dependencies, but not the Low Po dependence. RyR channels purified from amphibian skeletal muscle displayed the same three different calcium dependencies of native amphibian channels, as illustrated in Figure 2, indicating that this behavior is intrinsic to the RyR channel protein.

Figure 1. Effect of cis [Ca2+] on the activity of RyR-channels. The panels show the changes in Po as a function of cis [Ca2+] for channels from rat brain cortex (A), frog skeletal muscle (B), rabbit skeletal muscle (C), or rabbit cardiac muscle (D). Values are given as Mean ± SEM. For RyR-channels that presented the Low Po (filled diamonds), the MS (open circles) or the C (filled circles) calcium dependencies the solid line through the data was obtained by the best fit of the experimental points to the equations defined previously (Bull and Marengo, 1993; Marengo et al.1998).


Figure 2. Effect of cis [Ca2+] on the activity of RyR-channels purified from frog skeletal muscle. The panel shows the changes in Po as a function of cis [Ca2+]. Values are given as Mean ± SEM. For RyR-channels that presented the Low Po (filled diamonds), the MS (open circles) or the C (filled circles) calcium dependencies the solid line through the data was obtained by the best fit of the experimental points to the equations defined previously (Bull and Marengo, 1993; Marengo et al.1998). The inset shows an amplification of the Low Po calcium dependence.

Oxidation of RyR channels modifies their calcium dependence. As illustrated in Figure 3, a single native RyR channel from rat brain cortex that displayed Low Po calcium dependence (see top 3 traces) exhibited the MS calcium dependence following oxidization with 200 µM DTDP, as illustrated by the three central records of Figure 3. A second incubation with 200 µM DTDP changed the calcium dependence again, this time to the C type, as illustrated by the three lower traces of Figure 3. The same behavior was exhibited by native RyR channels with the Low Po calcium dependence from mammalian and amphibian skeletal muscle oxidized either with DTDP or thimerosal (Marengo et al., 1998). Likewise, RyR channels purified from amphibian skeletal muscle following oxidation changed from Low Po to MS calcium dependence (Figure 4).


Figure 3. Effect of sequential incubation with DTDP on the calcium dependence of a native neuronal RyR-channel. The three top current records indicate that this native channel displayed the Low Po calcium dependence. Following 7 min oxidation with 200 µM DTDP, the reaction was stopped and the calcium dependence of the channels was characterized as MS, as demonstrated by the middle set of current records. After a second incubation with DTDP the channel displayed the C behavior, as shown in the lower set of records. Dashed line, maximal current amplitude.

Figure 4. Effect of incubation with thimerosal on the activity of a RyR channel purified from frog skeletal muscle. The two left current records indicate that this native channel displayed the Low Po calcium dependence. Following oxidation with thimerosal, the reaction was stopped and the calcium dependence of the channel was characterized as MS, as demonstrated by the set of current records shown at right. Dashed line, maximal current amplitude; c, closed channel level.

In addition to increasing the activity of channels with Low Po calcium dependence, oxidation also increased the activity of native single RyR channels from brain cortex neurons, cardiac muscle, amphibian or mammalian skeletal muscle that spontaneously displayed the MS behavior. Following incubation with DTDP or thimerosal, all channels acquired the C calcium dependence, yet further oxidation of cardiac channels with the C calcium dependence produced irreversible loss of channel activity (Marengo et al., 1998). These results indicate that oxidation or RyR channels produced sequential modifications of their calcium dependencies, from Low Po to MS, from MS to C, as illustrated in Figure 5, and from C to irreversibly damaged channels.


Figure 5. Effect of cis [Ca2+] on the activity of oxidized RyR channels from muscle or brain. The panels show the changes in Po as a function of cis [Ca2+] for RyR-channels from rat brain cortex (A), frog skeletal muscle (B), rabbit skeletal muscle (C), or rabbit cardiac muscle (D). Values are given as Mean ± SEM. The solid lines for the MS (open circles) or the C (filled circles) calcium dependencies correspond to the theoretical fits for the calcium dependencies of native channels given in Figure 1. The dotted lines correspond to the calcium dependence exhibited by the channels before oxidation. Values are given as Mean ± SEM.

Reversibility of the effects of SH oxidation. To further test the effects of changing channel redox state on channel activity, we investigated whether SH-reducing agents 1) modified the calcium dependencies of native channels, and 2) reversed the changes in channel response to cis [Ca2+] produced by oxidation. Native RyR channels from cardiac muscle or amphibian skeletal muscle that spontaneously displayed the C calcium dependence changed their calcium dependence to the MS type after addition of SH-reducing agents (Marengo et al., 1998). Furthermore, glutathion (GSH) partially reversed the effects of oxidation on channel activity. The continuous Po record illustrated in Figure 6 shows a neuronal RyR channel that originally displayed the Low Po calcium dependence and that after oxidation with DTDP increased its activity to Po > 0.6 in 3.16 µM [Ca2+], indicating that it had acquired the C calcium dependence. Further addition of 5 mM GSH produced a decrease in channel Po (Fig. 6), indicating that the channel had acquired the MS calcium dependence (Marengo et al., 1998). However, no changes from the MS to Low Po calcium dependence were observed, even after extensive incubation of native or oxidized channels with GSH or b-mercaptoethanol. After incubation with SH reducing reagents all RyR channels studied displayed the same change from the C to the MS behavior, but did not change from the MS to the Low Po mode (Marengo et al., 1998). Presumably the SH groups involved in the MS to Low Po transition undergo irreversible oxidation in vitro.

Figure 6. Effects of SH-reducing agents on R y R channel activity. Time-course of changes in Po following addition of 5 mM GSH (arrow) to a neuronal RyR-channel in 3.16 µM [Ca2+], which had acquired the C calcium dependence following previous treatment with 100 µM DTDP. The values of Po, given as Mean ± SEM, were calculated in successive periods and are displayed as a function of time.

Oxidation of RyR channels overrides the inhibitory effect of Mg2+ on calcium release. Figure 7 illustrates the time course of calcium release from native and oxidized triads actively loaded with calcium and measured at different free [Mg2+]. All fluorescence signals increased with time following a single exponential function with a rate constant k, which was strongly dependent on free [Mg2+] in native triads, as illustrated in Figure 8. Increasing free [Mg2+] from 25 µM to 1 mM in native triads produced a substantial decrease in k, from 24 s-1 to 6.5 s-1, with 50% inhibition at a free [Mg2+] of 56.5 ± 37 µM (N = 5). In contrast, increasing free [Mg2+] to 1 mM did not inhibit calcium release in triads oxidized with 0.5 mM thimerosal, and k values in the range of 26 s-1 were obtained at all [Mg2+] tested (Fig. 8).

Figure 7. Effect of [Mg2+] on calcium release kinetics in native and oxidized triads. Triads actively loaded with calcium as described in the text were mixed (1:10) in a stopped flow fluorescence spectrometer with a solution containing Oregon Green 488 Bapta 5 N and the required [ATP] and [MgCl2] to obtain after mixing pCa 5, 1.4 mM free ATP and the indicated free [Mg2+]. Panel A: native triads. Panel B: triads oxidized with 0.5 mM thimerosal. The solid line represents the fitting to a single exponential function.

Figure 8. Effect of [Mg2+] on calcium release rate constants in native and oxidized triads. The rate constant k of the fluorescence increase was calculated from the single exponential fitting of experimental records such as those shown in Figure 7. The solid line represents the best fit to the experimental points of the equation:
k = kmax K05 / ([Mg2+] + K05
kmax is the value of k in the absence of Mg2+ and K05 represents the Mg2+ concentration that produced 0.5 kmax. The dashed line has no theoretical meaning. Solid symbols: native triads. Open symbols: oxidized triads. Data are given as Mean ± SEM for 5 experiments with native triads and 4 for oxidized triads.

The stimulatory effects of thimerosal on calcium release were fully reversed by DTT, yet incubation of native triads with 5 mM DTT alone did not modify the rate constants of calcium release at all [Mg2+] tested (data not shown).


All single RyR channels studied exhibited two or three calcium dependencies. We describe here how single RyR channels present in rabbit or frog skeletal muscle SR, rabbit cardiac muscle SR or endoplasmic reticulum vesicles isolated from rat brain cortex responded in steady-state conditions to changes in cis [Ca2+]. We found that all native or purified RyR channels studied displayed either two (cardiac muscle) or three (skeletal muscle, brain cortex) calcium dependencies. The presence of different RyR isoforms in these tissues does not account for the differences in calcium dependencies observed as channels isolated from mammalian skeletal or cardiac muscle, which have essentially only one RyR isoform (Table I), displayed either two or three different calcium dependencies.



Isoform Total cysteines References

RyR1, rabbit skeletal muscle 100 (3 TM, 2 L) Takeshima et al., 1989; Marks et al., 1989; Zorzato et al., 1990.
RyR2, rabbit cardiac muscle 89 (5 TM, 1 L) Otsu et al., 1990; Witcher et al., 1991.
RyR3, human brain 109* Nakashima et al., 1997; Leeb and Brenig, 1998.
RyR-alpha, bullfrog skeletal muscle 94* Oyamada et al., 1994.
RyR-beta; bullfrog skeletal muscle 97* Oyamada et al., 1994.

Key: TM stands for a transmembrane location and L for a luminal location. The * indicates that the location of cysteine residues on the protein has not been reported.

Oxidation of RyR channels modified their calcium dependence. In search of the possible causes for the different calcium dependencies found, we investigated whether changing their redox state affected the way in which RyR channels responded to cis [Ca2+] changes. We found that oxidation of SH residues modified the calcium dependencies of all single RyR channels studied, regardless of their origin, producing either the MS or the C mode (Marengo et al., 1998). In addition, SH reducing agents caused a decrease in channel activity of native or oxidized RyR channels with the C calcium dependence and induced the MS calcium dependence. Purified RyR channels displayed the same behavior, indicating that the observed changes are due to modifications of SH residues of the channel protein. The calcium dependencies obtained after oxidation or reduction were comparable to those exhibited spontaneously by the native channels, suggesting that SH oxidation or reduction produced the same channel states found in native conditions. Accordingly, we propose that the oxidation state of the channel protein is a decisive factor in determining the calcium dependence of the channel activity exhibited by any given isoform. In addition, thimerosal, which is water soluble at pH 7.2 (Elferink, 1999), increased channel Po only when added to the cis compartment. Thus it is likely that the SH groups whose redox state determine a given calcium dependence belong among the many cysteine residues of the large cytoplasmic domain of all RyR isoforms characterized to date (Table I). The changes in calcium dependence may involve molecular rearrangement of channel protein segments produced by oxidation of critical SH residues, which somehow increase the calcium affinity of cytoplasmic activating sites and hinder calcium binding to the inhibiting sites. Thus, the channel would exhibit Low Po calcium dependence when all critical SH residues are reduced. Partial oxidation of these residues would give rise to the MS calcium dependence, extensive oxidation would induce the C calcium dependence, and extreme oxidation would result in irreversible loss of channel activity. According to this view, the fact that cardiac RyR channels did not exhibit Low Po behavior, even after extensive treatment with SH reducing reagents, may be attributed to irreversible oxidation in the native vesicles of the putative SH residues that are critical for this calcium dependence. Alternatively, since not all SH residues are conserved in the different channel isoforms (Table I) the cardiac RyR isoform may lack the SH residues which would give rise to the Low Po behavior when reduced.

Oxidation of RyR channels decreased the inhibitory effect of Mg on channel activity. The results discussed above indicate that extensive oxidation of RyR channels incorporated in planar bilayers suppressed the inhibitory effect of 0.5 mM [Ca2+]. Since the inhibitory sites for Ca2+ seem to be the same as for Mg2+ (Meissner et al., 1986; Laver et al., 1997), we also investigated whether oxidation decreased the Mg2+ inhibition of RyR channels. For this purpose, calcium release from native and oxidized rabbit skeletal SR vesicles was determined at different free Mg2+ concentrations. We found in this work, as well as in a parallel study (Donoso et al., 2000), that SH oxidation reduced the Mg2+ inhibition of calcium release, further supporting the idea that Ca2+ and Mg2+ share the same inhibitory site on the RyR channels. Our results agree with a previous study reporting a decrease of Mg2+ inhibition by oxidation in single RyR channels from cardiac muscle incorporated in lipid bilayers (Eager and Dulhunty, 1999).

To mimic the conditions that presumably prevail in vivo during CICR, we investigated the inhibitory effects of Mg2+ using a releasing solution containing 1.4 mM free ATP and 10 µM [Ca2+]. We found that under these conditions Mg2+ is a potent inhibitor of calcium release from native triads actively loaded with calcium, with a K0.5 for Mg2+ in the range of 60 µM. These results imply that in skeletal muscle CICR should be inhibited in vivo, since the Mg2+ inhibitory sites would be saturated at the normal concentrations of Mg2+ present in muscle cells that range around 1 mM (Konishi, 1998). Although there are several reports describing the inhibitory effect of Mg2+ on CICR in SR vesicles, few have described inhibition constants for Mg2+, giving values ranging from 15 µM to >100 µM (Meissner et al., 1996; Moutin and Dupont, 1988; Carrier et al., 1991). A straightforward comparison of these values is not possible because in these studies different [Ca2+] and nucleotide concentrations, which in themselves affect channel activity, were used.

To our knowledge, this work and our more extensive study (Donoso et al., 2000) are the first reports to show that oxidation decreases the inhibitory effect of Mg2+ on skeletal RyR channels, allowing fast CICR even at 1 mM free [Mg2+]. It has been reported that phosphorylation decreases Mg2+ inhibition of single RyR channels from skeletal muscle incorporated in lipid bilayers (Hain et al., 1994). Yet in our conditions, native triads that were extensively phosphorylated during active calcium loading (Barrientos et al., unpublished observations) retained the high affinity Mg2+ inhibitory sites, indicating that endogenous phosphorylation did not overcome the inhibitory effects of Mg2+. However, oxidized triads were not inhibited by 1 mM Mg2+, indicating that following oxidation CICR would take place in vivo. It has been proposed that the inhibitory effect of Mg2+ on RyR channel activity decreases during their physiological activation by the voltage sensors (Lamb and Laver, 1998). It remains to be tested in vivo whether channel oxidation takes place during the activation of RyR channels by the voltage sensors, thereby allowing the amplification by CICR of the release process in skeletal muscle.

Physiological and pathological implications of the present results. The RyR channels of excitable cells mediate skeletal and cardiac muscle contraction and neuronal functions such as the intracellular processes underlying gene expression, spatial learning and synaptic plasticity (Gosh and Greenberg, 1995; Berridge et al., 1998; Balschun et al., 1999). Consequently, modifications of RyR channel activity should have important consequences regarding the function of muscle cells and neurons. It is known in this regard that abnormal RyR channels underlie certain muscle pathological conditions (Zucchi and Ronca-Testoni, 1997) and that alterations of RyR channels correlate with the pathological stages of Alzheimer’s disease (Kelliher et al., 1999). The current results indicate that SH oxidation in vivo should increase CICR through RyR channels by the combined effects of increasing channel activation by µM [Ca2+] and decreasing channel inhibition by mM Mg2+. Therefore, oxidative stress through the generation of free radicals (Vladez et al., this issue) should enhance CICR in neurons and muscle. A concomitant increase in reactive oxygen species and cytoplasmic free [Ca2+] has been observed in conditions such as hypoxia, reperfusion following ischemia, and neurodegenerative diseases (Bonfoco et al., 1995; Kaneko et al., 1994). The present results suggest that through the resulting enhancement of CICR, oxidation of RyR channels may underlie the increase in cytoplasmic [Ca2+] that has been observed in these pathological conditions.


The contribution of Paula Aracena in some of the calcium release experiments is gratefully acknowledged. This study was supported by Fondo Nacional de Investigación Científica y Tecnológica grant 8980009. The institutional support of Fuerza Aérea de Chile, I. Municipalidad de Las Condes, and a group of Chilean companies (AFP Provida, CODELCO, Empresas CMPC MASISA S.A.and Telefónica del Sur) to CECS is acknowledged. CECS is a Millenium Science Institute.

Received: January 3, 2000. Accepted: January 3, 2000-

Corresponding Author: Dr. Cecilia Hidalgo ICBM, Facultad de Medicina. Universidad de Chile, Casilla 70005, Santiago 7, Chile. Phone: (56-2) 678-6510. FAX: (56-2) 776-6916. e-mail:


ABRAMSON JJ, ZABLE AC, FAVERO TF, SALAMA G (1995) Thimerosal interacts with the Ca2+ release channel ryanodine receptor from skeletal muscle sarcoplasmic reticulum. J Biol Chem 270: 29644-29647         [ Links ]

BALSCHUN D, WOLFER DP, BERTOCCHINI F, BARONE V, CONTI A, ZUSCHRATTER W, MISSIAEN L, LIPP HP, FREY JU, SORRENTINO V (1999) Deletion of the ryanodine receptor type 3 (RyR3) impairs forms of synaptic plasticity and spatial learning. EMBO J 18: 5264-5273         [ Links ]

BERRIDGE MJ (1997) Elementary and global aspects of calcium signalling. J Physiol 499, 291-306         [ Links ]

BERRIDGE MJ, BOOTMAN MD, LIPP P (1998) Calcium - a life and death signal. Nature 395: 645-648         [ Links ]

BONFOCO E, KRAINC D, ANKARCRONA M, NICOTERA P, LIPTON SA (1995) Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci USA 92: 7162-7166         [ Links ]

BULL R, MARENGO JJ (1993) Sarcoplasmic reticulum release channels from frog skeletal muscle display two types of calcium dependence. FEBS Lett 331: 223-227         [ Links ]

BULL R, MARENGO JJ (1994) Calcium-dependent halothane activation of sarcoplasmic reticulum calcium channels from frog skeletal muscle. Am J Physiol 266 (Cell Physiol 35): C391-C396         [ Links ]

CARRIER L, VILLAZ M, DUPONT Y (1991) Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs. Biochem Biophys Acta 1064: 175-183         [ Links ]

CORONADO R, MORRISSETTE J, SUKHAREVA M, VAUGHAN DM (1994) Structure and function of ryanodine receptor. Am J Physiol 266 (Cell Physiol 35): C1485-C1504         [ Links ]

DONOSO P, RODRÍGUEZ P, MARAMBIO P (1997) Rapid kinetic studies of SH-oxidation induced calcium release from sarcoplasmic reticulum vesicles. Arch Biochem Biophys 341: 295-299         [ Links ]

DONOSO P, ARACENA P, HIDALGO C (2000) SH oxidation overrides Mg2+ inhibition of calcium induced calcium release in skeletal muscle triads. Biophys J 79: 279-286 (In press)         [ Links ]

EAGER KR, RODEN LD, DULHUNTY AF (1997) Actions of sulfhydryl reagents on single ryanodine receptor Ca2+ -release channels from sheep myocardium. Am J Physiol 272 (Cell Physiol 41): C1908-C1918         [ Links ]

EAGER KR, DULHUNTY AF (1999) Cardiac ryanodine receptor activity is altered by oxidizing reagents in either the luminal or cytoplasmic solution. J Membrane Biol 167: 205-214         [ Links ]

ELFERINK JGR (1999) Thimerosal. A versatile sulfhydryl reagent, calcium mobilizer, and cell function-modulating agent. Gen Pharmacol 33: 1-6         [ Links ]

FRANZINI-ARMSTRONG C, PROTASI F (1997) Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions. Physiol Rev 77: 699-729         [ Links ]

GOSH A, GREENBERG ME (1995) Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 268: 239-247         [ Links ]

HAIN J, NATH S, MAYRLEITNER M, FLEISCHER S, SCHINDLER H (1994) Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. Biophys J 67: 1823-1833         [ Links ]

HIDALGO C, JORQUERA J, TAPIA V, DONOSO P (1993) Triads and transverse tubules isolated from frog skeletal muscle contain high levels of inositol 1,4,5-trisphosphate. J Biol Chem 208: 15111-15117         [ Links ]

KANEKO M, MATSUMOTO Y, HAYASHI H, KOBAYASHI A, YAMAZAKI N (1994) Oxygen free radicals and calcium homeostasis in the heart. Mol Cell Biochem 139: 91-100         [ Links ]

KELLIHER M, FASTBOM J, COWBURN RF, BONKALE W, OHM TG, RAVID R, SORRENTINO V, ONEILL C (1999) Alterations in the ryanodine receptor calcium release channel correlates with Alzheimer’s disease neurofibrillary and b-amyloid pathologies. Neuroscience 92: 499-513         [ Links ]

KONISHI M (1998) Cytoplasmic free concentrations of Ca2+ and Mg2+ in skeletal muscle fibers at rest and during contraction. Jpn J Physiol 48: 421-38         [ Links ]

LAMB GD, LAVER DR (1998) Adaptation, inactivation and inhibition in ryanodine receptors. In: SITSAPESAN R, WILLIAMS AJ (eds) The structure and function of ryanodine receptors. London: Imperial College Press. pp: 269-290         [ Links ]

LAVER DR, BAYNES TM, DULHUNTY AF (1997) Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms. J Membrane Biol 156: 213-219         [ Links ]

LEEB T, BRENIG B (1998) cDNA cloning and sequencing of the human ryanodine receptor type 3 (RYR3) reveals a novel alternative splice site in the RYR3 gene. FEBS Lett 423: 367-370         [ Links ]

LIU G, PESSAH IN (1994) Molecular interaction between ryanodine receptor and glycoprotein triadin involves redox cycling of functionally important hyperreactive sulfhydryls. J Biol Chem 269: 33028-33034         [ Links ]

MARENGO JJ, BULL R, HIDALGO C (1996) Calcium dependence of ryanodine-sensitive calcium channels from brain cortex endoplasmic reticulum. FEBS Letters 383: 59-62         [ Links ]

MARENGO JJ, HIDALGO C, BULL R (1998) Sulfhydryl oxidation modifies the calcium dependence of ryanodine-sensitive calcium channels of excitable cells. Biophys J 74: 1263-1277         [ Links ]

MARKS AR, TEMPST P, HWANG KS, TAUBMAN MB, INUI M, CHADWICK C, FLEISCHER S, NADAL-GINARD B (1989) Molecular cloning and characterization of the ryanodine receptor/junctional channel complex cDNA from skeletal muscle sarcoplasmic reticulum. Proc Natl Acad Sci USA 86: 8683-8687         [ Links ]

MEISSNER G (1994) Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors. Ann Rev Physiol 56: 485-508         [ Links ]

MEISSNER G, DARLING E, EVELETH J (1986) Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides. Biochemistry 25: 236-244         [ Links ]

MOUTIN MJ, DUPONT Y (1988) Rapid filtration studies of Ca2+ -induced Ca2+ release from skeletal sarcoplasmic reticulum. Role of monovalent ions. J Biol Chem 263: 4228-4235         [ Links ]

NAKASHIMA Y, NISHIMURA S, MAEDA A, BARSOUMIAN EL, HAKAMATA Y, NAKAI J, ALLEN PD, IMOTO K, KITA T (1997) Molecular cloning and characterization of a human brain ryanodine receptor. FEBS Lett 417: 157-162         [ Links ]

OTSU K, WILLARD HF, KHANNA VK, ZORZATO F, GREEN NM, MACLENNAN DH (1990) Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. J Biol Chem 265: 13472-13483.         [ Links ]

OYAMADA H, MURAYAMA T, TAKAGI T, IINO M, IWABE N, MIYATA T, OGAWA Y, ENDO M (1994) Primary structure and distribution of ryanodine-binding protein isoforms of the bullfrog skeletal muscle J Biol Chem 269: 17206-17214         [ Links ]

PEREZ C, MARENGO JJ, BULL R, HIDALGO C (1998) Cyclic ATP-ribose activates caffeine sensitive calcium channels from sea urchin egg microsomes. Am J Physiol 274 (Cell Physiol 43): C430-C439         [ Links ]

PRABHU SD, SALAMA G (1990) Reactive disulfide compounds induce Ca2+ release from cardiac sarcoplasmic reticulum. Arch Biochem Biophys 282: 275-283         [ Links ]

TAKESHIMA H, NISHIMURA S, MATSUMOTO T, ISHIDA H, KANGAWA K, MINAMINO N, MATSUO H, UEDA M, HANAOKA M, HIROSE T, NUMA S. (1989) Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 339: 439-445         [ Links ]

VALDEZ L., LORES ARNAIZ S., BUSTAMANTE J., ALVAREZ S., COSTA L.E., BOVERIS A. (2000) Free radical chemestry in biological systems. Biol Res. 33: 65-70         [ Links ]

WITCHER DR, KOVACS RJ, SCHULMAN H, CEFALI DC, JONES LR (1991) Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity. J Biol Chem 266: 11144-11152         [ Links ]

WU Y, AGHDASI B, DOU SJ, ZHANG JZ, LIU SQ, HAMILTON SL (1997) Functional interactions between cytoplasmic domains of the skeletal muscle Ca2+ release channel. J Biol Chem 272: 25051-25061         [ Links ]

ZHANG L, KELLEY J, SCHMEISSER G, KOBAYASHI Y M, JONES LR (1997) Complex formation between junctin, triadin, calsequestrin, and the ryanodine receptor. Proteins of the cardiac junctional sarcoplasmic reticulum membrane J Biol Chem 272: 23389-23397         [ Links ]

ZHANG JZ, WU Y, WILLIAMS BY, RODNEY G, MANDEL F, STRASBURG GM, HAMILTON SL (1999) Oxidation of the skeletal muscle Ca2+ release channel alters calmodulin binding. Am J Physiol (276) (Cell Physiol 45): C46-C53         [ Links ]

ZORZATO F, FUJII J, OTSU K, PHILLIPS M, GREEN NM, LAI FA, MEISSNER G, MACLENNAN DH (1990) 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         [ Links ]

ZUCCHI R, RONCA-TESTONI S (1997) The sarcoplasmic reticulum Ca2+ channel/ryanodine receptor: modulation by endogenous effectors, drugs and disease states. Pharmacol Rev 49: 1-51         [ Links ]

Creative Commons License All the contents of this journal, except where otherwise noted, is licensed under a Creative Commons Attribution License