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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.35 n.2 Santiago  2002 

Biol Res 35: 183-193, 2002

Redox regulation of calcium release in skeletal and
cardiac muscle


1Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Casilla 70005, Santiago 7, Chile, and
Centro de Estudios Científicos, Valdivia, Chile.


In skeletal and cardiac muscle cells, specific isoforms of the Ryanodine receptor channels mediate Ca2+ release from the sarcoplasmic reticulum. These channels are highly susceptible to redox modifications, which regulate channel activity. In this work, we studied the effects of Ca2+ (endogenous agonist) and Mg2+ (endogenous inhibitor) on the kinetics of Ca2+ release from sarcoplasmic reticulum vesicles isolated from skeletal or cardiac mammalian muscle. Native skeletal vesicles exhibited maximal stimulation of release kinetics by 10-20 µM [Ca2+], whereas in native cardiac vesicles, maximal stimulation of release required only 1 µM [Ca2+]. In 10 µM [Ca2+], free [Mg2+] < 0.1 mM produced marked inhibition of release from skeletal vesicles but free [Mg2+] ­ 0.8 mM did not affect release from cardiac vesicles. Incubation of skeletal or cardiac vesicles with the oxidant thimerosal increased their susceptibility to stimulation by Ca2+ and decreased the inhibitory effect of Mg2+ in skeletal vesicles. Sulfhydryl-reducing agents fully reversed the effects of thimerosal. The endogenous redox species, glutathione disulfide and S-nitrosoglutathione, also stimulated release from skeletal sarcoplasmic reticulum vesicles. In 10 µM [Ca2+], 35S-nitrosoglutathione labeled a protein fraction enriched in release channels through S-glutathiolation. Free [Mg2+] 1 mM or decreasing free [Ca2+] to the nM range prevented this reaction. Possible physiological and pathological consequences of redox modification of release channels on Ca2+ signaling in heart and muscle cells are discussed.

Key terms: Redox state; Ryanodine receptors; sarcoplasmic reticulum; calcium release kinetics; Mg2+ inhibition; S-nitrosoglutathione.


The generation of Ca2+ signals in response to extracellular stimuli entails transient changes in cytoplasmic free Ca2+ concentration. Ca2+ signals can in turn initiate and modulate other signal transduction cascades, which include short-term responses such as muscle contraction and secretion, and long-term responses including cell growth and proliferation (Berridge, 2000; Carafoli, 2002).

Intracellular [Ca2+] plays a fundamental role in the regulation of numerous enzymatic activities, including kinases, phosphatases, proteases, phospholipases and endonucleases. Changes in [Ca2+] inside the nucleus modify gene expression, and an alteration of intracellular Ca2+ homeostasis is an early event in apoptosis and in the development of irreversible cell injury. Ca2+ signals are highly organized in space, frequency and amplitude (see Peterson, 2002); all these parameters convey specific information to cells. As an example, recent studies indicate that slow Ca2+ signals link membrane stimulation to regulation of gene transcription in skeletal myotubes (see Jaimovich and Carrasco, 2002).

Following stimulation, contraction of muscle cells begins with an increase in intracellular Ca2+ concentration generated by Ca2+ release from internal stores, the sarcoplasmic reticulum (SR). In skeletal and cardiac muscle cells, tissue specific isoforms of the Ryanodine receptor channels (RyR channels) mediate Ca2+ release from the SR. During muscle relaxation, the released Ca2+ is removed from the cytoplasm by tissue specific Ca2+ pumps (SERCA or PMCA) or Na+/Ca2+ exchangers.

The physiological mechanisms of activation of RyR channels differ in skeletal and cardiac muscle (Lamb, 2000). The RyR channels of cardiac muscle are activated directly by Ca2+ entering the cells through voltage-activated L-type Ca2+ channels (Bers, 2002). This process is a classical example of Ca2+-induced Ca2+ release (CICR), an amplification mechanism by means of which a localized [Ca2+] increase generated by Ca2+ entry induces Ca2+ release from the stores, significantly increasing the first signal. In skeletal muscle, in contrast, Ca2+ release from the SR does not require Ca2+ entry into cells. The RyR channels of skeletal muscle open following membrane depolarization, presumably by direct coupling with plasma membrane voltage sensors (Ríos and Pizarro, 1991). In fact, CICR in mammalian skeletal muscle has been questioned because this tissue does not present spontaneous Ca2+ sparks (Shirokova et al., 1998). The strong inhibition of skeletal RyR channels by Mg2+ observed in vitro may contribute to the lack of CICR observed in this tissue (Meissner et al., 1986; Moutin and Dupont, 1988; Donoso and Hidalgo, 1993).

Mounting evidence indicates that Ryanodine receptor channels are highly susceptible to redox modifications, and that channel redox state controls channel activity. Oxidation of SH groups induces the release of Ca2+ from SR vesicles (Trimm et al., 1986; Zaidi et al., 1989; Prabhu and Salama, 1990; Salama et al., 1992; Abramson et al., 1995), activates RyR channels incorporated in planar lipid bilayers (Abramson et al., 1995; Favero et al., 1995; Eager et al., 1997; Marengo et al., 1998), and modifies Ryanodine binding to SR membranes (Abramson et al., 1995; Favero et al., 1995; Aghdasi et al., 1997; Suko and Hellman, 1998). Highly reactive sulfhydryl groups of the channel protein participate in interactions between homotetrameric channel subunits (Wu et al., 1997), in the formation of high molecular weight complexes with triadin (Liu et al., 1994; Liu and Pessah, 1994) and in calmodulin binding (Zhang et al., 1999; Porter-Moore et al., 1999).

Each 565-kDa protein subunit of the skeletal muscle channel homotetramer protein contains 100 sulfhydryl residues (Liu et al., 1994). Of these, approximately 50 sulfhydryl residues are in the reduced state (Takeshima et al., 1989), and a few (10-12) are highly susceptible to oxidation /modification by a variety of sulfhydryl reagents, including H2O2, organic mercurial compounds, maleimides, and powerful oxidants such as diamide (Trimm et al., 1986; Zaidi et al., 1989; Prabhu and Salama, 1990; Salama et al., 1992; Koshita et al., 1993; Liu et al., 1994; Abramson et al., 1995; Aghdasi et al., 1997; Donoso et al., 1997; Marengo et al., 1998; Suko and Hellman, 1998; Menshikova et al., 2000; Menshikova and Salama, 2000; Donoso et al., 2000). Some of these reagents - diamide or H2O2 - promote RyR subunit crosslinking by means of sulfhydryl oxidation and formation of disulfide bonds (Aghdasi et al., 1997). Others - N-ethylmaleimide or thimerosal - are more likely to modify RyR channel sulfhydryls by alkylation without significant crosslinking to yield intersubunit aggregates (Wu et al., 1997; Elferink, 1999; Menshikova et al., 2000). We have found that thimerosal enhances single RyR channel activity in lipid bilayers (Marengo et al., 1998). Thimerosal also stimulates CICR kinetics from SR vesicles isolated from mammalian skeletal muscle (Donoso et al., 2000; Hidalgo et al., 2000). As indicated above, in the physiological concentration range (0.7-1 mM) Mg2+ abolishes CICR from vesicles isolated from mammalian skeletal muscle. Yet, oxidation with thimerosal of triad-enriched SR vesicles from rabbit skeletal muscle produces a significant decrease of the inhibitory effect of Mg2+ on CICR kinetics (Donoso et al., 2000).

In addition to modification by non-physiological redox reagents, RyR channels are also susceptible to modification by endogenous redox agents. This channel behavior has distinct physiological relevance. Endogenous redox species, such as O2 (Eu et al., 2000), H2O2 (Favero et al., 1995; Suzuki et al., 1998) and glutathione (GSH) (Zable et al., 1997; Sun et al., 2001b) modulate RyR channel activity. Changes in the GSH - glutathione disulfide (GSSG) ratio (Feng et al., 2000; Xia et al., 2000; Oba et al., 2002), or incubation with NO or NO donors also modify RyR channel activity (Xu et al., 1998; Suko et al., 1999; Salama et al., 2000). NO donors regulate channel activity apparently by S-nitros ylation of a few critical sulfhydryl residues (Eu et al., 2000; Sun et al., 2001a; b).

In the present work, we studied the effects of thimerosal and of the endogenous redox-active molecules, GSSG and S-nitrosoglutathione (GSNO), on CICR kinetics from SR vesicles isolated from skeletal or cardiac mammalian muscle. In agreement with previous observations on skeletal SR vesicles (Donoso et al., 2000), we found that thimerosal also stimulated CICR from cardiac vesicles. Both GSSG and GSNO, stimulated CICR from skeletal SR vesicles in a way similar to thimerosal, indicating that skeletal RyR channels are also susceptible to activation by endogenous redox agents (Aracena et al., 2001). Furthermore, GSNO in 10 µM free [Ca2+] labeled vesicles isolated from skeletal muscle through S-glutathiolation, as demonstrated by covalent incorporation of 35S from [35S]-GSNO into a protein fraction enriched in RyR channels. This reaction, which required 10 µM Ca2+ because it did not occur in nM free [Ca2+], was suppressed by 1 mM free [Mg2+]. We discuss the possible physiological and pathological implications of these findings.


Materials. All reagents used were of analytical grade. Protease inhibitors and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All fluorescent Ca2+ indicators were from Molecular Probes, Inc. (Eugene, OR, USA), and GSH, GSSG and DTT were from Calbiochem (La Jolla, CA, USA). [35S]-GSH and [3H]-Ryanodine were purchased from New England Nuclear (Boston, MA, USA).

Membrane preparations. SR vesicles enriched in triads were isolated from rabbit fast skeletal muscle in the presence of a combination of protease inhibitors, as described previously (Hidalgo et al., 1993). Cardiac sarcoplasmic reticulum vesicles were isolated from canine hearts as described (Inui et al., 1988). All procedures were done according to the "Position of the American Heart Association on Research and Animal Use" and the guidelines of the Animal Care Committee of the Faculty of Medicine, University of Chile. SR vesicles were stored at -80 °C for up to one month. The protein content of membrane fractions was determined according to Hartree (1972) using bovine serum albumin as standard.

Synthesis of GSNO. The synthesis of GSNO was performed as described by Rossi et al (1997). The amount of GSNO generated was determined spectrophotometrically (Smith and Dasgupta, 2000); yields of GSNO were routinely in the 90 to 95% range. Synthesis of [35S]-GSNO from [35S]-GSH was performed as above.

Incubation of vesicles with thimerosal, GSSG or GSNO. Ca2+ loaded vesicles were incubated at 25°C or 37°C with thimerosal, GSSG or GSNO for variable lengths of time, as specified in Figure and Table legends. Radioactive labeling was performed by incubation with 4 mM [35S]-GSNO for 5 min at 25ºC in the ionic conditions described in the text.

Calcium release kinetics. Ca2+ release kinetics were determined in a SX.18MV stopped-flow spectrofluorometer from Applied Photophysics Ltd. (Leatherhead, U.K.) following the procedures described in detail elsewhere (Donoso et al., 2000). Before inducing release, vesicles were actively or passively loaded with Ca2+ as described (Donoso et al., 2000). The increase in extravesicular [Ca2+] produced by release was determined by measuring the fluorescence of a Ca2+ indicator (1 µM) selected according to the [Ca2+] range of the release solution (Donoso et al., 2000). Release solutions contained (final concentrations): 1.2 mM free ATP, variable free [Ca2+] and [Mg2+], 20 mM Mops/Tris, pH 7.2.

Isolation of RyR-enriched fractions. Following incubation of SR vesicles with [35S]-GSNO, incorporation of 35S-radioactivity into a protein fraction enriched in RyR channels was studied. For this purpose, RyR channels were solubilized with CHAPS and further purified in non-reducing sucrose density gradients essentially as described (Lee et al., 1994). Parallel sucrose gradients were run with CHAPS-solubilized fractions obtained from membranes incubated either with [35S]-GSNO or with 4 mM GSNO plus 2 nM [3H]-Ryanodine to identify channel enriched fractions. Gradient fractions were analyzed for 35S or 3H radioactivity in a liquid scintillation counter. The protein concentration of CHAPS-containing gradient fractions was determined as described (Kaplan and Pedersen, 1985).


To collect data within a time-frame compatible with physiological release rates, Ca2+ release kinetics were measured in a stopped flow fluorescence spectrophotometer. All vesicular preparations displayed Ca2+ release time courses that followed either single or double exponential functions; each exponential function was characterized by its rate constant k, that is directly linked to channel activity (Donoso et al., 1995). The k values of double exponential functions differed in magnitude by at least 5-fold. Only the higher k values were considered when release followed a double exponential time course.

Calcium dependence of Ca2+ release

To study the Ca2+ dependence, release was measured at a constant free [ATP] of 1.2 mM and at different free [Ca2+] to cover the [Ca2+] range 0.1 µM to 0.3 mM. Figure 1 illustrates how changing free [Ca2+] modifies the relative values of k in cardiac (solid circles) and skeletal (open circles) SR vesicles. In both cases, a bell shaped curve characterized the Ca2+ dependence of the rate constant k.

Skeletal SR vesicles displayed a Ca2+ dependence with maximal relative values of k in the [Ca2+] range 10-20 µM (Figure 1, open symbols). The absolute values of k varied from 40 to 50 s-1 in this [Ca2+] range. Decreasing or increasing free [Ca2+] beyond this range produced a significant decrease in k, to values < 3 s-1. These results indicate that 10-20 µM [Ca2+] enhanced markedly Ca2+ release from skeletal SR vesicles. For this reason, free [Ca2+] within this range were used to measure the effects of increasing [Mg2+] on Ca2+ release from skeletal SR, as detailed below.

In cardiac SR vesicles the highest k values (113 ± 12.5 s-1; n = 6)# were obtained in 1 µM free [Ca2+] (Figure 1, solid symbols). A marked decrease in release rate constants was observed at lower or higher [Ca2+] values. Thus k had an average value of 5.1 s -1 at 0.1 µM [Ca2+] (n = 2), whereas at 10 µM [Ca2+] k was 11.0 ± 1.4 s-1 (n = 8).

These combined results show that even in the constant presence of 1.2 mM free [ATP], Ca2+ enhanced markedly Ca2+ release kinetics from skeletal and cardiac SR vesicles. However, activation and inhibition of release by Ca2+ was significantly shifted to the left in cardiac SR when compared to skeletal SR vesicles. Furthermore, the maximal absolute values of k were about two-fold higher in cardiac than in skeletal SR. Assuming these results reflect the behavior of RyR channels in vivo, cardiac muscle release would be most efficient in 1 µM free [Ca2+]. However, Ca2+ release in vivo occurs in the presence of ~1 mM free [Mg2+]. Accordingly, to have a better approximation of the physiological situation, we measured CICR kinetics from both skeletal and cardiac SR vesicles at various free Mg2+ concentrations.

Fig. 1. Calcium dependence of Ca2+ release in cardiac and skeletal SR vesicles. The effect of increasing extravesicular free [Ca2+] on Ca2+ release rate constants was determined in vesicles actively (cardiac) or passively (skeletal) loaded with calcium. Calcium release was measured as described in the text. The Ca2+ indicators Fluo 3, Calcium Green-2, Calcium Green 5N and Fluo 5N were used to study release at the different free [Ca2+] illustrated in the Figure (Donoso et al., 2000). Solid circles: relative rate constants in cardiac SR vesicles; the maximal k value was 113 s-1. Open circles: relative rate constants in skeletal SR vesicles, where the maximal k value was 50 s-1. Data represent Mean ± S.E from independent determinations done in 2-6 different preparations.

Effects of [Mg2+] on CICR from skeletal and cardiac SR

In skeletal SR, Ca2+ release was maximally stimulated by Ca2+ when working in the 10-20 µM free [Ca2+] range, whereas release from cardiac SR vesicles was maximally stimulated by 1 µM free [Ca2+]. However, in 1 µM free [Ca2+], Mg2+ competes with Ca2+ for the high affinity Ca2+ activation site (Laver et al., 1997), but this competition does not occur at 10 µM free [Ca2+]. In agreement with these results, we found that 0.1 mM free [Mg2+] produced a significant inhibition of release from cardiac SR when activated by 1 µM free [Ca2+], 1.2 mM ATP (data not shown). This inhibition may reflect the reported competition of Mg2+ with 1 µM free [Ca2+] for the high affinity Ca2+ activation site (Laver et al., 1997). For this reason, the effects of Mg2+ on CICR in subsequent experiments were studied in the presence of 10 µM free [Ca2+], 1.2 mM free ATP and variable free [Mg2+]. The results, shown in Figure 2 and Table I, indicate that in the presence of 10 µM free [Ca2+] Mg2+ was a very effective inhibitor of CICR in skeletal (open symbols) but not in cardiac SR vesicles (solid symbols). As indicated in Table I, the K0.5 value for Mg2+ inhibition in native skeletal SR was 45.6 ± 4.2 µM, whereas no significant inhibition was observed in native cardiac SR vesicles.

Fig. 2. Effect of [Mg2+] on Ca2+ release rate constants. The rate constants of Ca2+ release were obtained in the presence of different free [Mg2+] in 10 µM free [Ca2+] in cardiac (solid circles) and skeletal (open circles) SR vesicles. The solid line represents the best fit to the equation k= kmax K0.5/([Mg2+] + K0,5). In this equation kmax is the rate constant obtained in the absence of Mg2+ (10.2 s-1 in cardiac and 15.7 s-1 in skeletal SR vesicles) and K05 represents the [Mg2+] that reduced kmax by half. Data represent the Mean ± S.E. from independent determinations done in 3-7 different preparations.

Thimerosal diminished the inhibitory effect of Mg2+ on CICR from SR vesicles


Skeletal SR   Cardiac SR



Native vesicles

45.6 ± 4.2
> 1000

+ 0.25 mM Thimerosal

96.9 ± 11.8
> 1000

+ 0.5 mM Thimerosal

> 1000

Vesicles were pre-incubated at 25ºC with the thimerosal concentrations indicated in the Table. The times of incubation with 0.25 mM thimerosal were 1 min and 5 min for vesicles from skeletal and cardiac muscle, respectively. The time of incubation with 0.5 mM thimerosal was 10 min for vesicles from skeletal muscle. Before incubation with thimerosal, vesicles were passively (skeletal) or actively (cardiac) loaded with calcium. Ca2+ release was measured in the presence of varying free [Mg2+], 10 µM free [Ca2+] as detailed in the text. The values of K0.5 represent the free [Mg2+] that produced 50% inhibition of release rate constants. Data, obtained from at least 3 independent experiments in each case, are expressed as Mean ± S.E.; N.D. stands for not determined.

Oxidation with thimerosal stimulates CICR in skeletal and cardiac SR vesicles

We have reported previously that thimerosal stimulates CICR kinetics from skeletal SR vesicles (Donoso et al., 2000). In the absence of Mg2+, skeletal SR vesicles incubated with either 250 µM or 500 µM thimerosal displayed release rate constant values significantly higher than native vesicles, as illustrated in Figure 3 (top panel). Likewise, incubation with 250 µM thimerosal produced a significant increase in k values in cardiac SR vesicles (Figure 3, lower panel).

Fig. 3. Effect of oxidation with thimerosal on Ca2+ release rate constants. Calcium loaded vesicles were incubated with thimerosal for 5 minutes at 25ºC. Release was induced by mixing one volume of SR vesicles with ten volumes of a solution that produced after mixing 1.2 mM ATP, and 10 µM free [Ca2+]. Data represent the Mean ± S.E. from independent determinations done in 2-7 different preparations. The symbol ** indicates p<0.01 compared to native vesicles, calculated with the one-way ANOVA test with the Newman-Keuls post test.

We have also reported that increasing free [Mg2+] strongly inhibited CICR from native skeletal SR vesicles but was less effective in modulating CICR from vesicles oxidized with thimerosal (Donoso et al., 2000). As summarized in Table I, oxidation of skeletal SR vesicles with 250 µM thimerosal increased the K0.5 values for Mg2+ inhibition from 45.6 ± 4.2 µM to 96.9 ± 11.8. Increasing thimerosal concentration to 0.5 mM suppressed completely the inhibitory effect of Mg2+ on CICR in the free [Mg2+] range tested (up to 1 mM), with a K0.5 value > 1 mM. These results indicate that partial oxidation of SH groups decreased the affinity of the skeletal RyR channels for Mg2+ whereas more extensive oxidation completely suppressed this inhibition.

The K0.5 values for Mg2+ inhibition in native cardiac SR, or in vesicles oxidized with thimerosal, were also > 1 mM, as indicated in Table I. These results show complete absence of Mg2+-dependent CICR inhibition with native or oxidized cardiac SR vesicles in the free [Mg2+] range tested (up to 0.8 mM). Similar finding have been described in single native cardiac SR channels incorporated in planar lipid bilayers (Bull et al., 2001).

Effects of GSSG and GSNO on CICR from skeletal SR vesicles

Incubation of skeletal SR vesicles with GSSG or GSNO resulted in a significant increase in the k values of CICR measured in the absence of Mg2+, as illustrated in Figure 4 (top panel). In contrast, incubation with 10 mM GSH for up to 30 min at 25 °C had no effect on CICR kinetics (data not shown). Incubation of vesicles with GSSG or GSNO produced an increase of more than two-fold in the K0.5 values for Mg2+ inhibition of CICR, as illustrated in Figure 4 (lower panel). Incubation of SR vesicles with higher concentrations of GSSG or GSNO did not increase further the K0.5 values (data not shown).

These combined results indicate that CICR in skeletal SR vesicles is stimulated not only by thimerosal but also by the endogenous redox species GSSG and GSNO, even in the presence of high free [Mg2+]. It remains to be tested in vivo if oxidation of RyR channels with endogenous redox agents allows CICR in mammalian skeletal muscle.

Fig. 4. Effect of oxidation with GSSG or GSNO on Ca2+ release rate constants. Skeletal SR vesicles, passively loaded with calcium, were incubated either with 10 mM GSSG at 37ºC for 5 min or with 4 mM GSNO at 25ºC for 5 min. Under these conditions, maximal activation of CICR by 10 µM free [Ca2+] in the absence of Mg2+ was obtained. We measured Ca2+ release kinetics at different free [Mg2+], up to 0.45 mM, from either native or GSSG-/GSNO-treated vesicles as described in the text. The half-maximal inhibitory free Mg2+ concentration (K0.5) was calculated as described above. Data, presented as Mean ± S.E., were obtained from at least three independent experiments. The symbols ** and * indicate p<0.01 and p<0.5, respectively, compared to native vesicles. The values of p were calculated as described in the legend to Figure 3.

[35S]-glutathionyl labeling of skeletal SR protein fraction enriched in RyR channels

SR vesicles incubated with 4 mM [35S]-GSNO were solubilized with CHAPS, to investigate possible 35S-incorporation into a protein fraction enriched in RyR channels. Following fractionation of the soluble protein fraction in CHAPS-containing sucrose density gradients, RyR channels were found in a minor protein peak that co-migrated with a [3H]-Ryanodine peak (not shown). As shown in Table II, 35S- incorporation in this protein peak required 10 µM free [Ca2+], since it was not detected when the incubation of SR vesicles with [35S]-GSNO was performed in the presence of 2 mM EGTA (1 nM free [Ca2+]). In addition, Mg2+ inhibited S-glutathiolation, because 35S-incorporation did not occur in 10 µM free [Ca2+] and 1 mM free [Mg2+] (Table II). These findings suggest that S-glutathiolation of sulfhydryl residues only occurs under conditions that activate RyR channels.

Previous studies indicate that incubation of cardiac RyR channels with GSNO produces channel activation by poly-S-nitrosylation (Eu et al., 1998). Recent findings indicate that protein sulfhydryl residues can also undergo S-glutathiolation following incubation with GSNO (Tao et al., 2002). The present results are the first demonstration that incubation with [35S]-GSNO results in covalent incorporation of 35S, through S-glutathiolation, of a SR protein fraction enriched in RyR channels.


Glutarhionyl incorporation into RyR-
enriched fractions





10 µM free [Ca2+]

1.67 ± 0.05 (n = 6)

1 nM free [Ca2+]

< 0.1 (n = 3)

10 µM free [Ca2+]+1 mM free [Mg2+]

< 0.1 (n = 3)

Vesicles were pre-incubated at 2 mg of protein per ml for 10 min at 25°C in the solutions defined in the Table. 2 mM EGTA was used to reduce the free [Ca2+] to 1 nM. Following pre-incubation, vesicles were incubated with 4 mM [35S]-GSNO for 5 min at 25ºC, and subjected to RyR channel purification by sucrose-density gradient centrifugation (see Material and Methods). Data correspond to Mean ± S.E.

However, we cannot determine at this point whether the observed 35S-incorporation was due to S-glutathiolation of RyR channels or of an associated protein, that forms a complex with RyR channels under non-reducing conditions. In living cells, skeletal RyR channels form supramolecular complexes through interactions with a plethora of accessory proteins that modulate channel function (for reviews, see Franzini-Armstrong and Protasi, 1997; Mackrill, 1999). In particular, sulfhydryl groups of the channel protein participate in the formation of high molecular weight complexes with triadin (Liu et al., 1994; Liu and Pessah, 1994) and calmodulin (Zhang et al., 1999; Porter-Moore et al., 1999). Accordingly, some of these interactions may remain after channel solubilization with CHAPS and further fractionation in sucrose gradients under non-reducing conditions.


The results shown in this work suggest that redox modifications of RyR channels will affect Ca2+ signaling in mammalian cardiac and skeletal muscle cells. In both cases, oxidation produced a significant increase in Ca2+ release. However, the effects of oxidation were more dramatic in skeletal muscle, where oxidation with endogenous redox species decreased the inhibitory effect of Mg2+ on CICR. It remains to be tested in vivo whether Ca2+ release through the skeletal RyR channels - activated by the voltage sensors - is further amplified through CICR when the muscle is exposed to an increase in ROS or to other oxidative conditions. Likewise, whether channel oxidation enhances CICR through cardiac RyR channels in vivo remains an open question. Reversible activation of cardiac RyR channels by oxidation could be of relevance in the heart, especially when there is an increase in free radical production such as in ischemia/reperfusion situations. In any case, the present results suggest that sustained or uncontrolled oxidation of RyR channels may elicit an imbalance in Ca2+ homeostasis. If not compensated, this imbalance may trigger cell death through apoptosis or necrosis.


This study was supported by Fondo Nacional de Investigación Científica y Tecnológica grants 8980009 and 100642. The institutional support to the Centro de Estudios Científicos by a group of Chilean companies (Compañía del Cobre, Dimacofi, Empresas CMPC, MASISA, and Telefónica del Sur) is also acknowledged. The Centro de Estudios Científicos is a Millennium Institute.


#Data are expressed as Mean ± S.E.


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*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) 777-6916. e-mail:

Received: June 05, 2002. In revised form: June 24, 2002. Accepted: July 14, 2002

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