Biol Res 37: 539-552, 2004

ARTICLE

Redox regulation of RyR-mediated Ca²⁺ release in muscle and neurons

CECILIA HIDALGO¹, RICARDO BULL¹, M. ISABEL BEHRENS² and PAULINA DONOSO¹

¹ FONDAP Center of Molecular Studies of the Cell and Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Casilla 70005, Santiago 7, Chile.

² Departamento de Neurología y Neurocirugía, Hospital Clínico de la Universidad de Chile, Santiago, Chile.

Dirección para Correspondencia

ABSTRACT

Changes in the redox state of the intracellular ryanodine receptor/Ca²⁺ release channels of skeletal and cardiac muscle or brain cortex neurons affect their activity. In particular, agents that oxidize or alkylate free SH residues of the channel protein strongly enhance Ca²⁺-induced Ca²⁺ release, whereas reducing agents have the opposite effects. We will discuss here how modifications of highly reactive cysteine residues by endogenous redox agents or cellular redox state influence RyR channel activation by Ca²⁺ and ATP or inhibition by Mg²⁺. Possible physiological and pathological implications of these results on cellular Ca²⁺ signaling will be addressed as well.

Key words: Redox state; ryanodine receptors; sarcoplasmic/endoplasmic reticulum; calcium release; S-nitrosylation; S-glutathionylation.

INTRODUCTION

Two different receptors mediate Ca²⁺ release from intracellular stores: the inositol trisphosphate-gated calcium channels (see Foskett and Mak, this issue) and the ryanodine receptors/Ca²⁺ release channels (RyR channels) (see Danila and Hamilton, this issue). Both kinds of release channels originate Ca²⁺-induced Ca²⁺ release (CICR), a powerful mechanism for the amplification and propagation of Ca²⁺ signals initially generated by Ca²⁺ entry into cells. In particular, RyR-mediated CICR has a central role in cardiac muscle contraction (see Franzini-Armstrong, Farrell et al., and Mackenzie et al., this issue), neuronal function (see Carrasco et al., Friel, Verkhratsky, this issue) and secretion (Petersen, this issue).

Multiple cellular components, such as ATP, H⁺, Ca²⁺ and Mg²⁺, specific proteins, including kinases, phosphatases, and redox species regulate RyR channels (Fill and Copello, 2002; see also in this issue: Danila and Hamilton; Schneider and Rodney; Gyorke et al.; Rios and Zhou) and may thus affect RyR-mediated Ca²⁺ release. Many studies have reported RyR channel modifications by non-physiological redox compounds, such as reactive disulfides or thimerosal, or by pharmacological concentrations of hydrogen peroxide (for reviews, see Hamilton and Reid, 2000; Hidalgo et al., 2002; Pessah et al., 2002). These studies have contributed to the current understanding of how changes in RyR redox state affect Ca²⁺ release and have promoted current research into the action of redox compounds of physiological significance such as nitric oxide (NO) and superoxide anion (O₂^•-), probably the most relevant free radicals generated by biological systems. Enzymatic or non-enzymatic chemical reactions readily convert these free radicals into non-radical species of lower reactivity, including hydrogen peroxide and GSNO.

The family of RyR channels comprises three mammalian isoforms (RyR1, RyR2 and RyR3) codified by three different genes localized in three different chromosomes in humans (chromosome 19, 1 and 15 respectively). They were originally identified in skeletal muscle (RyR1), in heart muscle (RyR2), and in brain (RyR3) although it is now known that brain tissue expresses all three mammalian RyR isoforms (Coronado et al., 1994; Furuichi et al., 1994; Mori et al., 2000). Here, we will review recent data showing that redox modifications of the RyR channels present in skeletal muscle, cardiac muscle or brain exert a central role in regulating single channel properties and CICR from vesicles and cells. We also will discuss physiological and pathological implication of RyR redox regulation in cells.

REDOX REGULATION OF SKELETAL MUSCLE RYR CHANNELS

The RyR1 isoforms mediate Ca²⁺ release in adult mammalian skeletal muscle. Physiological activation of RyR1-mediated Ca²⁺ release in response to muscle depolarization is a very fast process (ms range) which does not require Ca²⁺ entry into cells. RyR1 channels open following membrane depolarization presumably by direct coupling with plasma membrane voltage sensors (Franzini-Armstrong, Rios and Zhou, this issue). Each of the four homologous 565-kDa RyR1 protein subunits contains 100 cysteine residues (Takeshima et al., 1989). In the native channel, ≈ 50 of these residues appear to be in the reduced state; of these, ≈ 10-12 are highly susceptible to oxidation/modification by exogenous sulfhydryl (SH) reagents (Sun et al., 2001a).

Modification of RyR channel activity by ROS and RNS

Endogenous redox active molecules, including O₂ (Eu et al., 2000) and glutathione disulfide (GSSG) (Sun et al., 2001a; Zable et al., 1997; Sun et al., 2001b; Aracena et al., 2003) enhance RyR channel activity. Likewise, hydrogen peroxide in vitro markedly activates RyR1 channels under redox control (provided by resting cytoplasmic and luminal GSH/GSSG ratios) (Oba et al., 2002a).

In certain conditions, NO or NO donors also enhance RyR1 channel activity, while in others they exert an inhibitory effect (Suko et al., 1999; Sun et al., 2001a; Sun et al.; 2001b; Eu et al., 2003; Sun et al., 2003). In particular, in situ generation of NO stimulates whole muscle contractility at low (physiological) pO₂ but inhibits contractility at higher pO₂ (Eu et al., 2003). S-nitrosoglutathione (GSNO) and other pharmacological NO donors stimulate RyR1 channel activity through S-nitrosylation of a few critical SH residues (Sun et al., 2001b, Sun et al., 2003). Furthermore, we have shown recently that 3 cysteine residues per RyR1 channel monomer undergo GSNO-induced S-glutathionylation, i.e. the formation of a mixed disulfide between a protein SH residue and glutathione; this modification decreases specifically channel inhibition by Mg²⁺ without affecting channel activation by Ca²⁺ (Aracena et al., 2003). We also found that S-nitrosylation enhances RyR1 channel activation by Ca²⁺ but does not affect channel inhibition by Mg²⁺ (Fig.1). We discuss below the implications of these findings for redox activation of CICR in skeletal muscle.

Redox potential

The skeletal muscle cytoplasm has a GSH/GSSG ratio >30: 1; this value yields a reduction potential in the range of -230 mV. Skeletal SR vesicles possess a large transmembrane potential difference of about 50 mV between the cytoplasm and the more oxidized SR lumen (Pessah, 2001). Changes in the GSH/GSSG ratio affect RyR1 channel activity (Oba et al., 2002b; Xia et al., 2000; Feng et al., 2000). A transmembrane redox sensor within the RyR1 channel complex confers tight regulation of channel activity in response to changes in transmembrane redox potential; hyperreactive SH residues present within the RyR1 complex (Liu et al., 1994) are essential components of this transmembrane redox sensor (Feng et al., 2000; Pessah, 2001).

NADH/superoxide anion

A recent report describes O₂^•- generation by a NADH oxidase activity that is presumably responsible for the activation of [³H]-ryanodine binding produced by 1 mM NADH in heavy SR vesicles and which copurifies with RyR1 channels (Xia et al., 2003). Both NADH and NAD⁺ activate skeletal RyR channels incorporated in planar bilayers, whereas NADPH and NADP⁺ are without effect; yet, in the presence of ATP both NADH and NAD⁺ are ineffective as skeletal RyR agonists (Zima et al., 2003). These results suggest that NADH interacts with the ATP binding site(s) of the channels. In addition, skeletal muscle homogenates contain a constitutively active non-phagocytic NAD(P)H oxidase complex that generates O₂^•- and contributes to ROS production (Javesghani et al., 2002). We have found a constitutive NAD(P)H oxidase in skeletal muscle transverse tubules that stimulates nearby Ca²⁺ release channels in triads through hydrogen peroxide generation (Aracena, Sánchez, and Hidalgo, unpublished observations). Accordingly, while only NADH can activate RyR1 channels by direct binding to the channel protein (Zima et al., 2003), both NADH or NADPH may activate RyR1 channels by serving as substrates of the transverse tubule NAD(P)H oxidase.

Redox studies in skeletal muscle fibers

In vitro Mg²⁺ exerts a strong inhibition on RyR1 channel-mediated Ca²⁺ release (Meissner et al., 1986; Moutin and Dupont, 1988; Donoso et al., 2000; Aracena et al., 2003). This Mg²⁺ inhibition may be responsible for the reported lack of sparks caused by spontaneous CICR in skeletal muscle fibers (Shirokova et al., 1998; see also Rios and Zhou, and Schneider et al., this volume). RyR1 redox modifications that enhance channel activation by Ca²⁺ and ATP (Marengo et al., 1998; Oba et al., 2002b; Aracena et al., 2003), and especially those that reduce the powerful inhibitory effect of Mg²⁺ (Donoso et al., 2000; Aracena et al., 2003) may stimulate CICR in skeletal muscle fibers. Yet, a definite demonstration of the relevance of RyR1 redox modification in the context of physiological gating of the channel during excitation-contraction coupling is still missing (Lamb and Posterino, 2003). It is known, however, that skeletal muscle generates ROS following physiological contraction (Bejma and Ji, 1999; Reid and Durham, 2002), as well as during heat stress conditions (Stofan et al., 2000). As described above, skeletal muscle homogenates contain a NAD(P)H oxidase enzyme that generates O₂^•- (Javesghani et al., 2002). Likewise, mitochondrial complexes I and III and cytoplasmic xanthine oxidase are major sources of superoxide anion in diaphragm muscle (Nethery et al., 1999; Stofan et al., 2000). Following muscle contraction, O₂^•- has been detected in the extracellular space (Reid et al., 1992; Stofan et al., 2000; Zuo et al., 2000). This free radical is not likely to diffuse as O₂^•- through cell membranes but may undergo in vivo enzymatic or non-enzymatic dismutation to H₂O₂, which is readily permeable. Exogenous H₂O₂ stimulates caffeine-induced contraction in skinned fibers but does not affect action potential-generated contractions (Posterino et al., 2003). Furthermore, H₂O₂ activates contraction in skinned skeletal muscle fibers without an apparent increase in cytoplasmic Ca²⁺ concentration (Andrade et al., 1998), despite the fact that H₂O₂ activates RyR1 channels in isolated SR vesicles (Favero et al., 1995). Furthermore, while hydrogen peroxide markedly activates skeletal RyR channels in vitro, externally applied H₂O₂ does not play an important role in the post-fatigue recovery process (Oba et al., 2002a). Yet, the concentration of oxygen, which modulates the response of RyR channels to NO in skeletal muscle cells (Eu et al., 2003), may also modulate their response to O₂^•- or H₂O₂. Thus, care must be used when trying to extrapolate the in vitro behavior of RyR channels to their behavior in cells.

Figure 1. Effect of SH modification on calcium release from skeletal and cardiac SR vesicles. Upper panel: Skeletal SR vesicles actively loaded with calcium were incubated at 25ºC with NOR-3 (50 mM for 10 min), GSNO (500 mM for 20 min) or GSSG (5 mM for 20 min). Calcium release was induced by mixing in a stopped flow spectrofluorometer one volume of vesicles with 10 volumes of a solution that after mixing yielded pCa 5, 1.2 mM free ATP and either 25 mM or 0.7 mM free [Mg2+]. Bars show the rate constants (k) calculated from non-linear regression of the exponential time course records of Calcium Green 5N fluorescence. For further details, see Aracena et al., 2003. Lower panel: Cardiac SR vesicles actively loaded with calcium were incubated with and without 250 mM thimerosal as described (Sánchez et al., 2003). Calcium release was induced by mixing in a stopped flow spectrofluorometer one volume of vesicles with 10 volumes of a solution that produced after mixing pCa 5 or pCa 6, plus 1.2 mM free ATP and either 17 mM or 0.7 mM free [Mg2+]. The rate constants of calcium release, calculated from non-linear regression of exponential fluorescent records as above, are shown as the ratio between k values obtained in vesicles incubated with thimerosal and native vesicles (kthim/knative)

Skeletal RyR1 channels are endogenously S-glutathionylated (Aracena, Hamilton, and Hidalgo, unpublished observations), as well as endogenously S-nitrosylated (Sun et al., 2003). Protein S-nitrosylation increases markedly following activation of the NOS enzymes present in different types of cells (Gow et al., 2002; Martínez-Ruiz and Lamas, 2004). RyR1 channel S-nitrosylation caused by endogenous NO generation by nNOS is likely responsible for the increase in skeletal muscle contractility (Eu et al., 2003). In addition to S-nitrosylation, S-glutathionylation is a reversible post-translational protein modification that modulates the activities of several signaling molecules (Rao and Clayton, 2002; Mallis et al., 2001; Pineda-Molina et al., 2001). Cellular oxidative stress markedly activates S-glutathionylation of several proteins, as shown by redox proteome analysis (Fratelli et al., 2002; Lind et al., 2002). We have found that RyR1 channels in C2C12 cells in culture were endogenously S-glutathionylated and that brief (1 min) K⁺-induced depolarization significantly enhanced RyR1 S-glutathionylation; this enhancement was suppressed by inhibitors of the NAD(P)H oxidase enzyme (Aracena, P., Gilman, C., Hamilton, S. L. and Hidalgo, C., manuscript in preparation). These preliminary results suggest that depolarization enhances NOX enzyme-dependent ROS generation, which increase RyR1 S-glutathionylation in skeletal muscle cells in culture. It remains to be investigated whether RyR1 modification by S-glutathionylation also increases following physiological activation of skeletal muscle fibers and whether this modification affects contractility as S-nitrosylation does (Eu et al., 2003).

REDOX REGULATION OF CARDIAC RYR CHANNELS

In analogy to RyR1 channels, RyR2 channels also possess a few highly reactive SH residues susceptible to redox modification at physiological pH (Xu et al., 1998). The redox status of single cardiac RyR2 channels is a crucial determinant of their activity. RyR2 channel redox modification affects the open probability of single channels incorporated in planar lipid bilayers (Boraso and Williams, 1994; Eager et al., 1997; Kawakami and Okabe, 1998; Marengo et al., 1998; Eager and Dulhunty, 1998; Eager and Dulhunty, 1999). It also affects Ca²⁺ release from SR vesicles (Prabhu and Salama, 1990; Sanchez et al., 2003) or isolated cardiomyocytes (Suzuki et al., 1998). A discussion of the effects of different redox agents on the function of RyR2 channels measured both in vitro or in cells follows.

ROS/RNS

Cardiac cells possess a cytoplasmic extramitochondrial NADH oxidase activity that is the major source of superoxide anions in the cytosol (Mohazzab et al., 1997). This enzyme may contribute to the burst in superoxide production observed after reoxigenation of ischemic tissue (Bolli et al., 1988). In physiological conditions, superoxide anion generated by this NADH-oxidizing activity may enhance RyR2-mediated calcium release since superoxide anions induce calcium release from isolated SR vesicles (Kawakami and Okabe, 1998). Furthermore, hydrogen peroxide induces calcium release from intracellular stores in isolated cardiomyocytes; this effect is more prominent in cells previously dialyzed with reducing agents (Suzuki et al., 1998; Gen et al., 2001), suggesting that prior reduction of SH residues enhances H₂O₂ induced Ca²⁺ release (Suzuki et al., 1998). In contrast, reducing agents such as DTT or GSH strongly inhibit calcium release in cardiomyocytes (Suzuki et al., 1998).

There are reports describing either positive or negative effects of NO on cardiac RyR function. Nitric oxide induces calcium release in isolated cardiac SR vesicles (Stoyanovsky et al., 1997) and increases the open probability (P_o) of purified cardiac RyR in bilayers (Xu et al., 1998). In contrast, NO generated in situ from L-arginine inhibits calcium release in isolated SR preparation and decreases the open probability of single channels fused in planar lipid bilayer (Zahradnikova et al., 1997). Differences in the effective concentrations of NO used in these experiments may explain these discrepancies. In fact, there is growing consensus that in isolated cardiomyocytes NO donors can either enhance or inhibit Ca²⁺ release, depending on the concentration of NO donor used and the degree of b-adrenergic stimulation (Ziolo et al., 2001; Massion et al., 2003). Furthermore, cardiac RyR2 channels are endogenously S-nitrosylated (Xu et al., 1998), and it has been proposed that nNOS is directly involved in RyR2 regulation since the neuronal isoform of NOS localizes to the SR (Xu et al., 1999). Studies in isolated cardiomyocytes from nNOS knock-out (nNOS^-/-) mice have shown that this isoform is an important determinant of cardiac contractility (Sears et al., 2003; Khan et al., 2003). Myocytes from neuronal NOS^-/- mice exhibit an increase in resting contractile state and an enhanced inotropic response to b-adrenergic stimulation (Khan et al., 2003; Ashley et al., 2002). The endogenous level of RyR2 nitrosylation in these NOS^-/- animals is not known, however.

Recent studies indicate that NADH inhibits calcium release from SR vesicles and single channel activity in lipid bilayers (Zima et al., 2003). NADH also suppresses spontaneous calcium release and wave propagation in permeabilized cardiomyocytes (Cherednichenko et al., 2004; Zima et al., 2004). The inhibitory effect of NADH is presumably due to a direct effect of NADH on RyR2 channels or closely associated proteins and does not seem to be related to an NADH oxidase activity (Zima et al., 2004), which according to earlier studies stimulates Ca²⁺ release through superoxide production (Mohazzab et al., 1997; see Griendling et al., 2000, for a review).

Thimerosal

In agreement with previous observations on skeletal SR vesicles (Donoso et al., 2000), we have found that thimerosal also stimulates CICR kinetics from cardiac vesicles. Calcium release from native cardiac SR vesicles shows bell-shaped calcium dependence, with maximal rate constants at pCa 6 when measured with a time resolution of milliseconds (Sanchez et al., 2003). Magnesium decreases the rate constants of calcium release at pCa 6 with a K_0.5 of 60 mM, probably by competition with Ca²⁺ for channel activation sites (Laver et al., 1997). At pCa 6, incubation of cardiac RyR2 channels with thimerosal increases > 2-fold the K_0.5 for Mg²⁺ inhibition, suggesting that removal by alkylation of critical SH residues enhances Ca²⁺ activation over Mg²⁺ inhibition. At pCa 5, release rate constants are about 10 times lower than at pCa 6 but are not inhibited by [Mg²⁺] up to 0.7 mM. These results suggest that cardiac RyR channel activation sites are saturated with Ca²⁺ at pCa 5 and cannot be competed with Mg²⁺, and confirm the low affinity for Mg²⁺ of the inhibitory sites (Laver et al., 1997). Incubation with thimerosal produces a three-fold increase in release rate constants at pCa 5, measured in 17 mM or in 0.7 mM [Mg²⁺] (Fig.1). This stimulation supports the above proposal that thimerosal targets critical SH residues that enhance Ca²⁺-induced activation of RyR2 channels, as it does in skeletal RyR1 channels (Donoso et al., 2000). Furthermore, subsequent reduction of RyR2 channels with DTT produces a strong inhibition of Ca²⁺ release (Sánchez et al., 2003). Similar inhibition of Ca²⁺ efflux by SH reducing agents has been described in isolated cardiac SR vesicles after incubation with reactive disulfide compounds (Prabhu and Salama, 1990) as well as in single channel experiments (Boraso and Williams, 1994; Marengo et al., 1998).

REDOX REGULATION OF BRAIN RYR CHANNELS

Transient elevations of cytoplasmic Ca²⁺ concentration in neurons play a central role in the regulation of several neuronal functions such as excitability, synaptic transmission, synaptic plasticity and gene expression (Simpson et al., 1995; Berridge, 1998; Berridge et al., 2000). This increase in calcium concentration is initially produced by Ca²⁺ influx through plasma membrane Ca²⁺ channels. A role for RyR channel-mediated Ca²⁺ release from neuronal intracellular stores as an amplification mechanism of the initial Ca²⁺ signal is emerging (Chameau et al., 2001; Bouchard et al., 2003; Pape et al., 2004; Gafni et al., 2004; Ouardouz et al., 2003; Verkhratsky, 2002; Verkhratsky and Petersen, 2002). It is now accepted that RyR channel-mediated release of Ca²⁺ from the ER to the cytoplasm is required for synaptic plasticity and gene expression in neurons (Berridge 1998; Berridge et al., 2000; Carafoli, 2002; Futatsugi et al., 1999; Carrasco et al., this volume; Verkhratsky, this volume). RyR-mediated calcium release may be also involved in neurodegeneration as discussed below.

Rat brain expresses the three known mammalian RyR genes (Furuichi et al., 1994). RyR2 is the most abundant isoform, expressed in almost all brain structures with the highest levels found in the Amons horn, the dentate gyrus of the hippocampus and the granular layer of the cerebellum. RyR1 is expressed mainly in the dentate gyrus of the hippocampus and the Purkinje cell layer of the cerebellum, whereas RyR3 is expressed mainly in the CA1 region of the hippocampus and in astrocytes (Furuichi et al., 1994; Giannini et al., 1995; Matyash et al., 2002; Mori et al., 2000). Despite the emerging importance of RyR-mediated calcium release for brain function, there is practically no information on how these different RyR isoforms contribute to the generation and/or regulation of the calcium signals that underlie diverse calcium-dependent neuronal functions. Furthermore, the properties and regulation of RyR channels from brain have been less studied than those of their skeletal or cardiac counterparts.

In neurons, physiological activation of RyR channels may operate via CICR or by depolarization-induced calcium release (DICR), as occurs in cardiac or skeletal muscle, respectively. Influx of Ca²⁺ through voltage-dependent L-type channels is not required in DICR. A functional interaction between RyR and DHPR has been described in cerebellar granule cells (Chavis et al., 1996), whereas complexes immunoprecipitated from solubilized rat brain membranes with antibodies against L-type channels contain RyR1 but not RyR2 (Mouton et al., 2001). Moreover, DICR was recently reported in hypothalamic neurons (De Crescenzo et al., 2004) and in spinal cord axons (Ouardouz et al., 2003).

CICR in synaptic terminals

Intracellular calcium stores responsive to caffeine and thapsigargin modulate presynaptic protein synthesis (Benech et al., 1999), suggesting that CICR plays an important role in protein synthesis under conditions where presynaptic calcium is elevated (Alkon et al., 1998). Furthermore, a role for presynaptic Ca²⁺ uptake and release in neurotransmission was recognized over two decades ago (Erulkar and Rahamimoff, 1978). More recently, it has become possible to record from both pre- and post-synaptic elements simultaneously (see Zucker and Regehr, 2002); the data obtained suggest that CICR contributes significantly to neuronal Ca²⁺ transients and neurotransmitter release. It is noteworthy that presynaptic CICR has been implicated in many different types of synapses: glutamatergic, cholinergic, dopaminergic and neuropeptidergic (for a review, see Bouchard et al., 2003). Not all studies have agreed on the role of presynaptic CICR since there is some discrepancy among studies, probably related to the different experimental methods used. Recently, however, the first direct demonstration of RyR-mediated mobilization of Ca²⁺ from intracellular stores induced by depolarization in the absence of Ca²⁺ influx was reported in hypothalamic neurons (De Crescenzo et al., 2004). A role for RyR-mediated Ca²⁺ release in postsynaptic terminals is discussed more extensively elsewhere in this issue (Carrasco et al, this volume).

Redox modulation of the activation of brain RyR channels by Ca²⁺ and ATP

CICR relies on the fact that RyR channels are activated by an increase in cytoplasmic Ca²⁺ concentration, from the sub-micromolar to the micromolar range; in cells, this activation occurs in the presence of ATP. We have studied at the single-channel level the activation induced by Ca²⁺ and ATP of native and oxidized RyR channels from rat brain endoplasmic reticulum. RyR channels from rat brain cortex incorporated in planar lipid bilayers display three different Ca²⁺ dependencies (Marengo et al., 1996). The most frequently observed response was bell shaped, with a maximal P_o < 0.1, followed by a bell-shaped calcium response characteristic of RyR1 channels and much less frequently by a sigmoidal response characteristic of RyR2 channels. Noteworthy, changes of the oxidation state of the channel protein determine the Ca²⁺ dependence of RyR channels derived from brain cortex or from skeletal or cardiac muscle (Marengo et al., 1998). Thus, we have been able to observe the three calcium responses in the same single channel incorporated in the bilayer by sequential modification of its redox state (Marengo et al., 1998). In brief, highly-reduced channels respond poorly to Ca²⁺ activation, whereas increasing channel oxidation state increases the channel response to mM [Ca²⁺] and decreases the inhibitory effect of higher [Ca²⁺]; reducing agents reverse all these changes (Fig. 2).

In addition, we found that ATP differentially activates RyR channels depending on their calcium response. More oxidized channels, which are more responsive to Ca²⁺, require less ATP to attain maximal activation (Bull et al., 2003). Figure 3 illustrates the effects of sequential oxidation of low activity channels on the apparent activation constants for Ca²⁺ and ATP and the inhibition constant for Ca²⁺.

Channel oxidation also induces coordination in sub-channel gating. At pCa 5 RyR channels reveal multiple subconductance states; after SH oxidation, the frequency of these substates decreases because the channel subunits gate in a concerted fashion, favoring closed and full open states (Bull et al., 2003). From the results, it was proposed that RyR channels from rat brain activated by ATP and calcium function as four independent subchannels in the reduced state.

Changes in the redox state of RyR channels from brain also affects channel modulation by other agonists such as Mg²⁺ (Humeres, A. and Hidalgo, C., unpublished observations) or glucosylceramide, a type of glycosphingolipid (Lloyd Evans et al., 2003). Interestingly, ceramides may be implicated in cell death caused by Alzheimer's disease (Cutler et al., 2004) and HIV dementia (Haughey et al., 2004). In this context, the recent study showing that glucosylceramide augments agonist-stimulated Ca²⁺ release via RyR channels through a mechanism that may involve the redox sensor of the RyR without a direct effect on Ca²⁺ release (Lloyd Evans et al., 2003) may be of relevance to understanding the development of pathological conditions.

Figure 2. Effect of SH modification on calcium dependence of brain RyR channels. Schematic representation of the three different responses in fractional open time (Po) to changes in cytoplasmic [Ca2+] of brain RyR channels incorporated in planar lipid bilayers. SH oxidation modified the calcium dependence from the low Po behavior to the medium (M) Po response, and from the M Po to the high Po response. Reduction with glutathione of channels with high or M Po behavior shifted the calcium dependence to the M Po or the low Po behavior, respectively.

Through either DICR or CICR, RyR channels have a pivotal role in the generation or amplification of calcium signals that are required for contraction of muscle cells and neuronal plasticity. Endogenous activation of RyR channels by cellular ROS/RNS may represent a physiological mechanism of communication between Ca²⁺ and redox signaling pathways. Thus, cells may use redox-modulated RyR-mediated Ca²⁺ release as an additional mechanism to either amplify or inhibit Ca²⁺ signals as needed for a specific response. In the case of neurons, generation of ROS such as hydrogen peroxide, which acts as a diffusible signal molecule in synaptic plasticity (Kamsler and Segal, 2004), could modify cellular processes that depend on RyR-mediated Ca²⁺ release from the ER, including long-term potentiation and long-term depression, and presumably, learning and memory (see Carrasco et al., this issue). Oxidative stress and alterations in Ca²⁺ homeostasis may contribute to neuronal apoptosis and excitotoxicity, which may underlie the pathogenesis of several neurodegenerative disorders (Mattson, 2000; Mattson and Chan, 2003; Toescu and Verkhratsky, 2003). In particular, RyR channels may be involved in the pathophysiology of neurodegeneration in Alzheimer's disease (Kelliher et al., 1999; Chan et al., 2000; Mungarro-Menchaca et al., 2002). As discussed here, oxidative stress is likely to enhance RyR-mediated CICR in neurons. Thus, redox modification of RyR channels may have both physiological and pathological consequences for neuronal function.

Figure 3. Effect of SH modification on the apparent calcium activation and inhibition constants of brain RyR channels. Single rat brain RyR channels were incorporated into planar lipid bilayers and changes in fractional open time (Po) values as a function of cytoplasmic [Ca2+] or [ATP] concentrations were determined, before and after channel oxidation in vitro. Before oxidation the channels usually displayed the low Po behavior; after treatment they attained the M or the high Po response to cytoplasmic [Ca2+ ]. Ka and Ki values for calcium were obtained by nonlinear curve fitting of Po vs [Ca2+] data to the equation: Po = Pomax * [Ca2+]n * Ki / (([Ca2+]n + Kan) * ([Ca2+] + Ki)) Ka values for ATP were obtained by nonlinear curve fitting of Po vs [ATP] data to a hyperbolic equation. Bars represent the K values obtained from the nonlinear fits and error bars the S.D. of fitted values.

ACKNOWLEDGEMENTS

This work was supported by FONDAP Center for Molecular Studies of the Cell, Fondo Nacional de Investigación Científica y Tecnológica (FONDECYT) 15010006, and by FONDECYT grants 1030449 and 1040717.

REFERENCES

ALKON DL, NELSON TJ, ZHAO W, CAVALLARO S (1998) Time domains of neuronal Ca²⁺ signaling and associative memory: Steps through a calexcitin, ryanodine receptor, K⁺ channel cascade. Trends Neurosci 21: 529-537

ANDRADE FH, REID MB, ALLEN DG, WESTERBLAD H (1998) Effect of hydrogen peroxide and dithiothreitol on contractile function of single skeletal muscle fibres from the mouse. J Physiol 509: 565-575

ARACENA P, SÁNCHEZ G, DONOSO P, HAMILTON SL, HIDALGO C (2003) S-glutathionylation decreases Mg²⁺ inhibition and S-nitrosylation enhances Ca²⁺ activation of RyR1 channels. J Biol Chem 278: 42927-42935

ASHLEY EA, SEARS CE, BRYANT SM, WATKINS HC, CASADEI B (2002) Cardiac nitric oxide synthase 1 regulates basal and beta-adrenergic contractility in murine ventricular myocytes. Circulation 105: 3011-3016

BEJMA J, JI LL (1999) Aging and acute exercise enhance free radical generation in rat skeletal muscle. J Appl Physiol 87: 465-470

BENECH JC, CRISPINO M, KAPLAN BB, GIUDITTA A (1999) Protein synthesis in presynaptic endings from squid brain: Modulation by calcium ions. J Neurosci Res 55: 776-781

BERRIDGE MJ (1998) Neuronal calcium signaling. Neuron 21: 13-26

BERRIDGE MJ, LIPP P, BOOTMAN MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1: 11-21

BOLLI R, PATEL BS, JEROUDI MO, LAI EK, MCCAY PB (1988) Demonstration of free radical generation in "stunned" myocardium of intact dogs with the use of the spin trap alpha-phenyl N-tert-butyl nitrone. J Clin Invest 82: 476-485

BORASO A, WILLIAMS AJ (1994) Modification of the gating of the cardiac sarcoplasmic reticulum Ca(2+)-release channel by H2O2 and dithiothreitol. Am J Physiol 267: H1010-H1016

BOUCHARD R, PATTARINI R, GEIGER JD (2003) Presence and functional significance of presynaptic ryanodine receptors. Prog Neurobiol 69: 391-418

BULL R, MARENGO JJ, FINKELSTEIN JP, BEHRENS MI, ALVAREZ O (2003) SH oxidation coordinates subunits of rat brain ryanodine receptor channels activated by calcium and ATP. Am J Physiol Cell Physiol 285: C119-C128

CARAFOLI E (2002) Calcium signaling: A tale for all seasons. Proc Natl Acad Sci USA 99: 1115-1122

CARRASCO MA, JAIMOVICH E, KEMMERLING U, HIDALGO C (2004) Signal transduction and gene expression regulated by calcium release from internal stores in excitable cells. Biol Res 37: 701-712 CHAMEAU P, VAN D, V, FOSSIER P, BAUX G (2001) Ryanodine-, IP3- and NAADP-dependent calcium stores control acetylcholine release. Pflugers Arch 443: 289-296

CHAN SL, MAYNE M, HOLDEN CP, GEIGER JD, MATTSON MP (2000) Presenilin-1 mutations increase levels of ryanodine receptors and calcium release in PC12 cells and cortical neurons. J Biol Chem 275: 18195-18200

CHAVIS P, FAGNI L, LANSMAN JB, BOCKAERT J (1996) Functional coupling between ryanodine receptors and L-type calcium channels in neurons. Nature 382: 719-722

CHEREDNICHENKO G, ZIMA AV, FENG W, SCHAEFER S, BLATTER LA, PESSAH IN (2004) NADH oxidase activity of rat cardiac sarcoplasmic reticulum regulates calcium-induced calcium release. Circ Res 94: 478-486

CORONADO R, MORRISSETTE J, SUKHAREVA M, VAUGHAN DM (1994) Structure and function of ryanodine receptors. Am J Physiol 266: C1485-C1504

CUTLER RG, KELLY J, STORIE K, PEDERSEN WA, TAMMARA A, HATANPAA K, TRONCOSO JC, MATTSON MP (2004) Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease. Proc Natl Acad Sci USA 101: 2070-2075

DANILA CI, HAMILTON SL (2004) Phosphorylation of ryanodine receptors. Biol Res 37: 521-525

DE CRESCENZO V, ZHUGE R, VELÁZQUEZ-MARRERO C, LIFSHITZ LM, CUSTER E, CARMICHAEL J, LAI FA, TUFT RA, FOGARTY KE, LEMOS JR, WALSH JV, JR (2004) Ca²⁺ syntillas, miniature Ca²⁺ release events in terminals of hypothalamic neurons, are increased in frequency by depolarization in the absence of Ca²⁺ influx. J Neurosci 24: 1226-1235

DONOSO P, ARACENA P, HIDALGO C (2000) Sulfhydryl oxidation overrides Mg(2+) inhibition of calcium-induced calcium release in skeletal muscle triads. Biophys J 79: 279-286

EAGER KR, DULHUNTY AF (1998) Activation of the cardiac ryanodine receptor by sulfhydryl oxidation is modified by Mg²⁺ and ATP. J Membr Biol 163: 9-18

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

EAGER KR, RODEN LD, DULHUNTY AF (1997) Actions of sulfhydryl reagents on single ryanodine receptor Ca(2+)-release channels from sheep myocardium. Am J Physiol 272: C1908-C1918

ERULKAR SD, RAHAMIMOFF R (1978) The role of calcium ions in tetanic and post-tetanic increase of miniature end-plate potential frequency. J Physiol 278: 501-511

EU JP, SUN J, XU L, STAMLER JS, MEISSNER G (2000) The skeletal muscle calcium release channel: Coupled O2 sensor and NO signaling functions. Cell 102: 499-509

EU JP, HARE JM, HESS DT, SKAF M, SUN J, CÁRDENAS-NAVINA I, SUN QA, DEWHIRST M, MEISSNER G, STAMLER JS (2003) Concerted regulation of skeletal muscle contractility by oxygen tension and endogenous nitric oxide. Proc Natl Acad Sci USA 100: 15229-15234

FARRELL EF, ANTARAMIAN A, BENKUSKY N, XHU X, RUEDA A, GÓMEZ AM, VALDIVIA HH (2004) Regulation of cardiac excitation-contraction coupling by sorcin, a novel modulator of ryanodine receptors. Biol Res 37: 609-612

FENG W, LIU G, ALLEN PD, PESSAH IN (2000) Transmembrane redox sensor of ryanodine receptor complex. J Biol Chem 275: 35902-35907

FILL M, COPELLO JA (2002) Ryanodine receptor calcium release channels. Physiol Rev 82: 893-922

FOSKETT K, MAK, DOD (2004) Novel model of inositol 1,4,5-trisphosphate regulation of InsP3 receptor channel gating in native endoplasmic reticulum. Biol Res 37: 513-519

FRANZINI-ARMSTRONG C (2004) Functional implications of RyR-DHPR relationships in skeletal and cardiac muscles. Biol Res 37: 507-512

FRIEL D (2004) Interplay between ER Ca²⁺ uptake and release fluxes in neurons and its impact on [Ca²⁺] dynamics. Biol Res 37: 665-674

FRATELLI M, DEMOL H, PUYPE M, CASAGRANDE S, EBERINI I, SALMONA M, BONETTO V, MENGOZZI M, DUFFIEUX F, MICLET E, BACHI A, VANDEKERCKHOVE J, GIANAZZA E, GHEZZI P (2002) Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc Natl Acad Sci USA 99: 3505-3510

FURUICHI T, FURUTAMA D, HAKAMATA Y, NAKAI J, TAKESHIMA H, MIKOSHIBA K (1994) Multiple types of ryanodine receptor/Ca²⁺ release channels are differentially expressed in rabbit brain. J Neurosci 14: 4794-4805

FUTATSUGI A, KATO K, OGURA H, LI ST, NAGATA E, KUWAJIMA G, TANAKA K, ITOHARA S, MIKOSHIBA K (1999) Facilitation of NMDAR-independent LTP and spatial learning in mutant mice lacking ryanodine receptor type 3. Neuron 24: 701-713

GAFNI J, WONG PW, PESSAH IN (2004) Non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95) amplifies ionotropic glutamate receptor signaling in embryonic cerebellar granule neurons by a mechanism involving ryanodine receptors. Toxicol Sci 77: 72-82

GEN W, TANI M, TAKESHITA J, EBIHARA Y, TAMAKI K (2001) Mechanisms of Ca²⁺ overload induced by extracellular H2O2 in quiescent isolated rat cardiomyocytes. Basic Res Cardiol 96: 623-629

GIANNINI G, CONTI A, MAMMARELLA S, SCROBOGNA M, SORRENTINO V (1995) The ryanodine receptor/calcium channel genes are widely and differentially expressed in murine brain and peripheral tissues. J Cell Biol 128: 893-904

GOW AJ, CHEN Q, HESS DT, DAY BJ, ISCHIROPOULOS H, STAMLER JS (2002) Basal and stimulated protein S-nitrosylation in multiple cell types and tissues. J Biol Chem 277: 9637-9640

GRIENDLING KK, SORESCU D, USHIO-FUKAI M (2000) NAD(P)H oxidase: Role in cardiovascular biology and disease. Circ Res 86: 494-501

GYORKE S, GYORKE I, TERENTYEV D, VIATCHENKO-KARPISNKI S, WILLIAMS SC (2004) Modulation of sarcoplasimc reticulum calcium release by calsequestrin in cardiac myocytes. Biol Res 37: 603-607

HAMILTON SL, REID MB (2000) RyR1 modulation by oxidation and calmodulin. Antioxid Redox Signal 2: 41-45

HAUGHEY NJ, CUTLER RG, TAMARA A, MCARTHUR JC, VARGAS DL, PARDO CA, TURCHAN J, NATH A, MATTSON MP (2004) Perturbation of sphingolipid metabolism and ceramide production in HIV-dementia. Ann Neurol 55: 257-267 HIDALGO C, ARACENA P, SÁNCHEZ G, DONOSO P (2002) Redox regulation of calcium release in skeletal and cardiac muscle. Biol Res 35: 183-193

JAVESGHANI D, MAGDER SA, BARREIRO E, QUINN MT, HUSSAIN SN (2002) Molecular characterization of a superoxide-generating NAD(P)H oxidase in the ventilatory muscles. Am J Respir Crit Care Med 165: 412-418

KAMSLER A, SEGAL M (2004) Hydrogen peroxide as a diffusible signal molecule in synaptic plasticity. Mol Neurobiol 29: 167-178

KAWAKAMI M, OKABE E (1998) Superoxide anion radical-triggered Ca²⁺ release from cardiac sarcoplasmic reticulum through ryanodine receptor Ca²⁺ channel. Mol Pharmacol 53: 497-503

KELLIHER M, FASTBOM J, COWBURN RF, BONKALE W, OHM TG, RAVID R, SORRENTINO V, O'NEILL C (1999) Alterations in the ryanodine receptor calcium release channel correlate with Alzheimer's disease neurofibrillary and beta-amyloid pathologies. Neuroscience 92: 499-513

KHAN SA, SKAF MW, HARRISON RW, LEE K, MINHAS KM, KUMAR A, FRADLEY M, SHOUKAS AA, BERKOWITZ DE, HARE JM (2003) Nitric oxide regulation of myocardial contractility and calcium cycling: Independent impact of neuronal and endothelial nitric oxide synthases. Circ Res 92: 1322-1329

LAMB GD, POSTERINO GS (2003) Effects of oxidation and reduction on contractile function in skeletal muscle fibres of the rat. J Physiol 546: 149-163

LAVER DR, BAYNES TM, DULHUNTY AF (1997) Magnesium inhibition of ryanodine-receptor calcium channels: evidence for two independent mechanisms. J Membr Biol 156: 213-229

LIND C, GERDES R, HAMNELL Y, SCHUPPE-KOISTINEN I, VON LOWENHIELM HB, HOLMGREN A, COTGREAVE IA (2002) Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis. Arch Biochem Biophys 406: 229-240

Liu G, Abramson JJ, Zable AC, Pessah IN (1994) Direct evidence for the existence and functional role of hyperreactive sulfhydryls on the ryanodine receptor-triadin complex selectively labeled by the coumarin maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin. Mol Pharmacol 45: 189-200

LLOYD-EVANS E, PELLED D, RIEBELING C, BODENNEC J, DE MORGAN A, WALLER H, SCHIFFMANN R, FUTERMAN AH (2003) Glucosylceramide and glucosylsphingosine modulate calcium mobilization from brain microsomes via different mechanisms. J Biol Chem 278: 23594-23599

MACKENZIE L, RODERICK HL, PROVEN A, BOOTMAN MD (2004) Inositol 1,4,5-trisphospate receptors in the heart. Biol Res 37: 553-557

MALLIS RJ, BUSS JE, THOMAS JA (2001) Oxidative modification of H-ras: S-thiolation and S-nitrosylation of reactive cysteines. Biochem J 355: 145-153

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

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

MARTÍNEZ-RUIZ A, LAMAS S (2004) S-nitrosylation: A potential new paradigm in signal transduction. Cardiovasc Res 62: 43-52

MATTSON MP (2000) Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1: 120-129

MATTSON MP, CHAN SL (2003) Neuronal and glial calcium signaling in Alzheimer's disease. Cell Calcium 34: 385-397

MATYASH M, MATYASH V, NOLTE C, SORRENTINO V, KETTENMANN H (2002) Requirement of functional ryanodine receptor type 3 for astrocyte migration. FASEB J 16: 84-86

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

MOHAZZAB H, KAMINSKI PM, WOLIN MS (1997) Lactate and PO2 modulate superoxide anion production in bovine cardiac myocytes: Potential role of NADH oxidase. Circulation 96: 614-620

MORI F, FUKAYA M, ABE H, WAKABAYASHI K, WATANABE M (2000) Developmental changes in expression of the three ryanodine receptor mRNAs in the mouse brain. Neurosci Lett 285: 57-60

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

MOUTON J, MARTY I, VILLAZ M, FELTZ A, MAULET Y (2001) Molecular interaction of dihydropyridine receptors with type-1 ryanodine receptors in rat brain. Biochem J 354: 597-603

MUNGARRO-MENCHACA X, FERRERA P, MORÁN J, ARIAS C (2002) Beta-amyloid peptide induces ultrastructural changes in synaptosomes and potentiates mitochondrial dysfunction in the presence of ryanodine. J Neurosci Res 68: 89-96

NETHERY D, STOFAN D, CALLAHAN L, DIMARCO A, SUPINSKI G (1999) Formation of reactive oxygen species by the contracting diaphragm is PLA(2) dependent. J Appl Physiol 87: 792-800

OBA T, KURONO C, NAKAJIMA R, TAKAISHI T, ISHIDA K, FULLER GA, KLOMKLEAW W, YAMAGUCHI M (2002a) H2O2 activates ryanodine receptor but has little effect on recovery of releasable Ca²⁺ content after fatigue. J Appl Physiol 93: 1999-2008

OBA T, MURAYAMA T, OGAWA Y (2002b) Redox states of type 1 ryanodine receptor alter Ca(2+) release channel response to modulators. Am J Physiol Cell Physiol 282: C684-C692

OUARDOUZ M, NIKOLAEVA MA, CODERRE E, ZAMPONI GW, MCRORY JE, TRAPP BD, YIN X, WANG W, WOULFE J, STYS PK (2003) Depolarization-induced Ca²⁺ release in ischemic spinal cord white matter involves L-type Ca2+ channel activation of ryanodine receptors. Neuron 40: 53-63

PAPE HC, MUNSCH T, BUDDE T (2004) Novel vistas of calcium-mediated signalling in the thalamus. Pflugers Arch 448: 131-138

PETERSEN O (2004) local and global Ca²⁺ signals: Physiology and pathophysiology. Biol Res 37: 661-664

PESSAH IN (2001) Ryanodine receptor acts as a sensor for redox stress. Pest Manag Sci 57: 941-945

PESSAH IN, KIM KH, FENG W (2002) Redox sensing properties of the ryanodine receptor complex. Front Biosci 7: a72-a79

PINEDA-MOLINA E, KLATT P, VÁZQUEZ J, MARINA A, GARCÍA DL, PÉREZ-SALA D, LAMAS S (2001) Glutathionylation of the p50 subunit of NF-kappaB: A mechanism for redox-induced inhibition of DNA binding. Biochemistry 40: 14134-14142

POSTERINO GS, CELLINI MA, LAMB GD (2003) Effects of oxidation and cytosolic redox conditions on excitation-contraction coupling in rat skeletal muscle. J Physiol 547: 807-823

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

RAO RK, CLAYTON LW (2002) Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation. Biochem Biophys Res Commun 293: 610-616

REID MB, SHOJI T, MOODY MR, ENTMAN ML (1992) Reactive oxygen in skeletal muscle II. Extracellular release of free radicals. J Appl Physiol 73: 1805-1809

REID MB, DURHAM WJ (2002) Generation of reactive oxygen and nitrogen species in contracting skeletal muscle: Potential impact on aging. Ann N Y Acad Sci 959: 108-116

RÍOS E, ZHOU J (2004) Control of dual isoforms of Ca²⁺ release channels in muscle. Biol Res 37: 583-591

SÁNCHEZ G, HIDALGO C, DONOSO P (2003) Kinetic studies of calcium-induced calcium release in cardiac sarcoplasmic reticulum vesicles. Biophys J 84: 2319-2330

SCHNEIDER MF, RODNEY GG (2004) Peptide and protein modulation of local Ca²⁺ release events in permeabilized skeletal muscle fibers. Biol Res 37: 613-616

SEARS CE, BRYANT SM, ASHLEY EA, LYGATE CA, RAKOVIC S, WALLIS HL, NEUBAUER S, TERRAR DA, CASADEI B (2003) Cardiac neuronal nitric oxide synthase isoform regulates myocardial contraction and calcium handling. Circ Res 92: e52-e59

SHIROKOVA N, GARCÍA J, RÍOS E (1998) Local calcium release in mammalian skeletal muscle. J Physiol 512: 377-384

SIMPSON PB, CHALLISS RA, NAHORSKI SR (1995) Neuronal Ca²⁺ stores: Activation and function. Trends Neurosci 18: 299-306

STOFAN DA, CALLAHAN LA, DIMARCO AF, NETHERY DE, SUPINSKI GS (2000) Modulation of release of reactive oxygen species by the contracting diaphragm. Am J Respir Crit Care Med 161: 891-898

STOYANOVSKY D, MURPHY T, ANNO PR, KIM YM, SALAMA G (1997) Nitric oxide activates skeletal and cardiac ryanodine receptors. Cell Calcium 21: 19-29

SUKO J, DROBNY H, HELLMANN G (1999) Activation and inhibition of purified skeletal muscle calcium release channel by NO donors in single channel current recordings. Biochim Biophys Acta 1451: 271-287

SUN J, XU L, EU JP, STAMLER JS, MEISSNER G (2001a) Classes of thiols that influence the activity of the skeletal muscle calcium release channel. J Biol Chem 276: 15625-15630

SUN J, XIN C, EU JP, STAMLER JS, MEISSNER G (2001b) Cysteine-3635 is responsible for skeletal muscle ryanodine receptor modulation by NO. Proc Natl Acad Sci USA 98: 11158-11162

SUN J, XU L, EU JP, STAMLER JS, MEISSNER G (2003) Nitric oxide, NOC-12, and S-nitrosoglutathione modulate the skeletal muscle calcium release channel/ryanodine receptor by different mechanisms. An allosteric function for O₂ in S-nitrosylation of the channel. J Biol Chem 278: 8184-8189

SUZUKI YJ, CLEEMANN L, ABERNETHY DR, MORAD M (1998) Glutathione is a cofactor for H2O2-mediated stimulation of Ca²⁺-induced Ca²⁺ release in cardiac myocytes. Free Radic Biol Med 24: 318-325

TOESCU EC, VERKHRATSKY A (2003) Neuronal ageing from an intraneuronal perspective: roles of endoplasmic reticulum and mitochondria. Cell Calcium 34: 311-323

VERKHRATSKY A (2002) The endoplasmic reticulum and neuronal calcium signaling. Cell Calcium 32: 393-404

VERKHRATSKY A (2004) Endoplasmic reticulum calcium signaling in nerve cells. Biol Res 37: 693-699

VERKHRATSKY A, PETERSEN OH (2002) The endoplasmic reticulum as an integrating signalling organelle: From neuronal signalling to neuronal death. Eur J Pharmacol 447: 141-154

XIA R, STANGLER T, ABRAMSON JJ (2000) Skeletal muscle ryanodine receptor is a redox sensor with a well defined redox potential that is sensitive to channel modulators. J Biol Chem 275: 36556-36561

XIA R, WEBB JA, GNALL LL, CUTLER K, ABRAMSON JJ (2003) Skeletal muscle sarcoplasmic reticulum contains a NADH-dependent oxidase that generates superoxide. Am J Physiol Cell Physiol 285: C215-C221

XU L, EU JP, MEISSNER G, STAMLER JS (1998) Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation. Science 279: 234-237 XU KY, HUSO DL, DAWSON T, BREDT DS, BECKER LC (1999) NO synthase in cardiac sarcoplasmic reticulum. Proc Natl Acad Sci USA 96: 657-662

ZABLE AC, FAVERO TG, ABRAMSON JJ (1997) Glutathione modulates ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Evidence for redox regulation of the Ca²⁺ release mechanism. J Biol Chem 272: 7069-7077

ZAHRADNIKOVA A, MINAROVIC I, VENEMA RC, MESZAROS LG (1997) Inactivation of the cardiac ryanodine receptor calcium release channel by nitric oxide. Cell Calcium 22: 447-454

ZIMA AV, COPELLO JA, BLATTER LA (2003) Differential modulation of cardiac and skeletal muscle ryanodine receptors by NADH. FEBS Lett 547: 32-36

ZIMA AV, COPELLO JA, BLATTER LA (2004) Effects of cytosolic NADH/NAD(+) levels on sarcoplasmic reticulum Ca(2+) release in permeabilized rat ventricular myocytes. J Physiol 555: 727-741

ZIOLO MT, KATOH H, BERS DM (2001) Positive and negative effects of nitric oxide on Ca(2+) sparks: Influence of beta-adrenergic stimulation. Am J Physiol Heart Circ Physiol 281: H2295-H2303

ZUCKER RS, REGEHR WG (2002) Short-term synaptic plasticity. Annu Rev Physiol 64: 355-405

ZUO L, CHRISTOFI FL, WRIGHT VP, LIU CY, MEROLA AJ, BERLINER LJ, CLANTON TL (2000) Intra- and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle. Am J Physiol Cell Physiol 279: C1058-C1066

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: chidalgo@med.uchile.cl

Received: April 7, 2004. Accepted: May 18, 2004.