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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.37 n.4 Santiago  2004 


Biol Res 37: 539-552, 2004


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


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


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.




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.

It is now well established that RyR channels are highly susceptible to modification by endogenous redox agents, including GSH, GSSG, NADH, reactive oxygen and nitrogen species (ROS and RNS), and by changes in the GSH/GSSG ratio. The redox sensitivity of skeletal RyR1 channels is due to the presence of highly reactive cysteine residues in the RyR1 molecule that have a pK value of 7.4 (Pessah et al., 2002) allowing RyR1 redox modification at physiological pH. Additionally, RyR1 channels exhibit a well-defined redox potential (Feng et al., 2000; Xia et al., 2000) and thus may be susceptible to redox potential changes in cells. For these reasons, modifications of RyR channels by endogenous agents or by changes in cellular redox state acquire distinct physiological relevance. Furthermore, if RyR channels serve a role as intracellular redox sensors (Eu et al., 2000), via redox induced Ca2+ release they are likely to connect cellular redox state with Ca2+ signaling cascades.

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.


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).


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.


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 (Po) 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).


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 K0.5 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 K0.5 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).


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 Po < 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.


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.


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


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