I. Introduction

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The ryanodine receptor (RyR)^c corresponds to the sarcoplasmic reticulum (SR) Ca²⁺ channel (Inui et al., 1987; Imagawa et al., 1987). Its structure and function have been reviewed recently (Fleischer and Inui, 1989; Lai and Meissner, 1989; McPherson and Campbell, 1993a; Sorrentino and Volpe, 1993; Coronado et al., 1994; Meissner, 1994; Ogawa, 1994; Sorrentino, 1995; Wagenknecht and Radermacher, 1995; Marks, 1996; Sutko and Airey, 1996), and only a few issues will be recalled here.

The RyR binds specifically the plant alkaloid ryanodine, which is the reason for its name. In striated muscle, RyRs are located at the triadic junctions between SR terminal cisternae and sarcolemmal T-tubules (Fleischer et al., 1985) and correspond to the "feet" structures observed in electron microscope images within the triads. However, RyRs also have been identified in SR structures that do not lie in contiguity with the sarcolemma, such as corbular and expanded junctional SR, and in intracellular membranes of other cells and tissues, such as brain, smooth muscle, endothelium, liver, and fibroblasts (Franzini-Armstrong and Jorgensen, 1994; Meissner, 1994).

The RyR has been purified, cloned, and sequenced from a variety of species, and several isoforms have been identified. Mammalian tissues express three isoforms, known as RyR1, RyR2, and RyR3. They include about 5000 (4872 to 5037) amino acid residues and are encoded by three different genes. In humans, the three genes are located on chromosomes 19, 1, and 15, respectively. RyR1 and RyR2 are expressed predominatly in skeletal muscle and in cardiac muscle, respectively (Marks et al., 1989; Takeshima et al., 1989; Nakai et al., 1990; Otsu et al., 1990; Zorzato et al., 1990). RyR3 has a wide tissue distribution (Ledbetter et al., 1994; Giannini et al., 1995), although it has been originally identified in brain (Hakamata et al., 1992) and is sometimes called "brain isoform." All three isoforms are actually expressed in brain, and the major brain isoform does not appear to be RyR3, but rather RyR2 (Witcher et al., 1992; McPherson and Campbell, 1993b; Murayama and Ogawa, 1996b). Alternative splicing variants of RyR1 and RyR2 have been identified, but their functional relevance remains to be established (Sutko and Airey, 1996). Two RyR isoforms, known as -RyR and -RyR, have been identified in fish, amphibian, and avian skeletal muscle (Airey et al., 1990, 1993b; Olivares et al., 1991; Sutko et al., 1991; Lai et al., 1992; Murayama and Ogawa, 1992), and they are the homologues of mammalian RyR1 and RyR3, respectively (Oyamada et al., 1994; Ottini et al., 1996). The overall identity of the RyR isoforms is of the order of 66 to 67%.

The RyR monomer has a sedimentation coefficient of about 30S and a molecular weight of about 560 kDa. The functional receptor is thought to be a homotetramer, which has a quarterfoil shape and a size of 22 to 27 nm on each side (Inui et al., 1987; Lai et al., 1988; Wagenknecht et al., 1989). The center of the quarterfoil includes a pore, with a diameter of 1 to 2 nm, that most likely represents the Ca²⁺ channel. There is structural and functional evidence that the central channel is connected to four radial channels included in the peripheral portion of each monomer (Wagenknecht et al., 1989; Ding and Kasai, 1996). Near its cytoplasmic end, the channel appears to be blocked by a mass, sometimes referred to as the "plug," that might be involved in the modulation of channel conductance. The pore region corresponds to the carboxy-terminal portion of each RyR monomer and includes, according to different suggested models, four (Takeshima et al., 1989; Nakai et al., 1990; Hakamata et al., 1992) or 10 to 12 (Zorzato et al., 1990; Otsu et al., 1990) transmembrane segments. Results obtained with site-specific antibodies (Grunwald and Meissner, 1995) support the four-transmembrane segment model, whereas cryoelectron microscopy data (Serysheva et al., 1995; Wagenknecht and Radermacher, 1995) favor the 10-transmembrane segment model. The rest of the molecule forms a large extramembrane region that corresponds to the foot structure, has a hollow appearance, and includes at least two domains in each monomer (Serysheva et al., 1995). Recent observations suggest that the channel opening is associated with a 4° rotation of the transmembrane with respect to the cytosolic region (Orlova et al., 1996).

The channel included in the RyR is a cation-selective channel with low cationic selectivity and large unitary conductance. With Ca²⁺ as current carrier, the maximum conductance was equal to 80 pS for the cardiac channel, and to 172 pS for the skeletal muscle channel, with a dissociation constant  3 to 4 mM (Smith et al., 1988; Lindsay and Williams, 1991). The maximum conductance was higher with monocations as current carriers, e.g., about 0.6/1 nS with Na⁺ and K⁺, respectively (Smith et al., 1988; Liu et al., 1989). Although in single salt solutions, channel conductance was higher for monocations than for Ca²⁺, in mixed salts, channel permeability was higher for Ca²⁺ than for monovalent cations. This finding has been interpreted on the basis of a model in which multiple ion-binding sites are arranged sequentially, assuming that Ca²⁺ binding is favored over monocation binding. In particular, a four-barrier, three-binding-site model might account for the experimental results (Tinker et al., 1992b; Tinker and Williams, 1992, 1993c).

The RyR mediates the efflux of Ca²⁺ from the SR or other intracellular stores. In striated muscle, it has a central role in excitation-contraction coupling, i.e., in the coupling between sarcolemmal depolarization and SR Ca²⁺ release.

There are at least two mechanisms that have been proposed to mediate excitation-contraction coupling. According to the direct-coupling model, SR Ca²⁺ release is produced by a direct interaction between the dihydropyridine and the RyRs. In particular, sarcolemmal depolarization is thought to produce a conformational change in the dihydropyridine receptor that is transmitted to the RyR and induces the release of Ca²⁺ from the SR (Rios and Pizarro, 1991; Rios et al., 1993; Schneider et al., 1994). In this model, the dihydropyridine receptor acts primarily as a voltage sensor rather than as a channel, because sarcolemmal calcium influx is not required for excitation-contraction coupling. Close contiguity between the ryanodine and dihydropyridine receptors has been shown by morphological studies (Block et al., 1988) and confirmed by biochemical investigations (Marty et al., 1994), although it is still uncertain whether other proteins that are closely associated with the dihydropyridine and RyRs (see below, II.A.7.) may play a role in the coupling process.

Alternatively, excitation-contraction coupling might be mediated by a process known as Ca²⁺-induced Ca²⁺ release (Fabiato, 1983). Because the SR channel is activated by an increase in cytosolic [Ca²⁺] (see below, II.A.1.a.), the sarcolemmal Ca²⁺ current, although insufficient to activate the contractile process directly, could induce further release of Ca²⁺ from the SR. This process may be favored by the existence of Ca²⁺ gradients in the cytosol, because Ca²⁺ ions entering the cell through the dihydropyridine receptor seem to have preferential access to the RyR, establishing a sort of "functional coupling" (Cannell et al., 1995; Sham et al., 1995).

The relative importance of these two mechanisms is still controversial. There is evidence that in skeletal muscle, the former (i.e., direct coupling) is sufficient to induce tension development, whereas in cardiac muscle, Ca²⁺ influx is necessary for contraction, and Ca²⁺-induced Ca²⁺ release is thought to be the dominant mechanism (Näbauer et al., 1989; Callewaert, 1992; Stern, 1992; Meissner, 1994). Consistently, morphological data suggest closer association of dihydropyridine and RyRs in skeletal muscle than in cardiac muscle (Sun et al., 1995). However, a large fraction of skeletal muscle RyRs are not associated with dihydropyridine receptors (Franzini-Armstrong and Jorgensen, 1994), and it has been suggested that Ca²⁺-induced Ca²⁺ release might also contribute to skeletal muscle activation (Anderson and Meissner, 1995; Yano et al., 1995b; Klein et al., 1996).

It is still uncertain whether the different modes of excitation-contraction coupling are related to differences in the RyR, in the dihydropyridine receptor, or in other components. Experiments performed in dysgenic myotubes with chimeric dihydropyridine receptors suggested that specific regions of the skeletal muscle dihydropyridine receptor (included between transmembrane repeats II and III) determine the appearance of skeletal-type excitation-contraction coupling (Tanabe et al., 1990). This conclusion has not been supported by the results of another study (Lu et al., 1994), in which peptides including the putative cytoplasmic loops between transmembrane repeats II and III of skeletal and cardiac dihydropyridine receptors were expressed in Escherichia coli, because both types of peptides activated the skeletal but not the cardiac RyR, suggesting that the type of excitation-contraction coupling was determined by the RyR.

1. Ca²⁺ release studies. In Ca²⁺ release experiments, SR vesicles are loaded with labeled or unlabeled Ca²⁺ by passive diffusion or by active uptake. Ca²⁺ release is then induced by exposing the SR to a release solution, and its kinetics are determined. If free Ca²⁺ concentration is assayed by metallochromic indicators, the increase in extravesicular Ca²⁺ concentration can be monitored after rapid mixing of the preparation with release buffer (stopped flow technique) (Yamamoto and Kasai, 1982; Kim et al., 1983; Nagasaki and Kasai, 1983; Ikemoto et al., 1989). If ⁴⁵Ca is used, residual vesicle radioactivity must be measured at different time points. This can be accomplished with rapid quenching (Meissner, 1984, 1988; Ikemoto et al., 1985; Meissner et al., 1986; Meissner and Henderson, 1987) or rapid filtration techniques (Dupont, 1984; Submilla and Inesi, 1987; Chiesi et al., 1988; Moutin and Dupont, 1988; Calviello and Chiesi, 1989).

2. Single-channel studies. SR vesicles or purified RyRs are incorporated into artificial lipid bilayers, which separate two ionic solutions. Incorporation of a channel in the bilayer is shown by the appearance of a current flowing between the two chambers (Coronado et al., 1992). The chamber to which channels are added is called cis chamber; the other one is called trans chamber. Channel incorporation is usually polar, so that the cytosolic face corresponds to the cis chamber. Current recordings show spontaneous openings and closures and are used to calculate the conductance of the channel and its open probability, that is, the fractional time during which the channel is open, henceforward designed as Po. Channel gating is described on the basis of mathematical models that assume the existence of one or more open state(s) and one or more closed state(s). Statistical techniques provide a detailed evaluation of channel gating. The usual approach (lifetime analysis) consists in determining the time constant of each state, which is linearly related to its mean lifetime (Ashley and Williams, 1990; Jackson, 1992). Increased current may be due to increased conductance of the open channel or to increased Po. The latter can be due either to increased lifetime of the open channel or to decreased lifetime of the closed channel, also referred to as increased frequency of channel opening.

3. [³H]ryanodine binding. The production of radiolabeled ryanodine (Pessah et al., 1985; Sutko et al., 1986) introduced a new approach in the study of RyR structure and function. High affinity binding of [³H]ryanodine to the RyR has been extensively characterized. In a variety of tissues, the dissociation constant (K_D) for [³H]ryanodine was in the low nanomolar range. The Hill coefficient was  1, and the kinetic K_D, i.e., the ratio of the dissociation and association constants, was close to the equilibrium K_D (Pessah et al., 1985, 1986; Michalak et al., 1988; Lai et al., 1989; McGrew et al., 1989; Holmberg and Williams, 1990a; Carroll et al., 1991; Pessah and Zimanyi, 1991). High affinity [³H]ryanodine binding was correlated to the functional state of the Ca²⁺ channel. Conditions that are associated with increased channel Po usually favored [³H]ryanodine binding, suggesting that ryanodine binds to the open channel. However, exceptions to this rule have been described, and this issue will be further discussed in section II.C.

4. Indirect studies. RyR function often has been evaluated indirectly. For instance, tension development by skinned cells or intact preparations after exposure to caffeine or after rapid cooling has been regarded as an index of SR Ca²⁺ release. Although such techniques may produce useful results, their limitations should be kept in mind. The contractile response can be affected by actions at the contractile protein level, and both caffeine and rapid cooling have multiple targets besides the RyR (Akera, 1990; Feher and Rebeyka, 1994). Similar considerations apply to the analysis of changes in intracellular Ca²⁺ concentration (Ca²⁺ transients), which are affected by other sarcolemmal or intracellular Ca²⁺ transport systems, and by Ca²⁺ binding to intracellular proteins.

	II. Modulation of the Ryanodine Receptor

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Many substances can modulate RyR function. In this section (section II.), endogenous (physiological) and exogenous (pharmacological) modulators are distinguished, and their mechanisms of action are discussed. For the sake of clarity, the former subsection also includes ions that are not physiological cell components, but whose action is related closely to that of endogenous ions. A comprehensive summary of the effects of the chief endogenous and pharmacological modulators on Ca²⁺ release, single-channel gating, and [³H]ryanodine binding is provided in tables 1 and 2, respectively.

A. Endogenous Modulators

1. Ions.

2. Nucleotides. Adenine nucleotides activate the RyR. Ca²⁺ release studies performed in skeletal muscle (Morii and Tonomura, 1983; Nagasaki and Kasai, 1983; Meissner, 1984; Meissner et al., 1986; Sumbilla and Inesi, 1987; Moutin and Dupont, 1988; Calviello and Chiesi, 1989; Wyskovsky et al., 1990; Donoso and Hidalgo, 1993) have shown that in the presence of adenine nucleotides, Ca²⁺ release occurred, even at nanomolar Ca²⁺ concentration and/or in the presence of Mg²⁺. The Ca²⁺-activation curve was shifted to the left, and the maximum rate of Ca²⁺ release was increased. In fact, full activation of Ca²⁺ release required the presence of both Ca²⁺ and nucleotides. The EC₅₀ for adenine nucleotides was in the millimolar range at all Ca²⁺ concentrations, and the Hill coefficient was close to 2 (Meissner et al., 1986). In cardiac muscle, the effect of adenine nucleotides was qualitatively similar, although less remarkable (Rousseau et al., 1986; Meissner and Henderson, 1987). The order of potency was adenosine 5'-(,-methylene)triphosphate (AMP-PCP) > cyclic AMP (cAMP) > adenosine diphosphate (ADP) > adenosine monophosphate (AMP), while nonadenine nucleotides, such as cytosine triphosphate (CTP), guanosine triphosphate (GTP), inosine triphosphate (ITP), and uridine triphosphate (UTP) were ineffective (Morii and Tonomura, 1983; Meissner, 1984). In cardiac muscle, adenosine and adenine were also effective (Meissner, 1984), whereas in skeletal muscle, Ca²⁺ release was produced by adenine but not by adenosine (Rousseau et al., 1988).

3. Cyclic adenosine diphosphate-ribose. Cyclic ADP-ribose (cADPR) is an endogenous metabolite of nicotinamide-adenine dinucleotide (NAD), which is thought to act as a second-messenger in several tissues (Clapper et al., 1987; Lee et al., 1989). In sea urchin eggs, nanomolar cADPR induced Ca²⁺ release from intracellular stores. Its action was independent from inositol 1,4,5-trisphosphate, was inhibited by ruthenium red and endogenous polyamines, and was potentiated by Ca²⁺, ryanodine, and caffeine (Galione et al., 1991, 1993a,b; Galione and White, 1994; Lee et al., 1993; Chini et al., 1995). On the basis of these observations, it has been suggested that cADPR activates the RyR. However, other findings have questioned this conclusion. In sea urchin eggs, Ca²⁺ release showed peculiar properties, because it was dependent on the presence of calmodulin (Lee et al., 1994, 1995; Tanaka and Tashjian, 1995), and it was not activated by ATP (Graeff et al., 1995). In addition, the cADPR derivative 8-amino-cADPR antagonized cADPR-induced, but not ryanodine-induced, Ca²⁺ release (Walseth and Lee, 1993). Finally, photoaffinity labeling studies showed that cADPR binds to two proteins of 100 kDa and 140 kDa (Walseth et al., 1993), and it is not known whether such proteins interact with the RyR or rather represent a novel type of Ca²⁺ channel. It should be stressed that RyR expression has not been extensively studied in sea urchin eggs. Antibodies raised against RyR1 identified a 380-kDa protein that has not been further characterized (McPherson et al., 1992). More recently, Ca²⁺-sensitive and caffeine-sensitive [³H]ryanodine binding has been described in a preliminary report, but no modulation by cADPR and ATP has been detected (Lokuta et al., 1996).

4. Lipid derivatives. In skeletal muscle, but not in cardiac muscle, palmitoyl carnitine and other long-chain (>C14) acyl carnitines induced SR Ca²⁺ release (El-Hayek et al., 1993; Dumonteil et al., 1994). The stimulation of Ca²⁺ release was slower than that produced by Ca²⁺ or ATP and had a lag of about 100 to 150 msec. Consistently, palmitoyl carnitine increased ryanodine binding at all Ca²⁺ concentrations (1 µM to 1 mM). In mammalian muscle, palmitoyl carnitine increased the B_max without affecting the K_D (El-Hayek et al., 1993), whereas in avian muscle, Dumonteil et al. (1994) reported increased affinity with unchanged B_max. In bilayer experiments, channel Po increased, due to an increased ratio of long-lived versus short-lived openings. These actions occurred at concentrations ranging from 5 to 100 µM (EC₅₀ = 10 to 15 µM), and their physiological or pathophysiological implications are uncertain, because the plasma palmitoyl carnitine concentration is of the order of 2 to 4 µM, but the cytosolic concentration might be higher (Dumonteil et al., 1994).

5. Endogenous polyamines. Palade (1987c) first reported that caffeine-induced and thymol-induced Ca²⁺ release were inhibited by endogenous polyamines such as spermine, spermidine, and putrescine. The IC₅₀ for spermine was in the 10 to 100 µM range, whereas spermidine and putrescine, which contain fewer amino groups, were less effective.

6. Phosphorylation. The RyR is the substrate of several protein kinases, namely cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), protein kinase C (PKC), and calmodulin-dependent protein kinase II (CaMK). There is evidence that junctional SR contains membrane-bound CaMK (Chu et al., 1990b).