The Ryanodine Receptor Family of Intracellular Calcium Release Channels

Vincenzo Sorrentino DIBIT, San Raffaele Scientific Institute 20 I 32 Milan, Italy and Institute of Histology School of Medicine University of Siena Siena, Italy

The Ryanodine Receptor Family of lntracellular Calcium Release Channels

1. Introduction Mobilization of calcium from cytosolic stores is a commonly utilized mechanism for signal transduction in eukaryotic cells. It is well documented that receptor activation generates an increase in intracellular calcium concentrations by increasing the levels of the second messenger molecule inositol1,4,5-trisphosphate ( InsP,) via phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol-4,5-biphosphate.InsP3 interacts in the cytosol with an intracellular receptor, which acts as a release channel for calcium stored in intracellular stores (Berridge, 1993a). Molecular studies have provided data on the nature and function of the intracellular calcium release channels. Two distinct classes of channels that mediate release of calcium from intracelM a r stores have been identified: one is sensitive to InsP3 and is referred to as the InsP, receptor family (Mikoshiba, 1993); the second is sensitive to a nonphysiological ligand, the plant alkaloid ryanodine (Ry), hence the name Ry receptors (RyR) (Coronado et al., 1994). These two classes of calcium release channels have general structural similarities: the active channels have a tetrameric structure in which four subunits assemble to form the functional channel (Lai et al., 1989; Maeda et al., 1991). Each subunit consists of a very large protein, in the order of Admnces in Pharmacology. Volume 33 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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300 kDa for the InsP3 receptors and of 500 kDa for the Ry receptors. Three different genes coding for InsP, receptors have been identified (Blondel et al., 1993; Furuichi et al., 1989; Maranto, 1993; Mignery et al., 1990; Ross et a1.,1992). Alternative splicing also appears to contribute to the diversity of InsP, receptors (Danoff et al., 1991; Nakagawa et al., 1991). Preliminary evidence suggests a difference in ligand binding affinity between the different isoforms (Sudhof et al.. 1991\. a s well as a different tissue-specific pattern of expression (Furuichi et al., 1990; Mignery et al., 1990; Ross et al., 1992; Yamamoto-Hino et al., 1994). As for the RyRs, three different molecules have been identified and the respective genes cloned. These are RyRl (Marks et al., 1989; Takeshima et al., 1989; Zorzato et al., 1990) and RyR2 (Nakai et al., 1990; Otsu et al., 1990), which are known as the calcium release channels responsible for calcium release in skeletal and cardiac muscle, respectively, and the more recently identified RyR3 (Giannini et al., 1992; Hakamata et al., 1992). We shall focus in this review on the RyRs. For reviews on InsP3 receptors see (Ferris and Snyder, 1992; Mikoshiba, 1993).

II. RyRs: A Three-Member Family of lntracellular Calcium Release Channels

Three Ry-sensitive calcium release channels have been identified (Coronado et al., 1994; McPherson and Campbell, 1993b). They are encoded by three genes, which are localized on different chromosomes in humans and mice. The gene for the human skeletal muscle RyR (RYRl) has been mapped to chromosome 19q13.1 (MacLennan et al., 1990; McCarthy et al., 1990), the cardiac human ryanodine receptor (RYR2) to chromosome 1 (Otsu et al., 1993, 1990), and the third RyR (RYR3) to the 1 5 q l 4 - q l 5 region of the human genome (Sorrentino et al., 1993). The murine homologs of these genes are localized on chromosome 7A2-7A3 (RYRl), 13A1-13A2 (RYRZ), and 2E5-2F3 (RYR3) (Cavanna et al., 1990; Mattei et al., 1994). The three RyR genes encode proteins with homology in their amino acid sequences but with a different tissue-specific pattern of expression (Sorrentino and Volpe, 1993). Ryanodine receptors/Ca2+ channels have been associated for several decades with muscle fibers. We shall therefore follow this historical perspective and only later shall we discuss recent developments in the field of RyR research that suggest a role for RyRs in nonmuscle, nonexcitable cells.

A. RyRI :The Skeletal Sarcoplasmic Calcium Release Channel The role of calcium as a second messenger in regulating muscle contraction has been recognized for a long time, and studies on the sarcoplasmic reticulum of skeletal muscle have provided an established example of how

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storage and release of calcium can be regulated (Franzini-Amstrong and Jorgensen, 1994). In skeletal muscle fibers, a basic functional structure can be identified, named the “sarcomere.” It consists of two components: myofibrils and membranous compartments. The myofibrillar apparatus is responsible for contraction and is regulated by the membrane compartments. Two separate sets of membrane compartments should be distinguished: the transverse (T) tubules and the sarcoplasmic reticulum (SR). The T-tubules are specialized invaginations of the plasma membrane that propagate the action potential into the interior of the muscle fiber. The SR is an internal membrane system, a specialized compartment of the endoplasmic reticulum (ER), that is present in muscle cells and is responsible for the uptake, storage, and release of intracellular calcium. These two membrane compartments are in contact through a junction called the triad (Flucher et al., 1993). In the triad, two terminal SR cisternae are located on the opposite sides of one T-tubule. The gap between the terminal SR cisternae and the T-tubule is approximately 15 nm wide. In this cleft, electron-dense structures, called junctional “feet,” are present: these have been shown to correspond to the calcium-gated channels that release calcium from the SR, the RyR (Inui et al., 1987b). In skeletal muscle fibers, calcium is released from the SR following depolarization of the plasma membrane and of the T-tubule (Fleischer and Inui, 1989). Activation of calcium release from the SR seems to require a direct contact between the voltage sensor on the T-tubule, the dihydropyridine receptor (DHPR), and the RyR/calcium release channel, located on the SR (Lu et al., 1994). In skeletal muscle, activation of calcium release from the SR following the action potential, the so-called excitation-contraction (E-C) coupling, does not seem to require a calcium influx through the DHPR, which is involved in E-C coupling probably only as a voltage sensor and not as a calcium channel. The skeletal muscle RyR has been purified in the past years on the basis of its high affinity for ryanodine (Fleisher et al., 1985; Pessah et al., 1986; Seifert and Casida, 1986). [‘HIRyanodine has been used to localize the channel during several purification steps, such as sucrose gradients or column chromatography (Campbell et al., 1987; Inui et al., 1987b). And in fact, the RyR from skeletal muscle can be resolved, following such procedures as a single band on polyacrylamide gels with an apparent molecular weight of about 400-500 kDa (Lai etal., 1988b). The native receptor binds (‘Hlryanodine with a K(, of 80 and a B,,, around 500 pmol/mg; this corresponds to a quarter of the maximum theorical [’Hlryanodine binding activity of the intact channel, suggesting that there is only one ryanodine binding site per four subunits, o r per functional channel (Lai et al., 1988b, 1989). The purified RyR has been the object of several electron microscopy studies in order to determine its structural organization. The RyR appears to have a fourfold symmetric structure similar to the one described for the “foot structure,” and compatible with the proposed tetrameric structure

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of the functional channel (Franzini-Amstrong and Jorgensen, 1994). The channel appears to have two distinct sides, one oriented toward the T-tubule and one anchored to the SR. As RyRs are relatively large molecules (15 nm thick and 25 nm wide), it is possible to slice the receptor in two, perpendicularly to the fourfold symmetry axis (Wagenknecht et al., 1989). This has revealed the presence of a central hole, perhaps corresponding to the actual channel. This central channel does not seem to extend to the surface of the receptor complex. There are, however, near the midplane of the reconstruction, four radial channels that extend from the central channel to reach the surface of the molecule. Incorporation of the purified RyR in lipid bilayers results in the reconstitution of a calcium-channel activity with properties similar to the one found in terminal SR cisternae, with a conductance of about lOOpS (Coronado et al., 1994; Ehrlich et al., 1994; Smith et al., 1988). The fraction of channelopen time, observed under these conditions, is reduced by lowering the free calcium concentration and is stimulated by ATP, similar to that observed in vesicles from the heavy SR (Ehrlich et al., 1994). Recently it has been shown that FKBP12, a cytosolic protein that binds the immunosuppressant ligand FK506, binds to and copurifies with the RyRl (Jayaraman et al., 1992). The effect of FKBP12 on RyRl function has been studied by coexpressing the two proteins in insect cells. The results indicate that, as a consequence of FKBP12 binding to RyRl, subconductance states are abrogated and the full conductance of the channel is increased (Brillantes et al., 1994).

B. RyR2: The Cardiac Sarcoplasmic Calcium Release Channel In contrast with E-C coupling in skeletal muscle, depolarization of cardiac sarcolemma and T-tubules is not sufficient to induce calcium release from the cardiac SR (Fleischer and Inui, 1989). Actually, in order to observe cardiac muscle contraction, an influx of calcium, usually through the cardiac DHPR, is required. This calcium influx is insufficient to directly activate myofilament contraction but is sufficient to induce a much larger calcium release from the sarcoplasmic reticulum. Thus, calcium influx through the cardiac DHPR activates calcium release from the sarcoplasmic reticulum in a process termed “calcium-induced calcium release” (CICR)(Fabiato, 1989). The juxtaposition of myofibrils and membranous network differs substantially in skeletal and cardiac muscle. In mammalian cardiac myofiber SR, there are three distinct regions: the network SR, which is uniformly distributed along the sarcomere and is highly loaded with calcium-ATPase and phospholamban, and the junctional and corbular SRs, where calsequestrin is located (Jorgensen et al., 1993; Junker et al., 1994). Ryanodine receptors are apparently present only in the latter two regions, which extend from the network SR. However, while the junctional SR is apparently con-

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nected to the sarcolemma or to the T-tubules, the corbular SR is removed from contact with surface structures. Thus, given the absence of contact between corbular SR and the sarcolemma, this compartment may be an elective site for CICR (,lorgemen et al., 1993; Junker et al., 1994). The RyR from cardiac sarcoplasmic reticulum has been purified and shown to be similar to the skeletal RyR (Anderson et al., 1989; Inui et al., 1987a). It also has a large tetrameric structure and forms, when incorporated into planar lipid bilayers, a calcium-activated calcium channel with conductance properties and pharmacological regulation similar to those of the skeletal RyR (Lai et al., 1988a). In accordance with the proposed model of activation, the cardiac RyR is more sensitive to calcium than the skeletal RyR (Bezprozvanny et al., 1991; Ehrlich et al., 1994). Gyorke and Fill (1993) have shown that, in contrast with what usually observed for ligandgated channels of the plasma membrane, cardiac RyR channels appear to adapt to local calcium levels, but maintain their ability to respond to a subsequent calcium increase (Gyorke and Fill, 1993). Recently it has been shown that the cardiac RyR is insensitive to one of the two peptides purified from the venom of the scorpion Pandinus imperator, which are able to activate calcium release from the skeletal RyR (Valdivia etal., 1991; Valdivia et al., 1992). This phenomenon may reflect differences in the structural properties of the receptors, as suggested by the different sensitivity of cardiac and skeletal RyRs to various agents, as described below. C. RyR3 The cDNA for a calcium release channel has been recently cloned and, by sequence homology, been identified as a novel RyR (Giannini et al., 1992; Hakamata et al., 1992). This gene has been named RYR3 following the general agreement to name the genes for the skeletal muscle and cardiac RyRs RYRl and RYR2, respectively. RyR3 was initially isolated from a cDNA library from mink epithelial lung cells treated with transforming growth factor+ (TGFP) (Giannini et al., 1992). As a matter of fact, expression of the RyR3 gene is specifically induced by treatment of these cells with TGFP, suggesting that the expression of intracellular calcium release channels may be modulated by signals, like growth factors, from the extracelM a r environment. The changes induced by extracellular signals could potentially affect the generation of intracellular calcium signals. In MvlLu cells treated with TGFP, expression of the RyR3 parallels expression of a calcium channel responsive to ryanodine, but not to caffeine (Giannini et al., 1992). More recent data indicate that lack of response to caffeine could be due to the limited amount of RyR3 expressed in these cells rather than to specific regulatory property of this isoform (D. Rossi and V. Sorrentino, unpublished results). Unfortunately no data on the pharmacology and the biochemistry of RyR3 are available. An interesting feature of the RyR3 gene is that the gene encoding this channel was expressed in almost all tissues analyzed.

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This means that, at variance with the RyRl and RyR2, RyR3 does not show a major association with a specific organ (Giannini et al., 1992). Such a finding is clearly interesting with respect to the expression of RyRs in other tissues aside from muscle cells and neurons (see discussion on tissue distribution).

111. Regulation of RyRs Calcium release in skeletal muscle is activated by the interaction of RyRl with the dihydropyridine receptor, without any requirement for calcium entry from extracellular fluids. In myocytes, RyR2 activation appears to be dependent on the entry of a limited amount of calcium through the cardiac DHPR, which induces the opening of RyR2, resulting in a massive release of calcium from the cardiac SR and subsequent muscle contraction. Calcium release through the ryanodine receptors is regulated by several compounds. Potentiators of sarcoplasmic reticulum calcium release include calcium a t micromolar concentrations, adenine nucleotides, caffeine, halothane, ryanodine at nanomolar concentrations, sulfhydryl reagents, and cyclic adenosine diphosphoribose (cADPR). Inhibitors include Mg2 , calcium at millimolar concentrations, ryanodine at micromolar concentrations, and Ruthenium red. The compounds that activate the channel, such as calcium, also stimulate [3H]ryanodinebinding, while compounds that inhibit calcium release, such as Mg2+ and Ruthenium Red, also prevent ['Hlryanodine binding. For an updated review on the pharmacology of these compound with respect t o RyRs, see Coronado et al. (1994) and Ehrlich et al. (1994). +

A. Cyclic Adenosine Diphosphoribose Over the past few years, a new molecule that is a strong activator of calcium release through RyRs has been identified and subsequently characterized. I t is a nucleotide derivative, cyclic adenosine diphosphoribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD '), originally identified by Hon Cheung Lee and his colleagues while studying calcium release by sea urchin egg microsomes (Clapper et al., 1987; Lee et al., 1989). cADPR is a potent calcium releasing agent, and it is postulated to be a new second messenger, as it is present in many and possibly all cells (Rusinko and Lee, 1989; Walseth etal., 1991). Levels of cADPR appear to be dependent on the presence of enzymes that synthesize and degrade this nucleotide (Lee and Aarhus, 1993). cADPR is synthesized from NAD ' by ADP-rybosyl cyclase, an enzyme purified from Aplysia as a soluble protein of 29 kDa (Click etal., 1991; Hellmich and Stumwasser, 1991). An activity that generates cADPR has also been detected in mammalian tissues (Walseth et al., 1991). In addition, the existence of enzymes that degrade cADPR has been suggested. The isolation and characterization from canine spleen microsomes of an enzyme that catalyzes the hydrolysis of cADPR to ADPR has been

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reported (Kim et al., 1993). Interestingly, the same enzyme also catalyzes the synthesis of cADPR from NAD, and it may be classified as an NAD glycohydrolase (NADase). The enzyme from Aplysia has been cloned (Click et al., 1991), and the amino acid sequence was found to share a significant degree of homology with CD38, a surface antigen of human lymphocytes (States et al., 1992). Cloning and expression of CD38 has yielded purified CD38 protein, which synthesizes cADPR from NAD' in vitro (Howard et al., 1993; Takasawa et al., 1993b). CD38 has, in addition, a cADPR hydrolase activity, which is missing in the Aplysia cyclase. The cADPR hydrolase activity of CD38 is inhibited by ATP, resulting in the increased production of cADPR. Although these data suggest a role for CD38 in the regulation of cADPR production, it must be considered that the catalytic site of CD38 is located in the external part of the cell membrane. Thus, the mechanism by which cADPR synthesized by CD38 on the extracellular side of the plasma membrane participates in activation of calcium release from intracellular stores remains to be elucidated (Malavasi et al., 1994). A cDNA encoding a novel protein with limited but significant homology to CD38 has been cloned (Kaisho et al., 1994). cADPR has been shown to be able to mobilize calcium in a number of cell types, including pituitary cells, dorsal root ganglion cells, and sea urchin eggs (Berridge, 1993b; Galione et al., 1993a; Lee and Aarhus, 1993; Lee et al., 1993). The calcium mobilizing activity of cADPR is blocked by ryanodine and caffeine (Galione et al., 1991; Lee, 1993), which d o not affect InsP,-activated calcium release (Galione et al., 1993a; Lee, 1991). The direct effect of cADPR has been shown by studies in lipid bilayers where it activates Ry receptors at very low concentrations (Meszaros et al., 1993). interestingly, RyRl and RyR2 seem to differ in their sensitivity to cADPR, with RyR2 being much more sensitive than RyRl (Meszaros et al., 1993; Morrisette et al., 1993). A major objection to the proposed role of cADPR as a second messenger for extracellular stimuli has been the relatively constant levels of cADPR observed in many mammalian cell types under resting conditions. Since the effects of calcium and cADPR are synergistic, it has also been suggested that cADPR may function as a cofactor for CICR by enhancing the sensitivity of RyRs to calcium. Results in favor of a role for ADPR as a second messenger come from studies with digitonin-permeabilized islet cells, where cADPR and calcium, but not InsP,, induced insulin secretion. In islet cells, glucose can induce an increase in the concentration of a calcium-releasing activity that was suggested to be an effect of cADPR (Takasawa et al., 1993a). Recent work from Galione's lab has linked cGMP to RyR stimulation via a metabolite of P-NAD+, which is likely to be cADPR (Galione et al., 1993b). In sea urchin eggs, dibutyryl cGMP, a membrane-permeable form of cGMP, was able to induce a large release of calcium after a latency period of 80 sec. The effect of cGMP was independent of the InsP,-activated pathway, but was abolished by treatment with drugs that block RyR, such

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as Ruthenium red and ryanodine. Calcium release induced by cGMP was dependent on a critical concentration of P-NAD+. The same authors also demonstrated an increased metabolism of NAD to ADPR in cells stimulated by cGMP (Galione et al., 1993b). The hypothesis that RyRs could be activated by a second messenger, possibly cADPR, similar to the activation of InsP,Rs, is of potential interest for understanding the mechanisms of activation of these channels in nonmuscle cells. +

B. Quanta1 Release Caffeine has been the most commonly used agonist to activate calcium release through Ry Rs (Rousseau and Meissner, 1989). Stimulation with caffeine has been shown to induce a transient increase in [Ca”],. However, repeated stimulation with low caffeine concentrations do not usually void the caffeine-sensitive calcium stores, which remain responsive to increased doses of caffeine. This “quantal” mode of response to an agonist is similar to the one observed for the InsP,R. Two possible models to explain this behavior have been suggested in the case of the InsP3R, and they may be valid for RyRs as well (Bootman, 1994). A steady-state model suggests that quantal release reflects the existence of intracellular calcium stores whose sensitivity to the agonist is homogeneous, but dependent on the calcium concentration within the lumen of the stores. In other words, high calcium levels in the stores would sensitize the receptors to open even at low concentrations of agonist (Cheek etal., 1991; Gilchrist et al., 1992). Upon channel activation, emptying of the store and the ensuring decrease in the intraluminal concentration of calcium reduces the sensitivity of the channel and thus stops calcium release. It will now take a higher concentration of agonist to activate the channel again. A second model suggests that the stores are heterogeneous with regard to their sensitivity to the agonist. In this model, a low concentration of agonist will recruit only a discrete fraction of calcium stores (Cheek, 1993, 1994). Higher concentrations, by also recruiting less sensitive stores, may be active on a larger fraction of calcium stores.

IV. Molecular Structure of the RyRs By analogy with the current model for the InsP,R, it has been proposed that three regions can be distinguished in the RyR molecule. The first one corresponds to the amino-terminal part, including approximately the first 1000 amino acids, which may contain a ligand-binding domain. The second region, that corresponds to the large central part of the molecule, may be involved in the modulation of calcium-release activity of the channel. The

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third part includes the last 1000 amino acids and contains the transmembrane domains (TMs) where the actual calcium pore is likely to be formed. The present model for ryanodine receptors suggests that each of the four monomers that join to form the calcium-gating channel is connected to the membrane of the intracellular calcium store by several transmembrane domains located in the COOH terminus. Analysis of the hydropathicity profiles has identified several amino acid stretches in the RyR sequence, which have the characteristics required for acting as T M (Lodish, 1988). A model with four TMs located in the 500 amino acids at the COOH end of the rabbit RyRl was initially proposed (Takeshima et al., 1989). Alternatively, the presence of 10 TMs, spanning the last 1000 amino acids, has been suggested (Zorzato et al., 1990). These TM regions, with the exception of T M 3 and TM4, are highly conserved among the different receptor isoforms and therefore the hydropathicity profiles of RyR2 and RyR3 are similar to the ones predicted for RyR1. In this review, when referring to the T M of RyRs we shall follow the model containing 10 TMs. In the MvlLu cell line, an alternative splicing mechanism removes the exon coding for the fifth T M in a significant fraction of the RyR3 mRNA (Giannini et al., 1992). The fifth T M of the RyR shows a strong homology with T M s of other channels such as the InsP,R and the acetylcholine receptor (Takeshima et al., 1989), supporting the idea that this domain may contain a region important for the structure or function of the channel. The functional significance of this spliced version of RyR3 is still unknown. It is not known whether the mRNA lacking the T M 5 is translated. If a protein product is formed, expression and the pharmacological characterization of the normal and spliced versions of RyR3 proteins will be required in order to understand the biological meaning of this unexpected finding. As we have previously mentioned the three RyRs, although quite similar in overall properties, present some significant differences in their sensitivity to agonists and antagonists of calcium release (see also Coronado et al., 1994; Ehrlich et al., 1994). These may reflect the differences observed in the three receptors at the protein-sequence level. In fact, although the three receptors display, a t the level of protein sequence, a high degree of homology, there are small defined areas where sequence homology between the different isoforms is poor (Sorrentino and Volpe, 1993). One such region ( D l , for divergency region 1) comprising residues 4254-4631 of RyRl (and the corresponding residues of RyR2 and RyR3) spans the intraluminal loop between the T M 3 and T M 4 domains and the large cytoplasmic loop between T M 4 and TM5, which includes the calcium regulatory region previously discussed (see discussion on regulatory domains). The other two divergent domains are D2 (residues 1342-1403 in RyR1) and D3 (residues 1872-1923 in RyRl). These domains may contain binding

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sites for the physiological modulators of the channel and may be responsible for some of the physiological and pharmacological differences of the three different isoforms. On this basis, it should be interesting to identify the putative function of the D2 domain, which is present in the sequence of the RyRl and RyR2, but absent in the RyR3 isoform. The existence of an alternative splicing mechanism in the processing of the RyRl mRNA has been recently reported (Zhang et af., 1993). An exon of 15 bp following G10437, encoding five amino acids (AGDIQ), may be spliced out in a fraction of RyRl mRNA in skeletal muscle. A similar mechanism is responsible for the deletion of 18 base pairs between G11572 and G11590. No experimental data are available on the functional relevance of the differentially spliced version of RyRs. It can be expected that they may introduce an additional level of complexity in the regulation of the calcium release activity of these channels, as shown for the InsP,Rs (Danoff et af., 1991).

A. Regulatory Domains The potential sites for the interaction with pharmacological and physiological modulators of the calcium release properties of RyRs have been inferred from the amino acid sequence of the receptors, but not all of them have been demonstrated to be functional in vivo. It has been proposed that the region between residues 4253 and 4499 of the rabbit RyRl is exposed on the surface of the native molecule. Potential calcium-, ATP-, and calmodulin-binding sites have been predicted by computer analysis of the amino acid sequence in this region of the RyR (Takeshima et af., 1989; Zorzato et af., 1990). The importance of this region is further supported by several experimental data. Antibodies directed against the region 4445-4586 in RyRl can reduce the open probability of the channel. Recently, Chen etal. (1992) used a 4sCa2+overlay of fusion proteins covering the length of the skeletal muscle RyR to determine that the region between amino acids 4478 and 4512 contains a calcium-binding site. An antibody against this region increases the open probability and opening time of purified RyRl incorporated in planar lipid bilayers, suggesting that this site may be involved in calcium-dependent regulation of the channel. The same authors have also reported that a second antibody against the peptide sequence PEPEPEPEPE (corresponding to aa 4489-4499), contained within the region mentioned above, has an inhibitory effect on calcium- or caffeinestimulated channel activities, but does not prevent ATP-induced activation of the channel (Chen et al., 1993b). In addition data from Treves et af. (1993) confirmed that the region between 4380 and 4625 is important for calcium-dependent regulation of channel activation, since antibodies against this region block calcium-induced activation of channel function (Treves et af., 1993). These data point to a major calcium-sensitive site lying in the cytoplasmic loop between T M 4 and TM5. This region corresponds to the

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divergent region D1, where the three RyRs share very low sequence homology. Differences in the amino acid sequences around this site may reflect the differences in the calcium sensitivities reported for the different isoforms. The presence of a potential regulatory domain in the region of RyR2 that comprises the S2809 has been suggested (Witcher et al., 1991). S2809 has been shown to be a relevant site for calcium/calmodulin-activated protein kinase (CaM kinase)-dependent phosphorylation of RyR2, as demonstrated by direct sequencing of the phosphorylated tryptic fragment after in vitro phosphorylation (Witcher et al., 1992). It has also been suggested that PKA is able to phosphorylate RyR2, but with a significantly lower efficiency when compared with CaM kinase. However, RyR2 phosphorylation by PKA was enhanced in the presence of isoproterenol, suggesting a possible mechanism for calcium release regulation by P-adrenergic stimulation in myocytes (Yoshida et al., 1992). Evidence that RyRl protein is phosphorylated has also been presented (Strand et al., 1993; Suko et al., 1993). Functional analysis of channel activity indicates that phosphorylation events may, directly or indirectely, affect the function of the RyRl (Wang and Best, 1992). Patch-clamp studies of sarcoplasmic membranes preparations have shown that the calcium-dependent inactivation of calcium release through RyRl can be reversed by treatment with phosphatase or with a peptide able to inhibit calcium/calmodulin-dependent protein kinase 11. No biochemical data are available on the putative phosphorylation sites of RyR3. Interestingly a potential PKA phosphorylation site (T1243 in mink RyR3), absent in RyRl and RyR2, is conserved in RyR3 cDNAs isolated from mink, rabbit, and chicken (unpublished observations). Adenosine triphosphate nucleotides (Meissner, 1984; Smith et al., 1985) and calmodulin (Meissner, 1986) can also affect RyRs activity (Coronado et al., 1994; Ehrlich et al., 1994). Several potential sites for ATP binding are present on each isoform, but only one of them seems to be well conserved (aa 2237-2242 of mink RyR3 and corresponding aminoacids of the rabbit RyRl and RyR2 protein sequences). Several potential sites for calmodulin binding have been described in RyRl and RyR2, but direct biochemical data are not available. A potential clamodulin binding site at residues 3472-3495 of the rabbit brain RyR3 has been reported by Hakamata et al. (1992). This sequence is also conserved in RyRl and RyR2. Recently, the site of binding of Ry on the RyRl has been identified using an azido derivative (Witcher et al., 1994). Photoaffinity labeling of a triad preparation with [3H]azido-Ry resulted in the covalent binding of these compounds to RyRl. Controlled trypsin digestion resulted in the accumulation of ['Hlazido-Ry in a fragment with a molecular weight of 76 kDa. This band was recognized by an antibody against the carboxylterminal part of RyR1. Previous work had determined that a 76-kDa fragment of RyRl was derived from the carboxyl-terminal part of the molecule, which probably contains the transmembrane domains of the molecule, as suggested by its insolubility (Chen et al., 1993a). Interestingly, with a simi-

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lar technical approach using a photoactivable derivative of ATP, it has been possible to identify a major ATP binding site in the RyRl protein (Zarka and Shoshan-Barmatz, 1993). This site also is localized within the 76-kDa fragment. The 76-kDa fragment may thus contain both Ry, ATP, and calcium-binding sites.

B. Mutations in the RYRl Gene of Patients with Malignant Hyperthermia and Central Core Disease Are Clustered in the Region Encoding the N-Terminal Domain of the Protein Identification of amino acid residues potentally important for functioning of these very large channels has benefitted from studies that have linked mutations in the sequence of the RYRl gene with malignant hyperthermia (MH) and Central Core Disease (CCD).M H is an inherited genetic myopathy triggered in humans by certain volatile anesthetics. M H has been ascribed to a defect in the calcium release channel of skeletal muscle SR (MacLennan and Phillips, 1992). In the SR from individuals affected by MH, calcium release can be induced by lower concentrations of calcium, ATP, and caffeine than in normal controls. Susceptibility to M H is associated with the q13.1 region of human chromosome 19 where the RYRl gene is located (MacLennan et af., 1990; McCarthy et al., 1990). A single point mutation in the RYRl gene, causing a substitution of a Cys for Arg‘l’, was initially identified as the possible cause of M H in swine and in some human M H families (Furuichi et af., 1994; Gillard et al., 1991). Another mutation, G l ~ ~ ~ ~ has A r been g , found in one family affected by M H (Gillard et af., 1992). CCD is an autosomal dominant myopathy characterized by the presence, in skeletal muscle biopsies, of areas depleted of mitochondria. These areas, or “cores,” are localized in the central regions of type-1 fibers and stain negative for oxidative enzyme activity. CCD has been associated with a predisposition to M H and with chromosome 19q13, where the RYRl gene is localized. Sequence analysis of RyRl cDNA from individuals with CCD has led to the identification of three additional mutations: Arg2434His, Arg’63Cys, and Ile4’”Met (Quane et af., 1993; Zhang et al., 1993). Interestingly, the Arg“Wys mutation, originally detected in CCD patients, has also been detected in two unrelated M H patients with no signs of “central cores” o r myopathy. In a similar fashion, the Argz434Hismutation has been found in members of a family that had been diagnosed with CCD and had a positive M H contraction test. M H is caused by mutations that enhance the sensitivity of the channel to stimulators. This hypersensitivity results in enhanced rates of Ca release from the SR and, as a consequence, in sustained muscle contraction and increased glycolytic and aerobic metabolism, as observed following anaesthe-

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sia. The pathogenesis of CCD is less clear, although it is possible that alterations in excitation-contraction coupling due to mutations in the RyRl could be responsible for the muscle weakness observed in CCD patients. It remains to be understood how an identical mutation in the RyRl can be present in a patient with CCD and also in M H patients, who do not present any sign of “central core” or of myopathy. It is interesting to note that, with the exception of the Arg2434Hismutation, the mutations linked to a pathological activity of the RyRl are clustered in a region of less than 500 amino acids in the N-terminal region of the protein. The possible function of this region of the RyR is suggested by comparative studies with the sequence of the InsP3R. These studies have shown that the RyRl region around Arg6” is homologous to the InsP, binding region of the InsP, receptor and may be involved in the binding of specific channel activators. The functional importance of this part of the RyR protein is underscored by its conservation in the different RyR isoforms and in different species.

V. Tissue Distribution and Cellular Localization RyRs were first identified and studied in muscle tissues. The RyR from skeletal muscle was subsequently found to differ in its properties from the isoform detected in cardiac muscle cells. Accordingly, a gene (RYR1)coding for the skeletal muscle specific isoform was found to be expressed in fastand slow-twitch muscle, while a cardiac specific gene (RYRZ) was cloned from a heart cDNA library. These two isoforms maintain their tissue-specific expression both during ontogenesis and in adult life. The more recently identified RYR3 gene is expressed in several tissues in addition to skeletal muscle and heart (Giannini et al., 1992). Although RyR3 is expressed in all skeletal muscles studied, it still remains to be demonstrated whether RyRl and RyR3 molecules are coexpressed in the same muscle fibers or are present in different types of fibers. In heart, RyR3 mRNA is preferentially expressed in the conductive tissue (Gorza et al., in press). Both InsP3- caffeine-sensitive intracellular calcium release pools have been identified in peripheral and central neurons (Berridge, 1993a). These pools present a differential distribution in the central nervous system (CNS) (Verma et al., 1990). The CA3 region of the hippocampus and dentate gyrus show higher levels of CICR, while CA1 appears to be more enriched in InsP3-sensitive pools. A different sensitivity to InsP, and CICR can also be observed in different areas of the CNS such as the corpus striatum, olfactory bulb, and cerebellum (Verma et al., 1992). The presence of InsP3R in several regions of the CNS has been long established (Ferris and Snyder, 1992; Furuichi et al., 1989; Mikoshiba, 1993). The expression of RyRs in the CNS has also been the subject of intense investigation (McPherson and

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Campbell, 1993b). RyR2 mRNA was initially detected in rabbit brain by Northern hybridization and subsequently by in situ hybridization (Lai et al., 1992; Nakai et al., 1990). Purification of RyRs from rabbit brain has yielded a molecule that closely resembles the cardiac isoform (McPherson et al., 1991; McPherson and Campbell, 1993a). Using antibodies specifically directed against the cardiac isoform, RyR2 was identified in several regions of the rat brain, including the hippocampus, cortex, caudate-putamen, septum, amygdala, olfactory bulb, thalamus hypothalamus, and cerebellum (Sharp et al., 1993). By immunoblot analysis and immunohistochemistry using isoform-specific anti-RyR antibodies, RyRl expression was found to be limited to Purkinje cells in the cerebellum in contrast with the wider pattern of expression of the RyR2 in mouse brain (Kuwajiima et al., 1992). RyR3 is also widely expressed in brain (Hakamata et al., 1992). The pattern of expression of the three RyRs in the CNS has been confirmed by a more detailed characterization of the distribution of the three RyRs in mouse brain by in situ hybridization studies, showing that all three genes are expressed in mouse forebrain and cerebellum (Giannini et al., 1995). In the cerebellum, RyRl expression is restricted to the Purkinje cells, while both RyR2 and RyR3 are expressed in the granular cell layer. All three RyRs were detected in cerebrum. In the hyppocampus, RyRl is prevalent in the dentate gyrus, and at lower levels, in Ammon’s horn, while RyR2 is abundantly expressed in the dentate gyrus and in CA1 pyramidal cells. On the contrary, a positive signal for RyR3 appears to be stronger in the CA1 region of Ammon’s horn than in the dentate gyrus. RyR3 and, at lower levels, RyRl are expressed in the striatum. RyR3 was also detected in the thalamic and hypothalamic area. Outside the heart and CNS, expression of cardiac RyR2 has been reported in the stomach (Nakai et al., 1990) and, more recently, in vascular and endocardial endothelium (Lesh et al., 1993). In mink, RyR3 mRNA was detected by RNase protection assay in the skeletal muscle, jejunum, ileum, kidney, lung, stomach, and spleen (Giannini et al., 1992). In several tissues, expression of RyR3 was correlated with the presence of smooth muscle in organs such as aorta, esophagus, taenia coli, urinary bladder, ureter, and uterus (Hakamata et al., 1992). The analysis, via RNase protection and in situ hybridization, of the expression of the three isoforms of RyRs in mouse tissues indicates that all three RyRs have a wider distribution than previously appreciated (Giannini et al., 1995). RyRl transcripts were detected in spleen, stomach, submaxillary gland, gut, thymus, testis, adrenal gland, and ovary. RyR2 was expressed in lung, esophagus, gut, stomach, thymus, adrenal gland, and ovary. In agreement with previous data, RyR3 transcripts were present in lung, esophagus, spleen gut, kidney, stomach, submaxillary gland, testis, adrenal glands, and ovary.

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VI. Ryanodine Receptors in Nonmammalian Species Mammalian skeletal muscles express high levels of the skeletal RyRl and such minute amounts of RyR3 (Giannini et al., 1995) that, for a long time, the presence of a second RyR isoform in mammalian skeletal muscle went undetected. In contrast, in several nonmammalian vertebrates, including birds, fish, and amphibians, the existence of two skeletal muscle RyR isoforms, both expressed a t high levels, has been acknowledged for many years (Airey et al., 1990; Olivares et al., 1991). The two isoforms, a and p, have been distinguished on the basis of their mobility on polyacrylamide gels and immunological properties. In chicken, both differed from the cardiac isoform, with the a-form bearing some similarity to the mammalian skeletal . the crooked-neck dwarf (cn) chicken, a mutant RyRl (Airey et al., 1 9 9 3 ~ )In strain with a defect in the development of skeletal muscle, the expression of the a-isoform appears to be decreased, suggesting a requirement for this isoform in muscle development (Airey et al., 1993a,b). Recently, two cDNAs have been cloned from bullfrog skeletal muscle mRNA (Oyamada et al., 1994). One cDNA codes for a protein showing similarity with the amino acid sequence of the tryptic fragments of the purified a-isoform. The entire primary sequence of the protein encoded by this cDNA has significant homology to the mammalian RyR1. The second cDNA, whose predicted protein is homologous to tryptic fragments of the purified p-isoform, shows high homology with the mammalian RyR3. Two different cDNAs corresponding to chicken RyRs have been cloned from a chicken skeletal muscle cDNA library (L. Ottini, G. Marziali, A. Conti, A. Charlesworth and V. Sorrentino, submitted for publication). In agreement with the results on the a- and p-isoforms in frogs, analysis of the two chicken nucleotide sequences indicates they are the chicken homologs of the mammalian RyRl and RyR3, respectively. The relationship of these two cDNAs to the a- and @proteins has been established by raising antibodies against the recombinant proteins obtained by expressing in Escherichia colispecific regions of the two chicken cDNAs. Antibodies for RyRl and RyR3 were able to recognize the chicken a- and @-isoforms, respectively. A recent investigation on the expression of the mammalian skeletal RyR isoforms has shown that, in lower vertebrates, both a- and p-isoforms are expressed in most muscles, though in some muscles only the a-isoform can be detected (O’Brien et al., 1993). Surprisingly, among reptiles, crocodiles and turtles express both a- and p-isoforms, while lizards and snakes express predominantly the a- isoform, as in mammals. In birds and fishes, the aisoform alone is expressed in the extraocular muscle, one of the fastest contracting muscles in vertebrates, while both a- and p-isoforms are present in the other muscles. The a-isoform alone has also been detected in the swim-bladder muscle of toad fish, another muscle capable of contracting at

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a very high frequency. Single-channel recordings of preparations of sarcoplasmic reticulum from chicken skeletal muscle have shown that two populations of channels with different regulatory properties can be observed (Percival et al., 1994). It is possible that usage of either a-alone or of aand p-isoforms together may reflect the specific contraction requirements of a given muscle. RyRs have also been described in nonvertebrate species. In lobster skeletal muscle, a single RyR has been observed (Seok et al., 1992). The lobster RyR has been purified and characterized biochemically. It has no immunological cross-reactivity with the mammalian skeletal or cardiac isoforrns. The calcium-release properties of the lobster channel differs from those present in mammals. The lobster channel is poorly responsive to stimulation with 5 m M adenine nucleotides and 10 m M caffeine and is not readily inhibited by millimolar Mg2 and micromolar Ruthenium red concentrations. Lobster Ry R, along with the mammalian channel, is inhibited by the local anesthetic tetracaine. The existence of a protein that binds ryanodine has been demonstrated in C. elegans (Kim et al., 1992). This protein has been partially purified and the single-channel properties of such preparations have been studied in planar bilayers, confirming the existence, in C. elegans, of a RyR with functional properties comparable to those of the mammalian RyRs. More recently, a partial sequence of a C. elegans gene with homology to the mammalian RyRs has also been reported (Waterston et al., 1992). A partial cDNA corresponding to a Drosophila homolog of the RyR (dry)has been cloned (Hasan and Rosbash, 1992). This gene is expressed in the mesoderm of early stage-9 embryos and, later on, in somatic muscles. In adult flies the dry gene is expressed in tubular muscles and neuronal tissues. The entire full-length sequence of a Drosophila RyR has also been reported (Takeshima et al., 1994). The gene maps to band 44F, while the partial cDNA clone dry has been mapped to 76C-D. +

VII. Conclusion Our knowledge about the contribution of RyR to calcium signaling is extending from its original field of interest, namely, muscle physiology, to several other areas. Along with their fundamental role in regulating skeletal and cardiac muscle contraction, a functional involvement of RyRs in different CNS activities, such as long-term potentiation (LTP) and long-term depression (LTD) has been proposed. Over the past few years, results obtained by molecular and pharmacological studies have provided evidence suggesting that these channels are present and functional in many other cell types, in addition to muscle and neuron cells. With regard to the activation of RyRs, much information has been gathered indicating that cADPR, a

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strong activator of RyR activity, is expressed in almost all cells, and enzymes capable of synthesizing and degrading cADPR are also present. Much attention is focused on studying the metabolism of cADPR. Both molecular biology and biochemistry have provided new hints to understanding the structure/function relationship of RyRs. It can be expected that such a large convergence of studies will certainly provide answers to many of the questions that are still left open.

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