I. Introduction

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It has been 25 years since the seminal review by Jenden and Fairhurst, The Pharmacology of Ryanodine (Jenden and Fairhurst, 1969), appeared in this Journal. During this time, we have learned a great deal about ryanodine and related compounds, and about ryanodine receptor (RyR)^c calcium release channels, the family of proteins that bind and are modified by these ligands. The interest in the pharmacology of the RyRs has increased as these proteins have been shown to serve as intracellular Ca²⁺ release channels in a variety of tissue and cell types (Sutko and Airey, 1996). Moreover, as discussed in this review, several important questions remain concerning the role of RyR-mediated Ca²⁺ release events in different cell types, and the channel properties of different RyR isoforms. Resolution of these questions will require, at least in part, the use of pharmacological agents that modify the activity of the RyRs in both channel property- and isoform-specific ways. Recent data suggest it will be possible to develop such agents. Compounds related to ryanodine, termed ryanoids, exhibit agonist- and antagonist-like actions on RyR channels and, if not provided by nature, have the potential to be made RyR isoform-specific by chemical engineering. In addition, new agents that affect RyR channels, many of which are chemically unrelated to ryanodine, are being identified at a rapid rate.

In this article, we focus on the present state of RyR pharmacology and consider the aspects of RyR function that require the development of RyR isoform-specific RyR channel modifiers. Ryanodine and related compounds will receive much of our attention, as they remain the prototypic and most potent modulators of the RyR family of proteins. We will also limit our consideration to vertebrate RyRs. As noted below, the ryanoids are commercially important pesticides, and the effects of these agents on insects have been discussed previously (Crosby, 1971; Pessah, 1990; Jefferies and Casida, 1994).

	II. Ryanodine: Historical Aspects

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Ryanodine is a natural product found in members of the genus Ryania, which grow as shrubs or slender trees in several tropical locations in Central and South America, including Trinidad, and the Amazon basin. In addition, a closely related compound, ryanodol (2) has been isolated from the wood of Persea indica (Gonzalez-Coloma et al., 1993). It has long been appreciated that wood from trees in the genus Ryania contains toxic components. Crude extracts were used by local natives to poison arrowheads (Crosby, 1971). In the 1940s before the widespread use of synthetic chlorinated hydrocarbon and organic phosphate insecticides, E.F. Rogers, K. Folkers and colleagues at Merck and Co., and members of the Department of Entomology at Rutgers University conducted a cooperative survey of plant materials for new insecticides. This work revealed that extracts of the stem and roots of Ryania speciosa Vahl had promising insecticidal activity. Rogers et al. (1948) purified a compound from stem wood that they designated ryanodine, which had 700 times the insecticidal potency of the starting material.

Powdered Ryania wood was marketed initially as an insecticide by the Penick Corporation, but became less profitable as synthetic compounds, such as DDT and parathion, came into wide usage during the 1950s and 1960s. The detrimental environmental impact of the latter insecticides and the increased desire for organically grown produce led to the reintroduction of Ryania wood as an insecticide by Tom and Lorraine Harding of Progressive Agri-Systems (DeVault, 1983). A mixture of Ryania wood, rotenone, and pyrethrum has proven to be particularly effective. Ryanodine retains its value as an insecticide for several reasons. First, it is as effective as DDT or parathion against crop-damaging insects, such as the Codling moth (apples) and the European corn borer. Second, it is biodegradable, with a relatively short half-life, permitting it be applied close to harvest and avoiding the environmental concerns associated with persistence. Third, in several cases, the selectivity of ryanodine's insecticidal actions is fortuitous in that crop-damaging insects are affected, while beneficial insects are spared. These properties led ryanodine, rotenone, and pyrethrum to be identified as acceptable alternative insecticides by Rachel Carson in her book, "Silent Spring" (Carson, 1962).

The availability of purified ryanodine suitable for biomedical research largely has been due to the efforts of a few individuals. These include Dr. E.F. Rogers (Merck Sharp and Dohme Research Laboratory, West Point, PA), Dr. Ronald Harmetz (Penick Corporation), and Lorraine and Tom Harding (Progressive Agri-Systems). A debt of gratitude is owed to these individuals, who have selflessly worked to keep ryanodine preparations suitable for research available to the biomedical community during times when it was not an economically viable product.

Finally, ryanodine is also of historical significance because its properties and biological actions have permitted the identification and molecular characterization of a family of intracellular Ca²⁺ release channels, now commonly termed the ryanodine receptors.

	III. The Ryanodine Receptor Ca2+ Release Channels

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The RyRs are a family of Ca²⁺ channels that participate in the release of Ca²⁺ from intracellular stores. To date, the most notable of these stores are the sarcoplasmic reticulum (SR) in muscle and the rough endoplasmic reticulum (RER) in nonmuscle cells (Sutko and Airey, 1996). In addition, recent data suggest a greater variety in the way cells use these proteins, as RyRs have been reported to release Ca²⁺ from stores associated with the nucleus (Gerasimenko et al., 1995) and to be used to sense extracellular Ca²⁺ in osteocytes (Zaidi et al., 1992, 1995). In this section, we consider RyR distribution, structure, and function to the extent necessary to describe specific issues that would benefit from the development of new pharmacological agents that affect RyR function. Readers desiring more information about these topics are referred to several recent reviews of the molecular and functional aspects of the RyRs (Williams, 1992; McPherson and Campbell, 1993; Ogawa, 1994; Coronado et al., 1994; Meissner, 1994; Sutko and Airey, 1996).

RyRs were initially observed in skeletal muscle, where they were visualized in electron micrographs as large electron-dense masses situated along the face of the SR terminal cisternae, which is closely apposed to transverse tubule membranes to form a structure known as the triad junction (Franzini-Armstrong, 1970, 1972). Based on their appearance, the RyRs were termed triad junctional foot proteins (Franzini-Armstrong, 1970, 1972, 1975). The RyRs gained their present name after they were found to be the proteins that bind [³H]ryanodine (Pessah et al., 1985; Inui et al., 1987, 1988; Campbell et al., 1987; Lai et al., 1988), an agent known from earlier studies to alter SR calcium release events (Fairhurst, 1974; Fairhurst and Hasselbach, 1970).

The RyR family is proving to be extensive. Three genes encoding different RyR isoforms have been described in mammalian tissues. They have been cloned and sequenced from skeletal muscle (ryr1), heart (ryr2), and brain (ryr3) (Takeshima et al., 1989; Marks et al., 1989; Zorzato et al., 1990; Nakai et al., 1990; Otsu et al., 1990; Giannini et al., 1992; Hakamata et al., 1992). Sequence homologs of the three mammalian RyR isoforms are expressed also in nonmammalian vertebrates, where they have been termed  (RyR1),  (RyR3) and cardiac (RyR2) (Airey et al., 1990, 1993c; Olivares et al., 1991; Murayama and Ogawa, 1992; Lai et al., 1992; Dutro et al., 1993; O'Brien et al., 1993; Ogawa, 1994; Oyamada et al., 1994; Jens et al., 1995; Conti et al., 1996; Ottini et al., 1996). In addition, two alternatively spliced variants of RyR1 and one variant of RyR2 have been identified (Nakai et al., 1990; Zorzato et al., 1994; Futatsugi et al., 1995). These splice variants involve deletions of five (Ala (3481)-Gln (3485)) (Zorzato et al., 1994; Futatsugi et al., 1995) and six (Val (3865)-Asn (3870)) (Futatsugi et al., 1995) amino acids within a region of the RyR1 molecule that contains several putative regulatory sites. These variants are expressed in both a tissue- and a developmental stage-specific manner, suggesting that they may have different functional properties and/or may be regulated in different ways (Futatsugi et al., 1995).

There also may be RyR isoforms that are distinct from the mammalian and nonmammalian vertebrate isoforms described in the preceding paragraph. For example, a ryanodine binding activity in mammalian liver exhibits distinctive properties (Shoshan-Barmatz, 1990; Shoshan-Barmatz et al., 1991; Bazotte et al., 1991; Lilly and Gollan, 1995). A RyR purified from lobster muscle requires much greater Ca²⁺ concentrations for channel activation than the vertebrate isoforms characterized to date (Olivares et al., 1993; Seok et al., 1992). The sequence of a RyR from Drosophila tissues is the most divergent identified to date (Hasan and Rosbach, 1992; Takeshima et al., 1994b), exhibiting < 50% homology with the vertebrate isoforms (Takeshima et al., 1994b).

A truncated version of the skeletal muscle RyR (RyR1), which is translated using an alternate start site within the RyR1 mRNA, has been found in mammalian brain (Takeshima et al., 1993). Although the functional properties of this protein have not yet been described, the protein consists of the C-terminal ~75 kDa of RyR1, a region thought to contain the Ca²⁺ channel domain. Consequently, this protein also may be a Ca²⁺ channel. As described below, two different approaches starting with the intact RyR have localized the high affinity ryanodine binding site to the region contained in the truncated RyR. However, cells expressing the truncated protein did not exhibit detectable [³H]ryanodine binding activity (Takeshima et al., 1993). Therefore, it is unclear whether the C-terminal 75 kDa protein alone is sufficient to bind ryanodine.

The channel properties of the mammalian RyR1 and RyR3 isoforms, the RyR and RyR isoforms in chickens, frogs, and fish, and the RyR expressed in lobster muscle have been investigated in vitro (Seok et al., 1992; Bull and Marengo, 1993; Olivares et al., 1993; Percival et al., 1994; O'Brien et al., 1995). Significant differences in the gating and activation of the channels associated with these proteins have been described, indicating that the diversity of this family of channels underlies different roles for each member in intracellular Ca²⁺ signaling.

The RyRs have broad phylogenetic and tissue distributions. Biochemical, molecular or pharmacological evidence for the presence of RyRs has been found in invertebrates (lobster, Drosophila and C. elegans), as well as vertebrates (mammals, birds, reptiles, fish, and amphibians) (Sutko and Airey, 1996). Some vertebrate cell types that express RyRs include neurons in both the central and peripheral nervous systems, smooth muscle, endothelial cells, adrenal chromaffin cells, hepatocytes, osteocytes, eggs, and pancreatic cells (Sutko and Airey, 1996). In many of these cells, the identity of the specific RyR isoforms expressed, the intracellular distributions of these proteins and the cellular responses elicited by RyR-mediated Ca²⁺ release events are not known and represent important topics for future study.

Other complexities involving RyR-mediated Ca²⁺ release signals are that the RyRs expressed within a cell are localized to different sites where they may have different functions, or where more than one RyR isoform is co-expressed. For example, RyRs expressed in skeletal muscle cells may be have both extrajunctional and junctional distributions (Dulhunty et al., 1992). The RyR expressed in cardiac muscle (RyR2) is localized to different regions of the SR. One region involves junctions between the SR and the surface or t-tubule membranes, while a second region, termed corbular SR, consists of SR membranes that form terminal, junctional-like structures in the interior of the cell that do not form junctions with another membrane (Jewett et al., 1973; Jorgensen et al., 1993; Carl et al., 1995). Localization to specialized regions within the cell is likely to confer different properties on the Ca²⁺ release events mediated by each population of RyRs. Freeze-fracture studies of toadfish swim bladder muscle, which expresses a single or predominant RyR (O'Brien et al., 1993), have revealed that the RyRs in this muscle are arranged so that they differ in their potential to physically interact with another protein, the dihydropyridine receptor (DHPR) (Block et al., 1988; Franzini-Armstrong and Jorgensen, 1994). This observation suggests the existence of two types of RyRs, distinguished by the protein species with which they associate, that may differ in the manner in which they are activated to release Ca²⁺.

Biochemical evidence for the co-expression of two molecularly distinct RyR isoforms, which were termed  and , was obtained for nonmammalian vertebrate skeletal muscles from chickens, frogs, and fish (Airey et al., 1990, 1993c; Olivares et al., 1991; Lai et al., 1992; Murayama and Ogawa, 1992; O'Brien et al., 1993, 1995). Subsequently, data from RNase protection and in situ hybridization analyses (Giannini et al., 1992, 1995; Takeshima et al., 1995), reverse transcriptase-polymerase chain reaction studies (Ledbetter et al., 1994), Western blot analysis (Conti et al., 1996), and functional studies of muscles from dyspedic mice in which expression of the primary skeletal muscle RyR isoform (RyR1) has been eliminated by homologous recombination (Takeshima et al., 1994a, 1995), indicate that two RyR isoforms, RyR1 and RyR3, are co-expressed in several mammalian muscle and nonmuscle tissues. A striking difference exists in the relative quantities of the two RyRs co-expressed in different vertebrate skeletal muscles. Similar and abundant levels of the RyR and RyR isoforms are found in many, but not all, nonmammalian vertebrate muscles, whereas the RyR or RyR3 isoforms are expressed at much lower levels than the RyR or RyR1 isoforms in some nonmammalian vertebrate muscles and in the mammalian muscles studied to date (Conti et al., 1996). The expression of these proteins at markedly different levels may be a rule for mammalian tissues, because it has proven difficult to detect RyR3 protein in them using either immunostaining or Western blot analysis.

Several observations indicate that both of the RyR isoforms co-expressed in nonmammalian vertebrate and mammalian muscles can function as Ca²⁺ release channels. Studies of purified avian RyRs and RyRs conducted in vitro demonstrate that both isoforms contain Ca²⁺ channels, but that they differ in their gating properties (Percival et al., 1994). Investigations of the ryanodine-sensitive channel activities in frog and fish SR membranes yield similar conclusions concerning the RyRs and RyRs expressed in these species (Bull and Marengo, 1993; O'Brien et al., 1995). Avian and frog RyRs and RyRs also differ in the extent to which they are phosphorylated by the cyclic adenosine monophosphate-dependent protein kinase and the Ca²⁺/calmodulin-dependent protein kinase II, and the avian isoforms differ in the extent to which they bind calmodulin, indicating that the channel activities associated with these proteins have the potential to be regulated differently (Airey et al., 1993c). Moreover, the avian RyRs and RyRs are expressed initially at different times during embryonic muscle development (Sutko et al., 1991), which also suggests that the isoforms have unique functions. Like the avian RyR isoforms, both the RyR1 and RyR3 isoforms expressed in mouse skeletal muscle can serve as SR Ca²⁺ release channels (Takeshima et al., 1994a, 1995). This is demonstrated by data from two genetically altered animals, Crooked Neck Dwarf (cn) mutant chickens and dyspedic mice, which indicate that the avian RyR and the mammalian RyR3 isoform release SR Ca²⁺ in situ (Airey et al., 1993a,b; Ivanenko et al., 1995; Takeshima et al., 1995). Cn/cn and dyspedic muscles are null for the RyR and RyR1 isoform, respectively, yet ryanodine-sensitive Ca²⁺ transients and contractions can be elicited from both by electrical stimuli or caffeine. This Ca²⁺ release can be attributed to the RyR and RyR3 isoforms expressed in the mutant muscles. The absence of the RyR or the RyR1 isoforms in these mutant animals results in a skeletal muscle dysgenesis that is lethal in both cases (Airey et al., 1993a,b; Takeshima et al., 1994a).

RyRs can be localized to specific cellular sites in different cell types, and, in some cases, more than one RyR isoform may be co-expressed within the same cell. Thus, important questions exist as to the intracellular distributions of these proteins and the functional contributions made by each isoform in different tissues. The distributions of RyRs within a cell are determined most directly by immunostaining with RyR isoform-specific antibodies. However, a limitation of this method is that it does not permit RyRs that are active as Ca²⁺ release channels to be distinguished from those that are not functionally active, e.g., ones being synthesized, trafficked, or degraded. For example, RyR and inositol trisphosphate (IP₃) receptors colocalize to RER membranes in the soma of avian cerebellar Purkinje neurons, but exhibit a differential distribution in the endomembranes in the dendrites and dendritic spines in these neurons (Walton et al., 1991; Sharp et al., 1993). These observations lead to the question of whether the RyRs in either or both of these locations are active as Ca²⁺ release channels. Similarly, functionality is an issue when the potential contributions made by different RyR populations is investigated in cell types that co-express more than one RyR isoform.

An approach to this question is to use a probe that only recognizes active RyR channels. Ryanodine binds with high affinity to the RyRs when they are in the conformation associated with an open state of the channel and, therefore, has the potential to serve as such a probe. Moreover, ryanodine can be labeled at the 21-position with bulky substituents, such as the fluorophore Bodipy (125), without a significant decrease in high affinity binding (Welch et al., 1994). Thus, at least in theory, comparison of the distribution of RyRs labeled by anti-RyR antibodies with that obtained for RyRs labeled by a fluorescent ryanodine derivative under appropriate cellular activation states has the potential for identifying RyRs that are active as Ca²⁺ release channels. Moreover, the use of a fluorescent group such as eosin, which is an efficient producer of free radicals, would permit utilization of the photo-oxidation labeling approach developed by Deerinck et al. (1994) and detection of active RyRs at the EM level. In practice, the use of fluorescently labeled ryanoids will require the development of conditions that minimize nonspecific binding of these derivatives. If the latter can be achieved, it will be of interest to develop fluorescent ryanodine derivatives that bind in a RyR isoform-specific manner (see next paragraph).

In many cell types, the role of RyR-mediated Ca²⁺ release events is not well understood (Sutko and Airey, 1996). Moreover, in cases in which two RyRs are co-expressed or a single RyR isoform is localized to different regions of the cell, it is unclear whether (and how) the different RyR populations interact to generate a Ca²⁺ signal. A pharmacological approach to these questions will not only be useful, but offers a significant advantage for dissecting the roles of co-expressed RyRs. As discussed below, the large size of the RyRs suggests they may have functions in addition to serving as Ca²⁺ release channels. The use of genetic techniques, such as homologous recombination or anti-sense, to prevent expression of a RyR, eliminates the physical presence, as well as the function, of the protein. Therefore, this approach may result in changes that are not simply due to the absence of RyR-mediated Ca²⁺ release events. For example, properties other than the activity of the RyRs as Ca²⁺ channels may influence aspects of cellular organization, such as the localization of other proteins. A more straightforward approach is to use RyR isoform-specific modifiers to alter the channel function of a RyR in a predictable manner under otherwise normal in situ conditions.

Definition of the contributions made by populations of the same RyR isoform that are localized to different sites within a cell, may prove in a practical sense to be a more difficult problem. Resolution of this issue is likely to require the use of several approaches, such as selectively disrupting the mechanism(s) used to activate the RyR channels in each population, the use of Ca²⁺ indicators localized to different regions in the cell (Etter et al., 1996), of spatially restricted techniques for sampling changes in Ca²⁺ (Cheng et al., 1993; Escobar et al., 1994), and of kinetic analyses to dissect the contributions made by each RyR population to spatially averaged changes in cell Ca²⁺.

With the possible exception of the truncated version of RyR1 expressed in mammalian brain described above, the vertebrate RyRs identified to date are homotetramers comprised of subunit polypeptides with molecular masses of 500 to 600 kDa (McPherson and Campbell, 1993; Meissner, 1994; Ogawa, 1994; Coronado et al., 1994). The massive size of the RyRs may make them physically the largest ion channels. Their closest relatives, the IP₃ receptors, are a little more than one-half as large (Mignery et al., 1989; Nakagawa et al., 1991; Südhof et al., 1991). The RyR isoforms are similar in their overall topology and in that the ion channel forming membrane spanning regions are highly conserved and appear to be localized to the carboxyl terminal 20% of the protein. This is consistent with the size of most other ion channel proteins. In each case, the remaining amino-terminal region of the protein, which consists of more than 80% of the mass of the RyRs, forms a large cytoplasmic foot domain that assumes a quarterfoil shape (Inui et al., 1987; Saito et al., 1988; Lai et al., 1988; Radermacher et al., 1992, 1994; Serysheva et al., 1995).

The three-dimensional topology of the RyR1 isoform has been established in greater detail using cryo-electron microscopy (Radermacher et al., 1994; Serysheva et al., 1995, 1996; Wagenknecht and Radermacher, 1995). These latter studies have yielded reconstructed images that exhibit a four-fold symmetry consistent with the homotetrameric organization of the native protein. An open region extends through the central region of the molecule and may represent the ion conducting channel. In some reconstructions, this pore appears to be plugged at the SR lumenal surface of the RyR, perhaps representing a closed state of the channel. Consistent with this possibility, this plug was not observed in RyRs frozen under conditions that result in channel activation (Radermacher et al., 1994; Serysheva et al., 1995, 1996; Wagenknecht and Radermacher, 1995; Orlova et al., 1996). The upper part of the cytoplasmic domain of the RyR is loosely packed and contains a large percentage of solvent space. As it enters this region of the receptor, the central pore becomes less well defined. The lattice-like structure suggests that each of the subunits may have the potential to undergo significant changes in conformation. Such changes could be involved in the transmission of activation signals received at the cytoplasmic surface of the RyR to the domains of the protein involved in channel gating, which may be located in regions of the molecule close to the SR lumen. Consistent with this scenario, binding sites for agents that affect RyR channel activity, such as calmodulin and the 12 kDa FKBP binding protein (FKBP-12), have been localized to the surface regions of the cytoplasmic domain (Wagenknecht et al., 1994, 1996). In addition, as summarized in detail in a recent review (Ogawa, 1994), putative binding domains have been identified for several regulators of the RyR channel within the context of the primary structure of the RyR. These include Ca²⁺, adenine nucleotides, and calmodulin. The effects of these agents on RyR channel properties have extensive interactions with each other. For example, calmodulin activates the RyR1 channel in the presence of submicromolar Ca²⁺, but inhibits channel activity when Ca²⁺ is increased into the micromolar range (Ikemoto et al., 1995; Tripathy et al., 1995). The presence of adenine nucleotides increases the extent to which the RyR1 and avian RyR isoforms can be activated by Ca²⁺ in vitro (Smith et al., 1988; Percival et al., 1994). In contrast, under similar conditions, the RyR2 and avian RyR isoforms are activated to a greater extent by Ca²⁺ in the absence of adenosine triphosphate (ATP) (Anderson et al., 1989).

As noted in the preceding paragraph, the massive size of the RyRs has also permitted visualization of two proteins that modify RyR channel properties, calmodulin and FKBP-12, bound to the surface of the receptor. It may be possible to extend this analysis to map the binding sites for non-protein ligands of the RyR, such as ryanodine. The latter will depend though on the nature and the position of the binding site within the structure of the protein and whether a suitable electron dense group can be attached to the ligand without inhibiting its ability to bind to the RyR. In any case, when used in conjunction with information from primary RyR sequence analysis and site-directed mutagenesis, this mapping approach should establish landmarks for determining how the primary sequence of the RyR is folded to achieve the secondary and tertiary structures of the native protein. In addition, this approach should yield important insights into how different domains of the RyR interact to regulate channel activity.

The importance of the large size of the RyRs is not well understood. Although they are found in a variety of tissue and cell types (Sutko and Airey, 1996), most of our knowledge of RyR structure and function comes from studies of skeletal and cardiac muscles. The RyRs in these muscles are localized primarily to membrane junctions and, in particular, to the space formed by the apposition of the SR membrane with either the surface membrane of the cell (peripheral coupling sites) or with the invaginating t-tubular membrane (triad or diad junctions). Within the context of the junctional structure, the large size of the RyRs may be needed to permit these proteins to participate in the organization of the junction, and/or to span the junctional gap and interact with components of the surface or t-tubular membranes. For example, recent studies indicate that the cytoplasmic loop between the SII and SIII transmembrane repeats in the ₁-DHPR subunit is one such component (Tanabe et al., 1990; Lu et al., 1994, 1995; El-Hayek et al., 1995a). However, functional RyRs may be localized outside of the junctional structures in both cardiac and skeletal muscles (Jorgensen et al., 1993; Carl et al., 1995). Moreover, it is unclear whether the RyRs expressed in nonmuscle tissues exist within a structural format that necessitates their large size. Thus, it will be of interest to learn whether the significant mass of this protein has evolved to give the RyR channel specific functional and/or regulatory properties. Expression of RyRs having N-terminal truncations in homologous systems, such as those produced by genetic knockouts (Takeshima et al., 1994a; Nakai et al., 1996), should provide insights into the functional significance of the large size of these proteins.

Ryanodine and related compounds have complex effects on the conductance and the gating of single RyR channels. Studies with skeletal muscle, particularly those conducted by Fairhurst and Hasselbach (1970) and by Fairhurst (1974), and the work of Hilgemann (Hilgemann, 1983, 1986) using cardiac muscle preparations demonstrated that the actions of ryanodine result in an increase in the Ca²⁺ permeability of the SR. In addition, ryanodine can decrease SR Ca²⁺ permeability (Sutko et al., 1979, 1985; Jones et al., 1979; Fabiato, 1985; Fleischer et al., 1985; Meissner, 1986; Lattanzio et al., 1987). Therefore, it is not surprising that, once the RyR was identified, ryanodine and related compounds were found to have complex effects on the conductance and gating of its channel.

Three general effects on the activity of RyR channels have been observed for ryanodine. At submicromolar concentrations, it has been reported to increase channel activity with openings to a full conductance state (Pessah and Zimanyi, 1991). It should be noted that this change has not been observed as consistently as the two effects described next. Also, at submicromolar concentrations, ryanodine causes the channel to exhibit partially conducting or subconductance states (Rousseau et al., 1987). This effect is observed consistently and has become a signature for a ryanodine-modified RyR channel. Multiple subconductance levels have been observed (Buck et al., 1992; Liu et al., 1989; Kwok and Best, 1990; Pessah and Zimanyi, 1991; Ding and Kasai, 1996), but one that is ~ 50% of the full conductance level is the most common. Both of the first two effects would contribute to the ryanodine-induced increase in SR Ca²⁺ permeability noted above. At micromolar or greater concentrations, ryanodine produces a closed state of the channel (Meissner, 1994). This effect is also consistently observed and accounts for the decrease in SR Ca²⁺ permeability caused by ryanodine. When defined in single channel experiments, the concentration dependence of these effects by ryanodine must be considered as estimates, because an insufficient number of trials have been conducted to accurately determine the concentration of ryanodine binding when a single RyR protein is involved.

It is not clear whether ryanodine alters the conductance state of the RyR channel by stabilizing a specific conformation of the channel via allosteric effects, or whether it physically interferes with the flux of ions through the pore of the channel. As discussed below in Section V., recent data obtained from analysis of the effects of ryanoids on the conductance of the RyR2 channel favor an allosteric mechanism (Welch et al., 1997); however, additional studies are needed to resolve this issue.

Given the complexities of the effects by ryanodine on RyR channel function, it is not surprising that investigations of the binding of this ligand to RyRs have yielded complex results. There is general agreement that [³H]ryanodine binds to sites on the RyRs exhibiting high (K_D ~1 to 10 nM) and low (K_D ~1 to 10 µm) affinities. It is also generally accepted that high affinity binding results in RyR channel activation and in channels exhibiting conductance substates, and that low affinity binding causes channel inhibition. The functional consequences of ryanodine binding exhibit use-dependence. For example, following infusion of ryanodine into rats, contracture developed in muscles receiving electrical stimuli, but not in their nonstimulated counterparts (Procita, 1956). Moreover, the latter muscles showed no signs of having been exposed to ryanodine if this agent was washed out before activation of the muscle. This indicated that ryanodine did not bind to RyRs in the inactive muscle. Consistent with this finding, high affinity [³H]ryanodine binding is observed under conditions that are associated with activation of the RyR channel, indicating that ryanodine binds to a RyR conformation associated with an open state of the channel. This property has proven useful, because it permits [³H]ryanodine binding to be used as an index of channel activation (Hawkes et al., 1992; Meissner and El-Hashem, 1992).

Several observations indicate that the tetrameric form of the RyR is required to bind ryanodine with high affinity. This is demonstrated by the inability of the individual subunits dissociated by exposure of the tetramer to the detergent Zwittergent 3-14, instead of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) (which maintains the RyR as a tetramer) to bind [³H]ryanodine (Lai et al., 1989). A caveat concerning the latter studies is that it was not shown that the separated monomers could be reassembled to form a tetrameric RyR capable of binding ligand. Consequently, denaturation of the monomer may have masked its ability to bind ligand. Also, it has been found that under appropriate conditions, the use of covalent cross-linking to stabilize the RyR tetramer does not interfere with high affinity ryanodine binding, indicating further that the tetrameric form of the RyR is relevant for ligand binding (Lai et al., 1989; Carroll et al., 1991; Shoshan-Barmatz et al., 1995). The inability of RyR monomers to bind ryanodine suggests that the binding site is created by the correct juxtaposition domains contributed by more than one subunit, or alternatively, that each monomer may contain a binding site, but the site only assumes the conformation appropriate for ligand binding in the context of the molecular organization achieved in the tetramer.

Results obtained for [³H]ryanodine binding to purified RyRs suggest stoichiometries (moles of [³H]ryanodine/mole of RyR tetramer) of 1:1 for the high affinity binding process (Lai et al., 1988, 1989; Carroll et al., 1991; Pessah and Zimanyi, 1991; Wang et al., 1993). Stoichiometries of either 3:1 (Lai et al., 1989) or 1:1 (Wang et al., 1993) have been reported for low affinity ryanodine binding. This difference in the number of low affinity sites has led to two different models of ryanodine binding to its receptor. The first model involves four initially identical interacting binding sites per RyR tetramer that can serve as either high or low affinity sites (Lai et al., 1989; Carroll et al., 1991; Pessah and Zimanyi, 1991) and will be termed the interconvertible site model (ISM). The second model proposes two nonidentical or distinct interacting ryanodine binding sites per RyR tetramer that subserve high and low affinity ryanodine binding, respectively (Wang et al., 1993). The model will be termed the distinct site model (DSM). In the ISM, the four sites are considered initially to be equivalent and capable of binding ryanodine with high affinity when the RyR assumes a conformation associated with an open state of the channel. The binding of ryanodine to one site exerts a negatively cooperative effect on the remaining sites, lowering their affinity for ryanodine. In one version of the ISM, binding to the first site produces an equivalent reduction in the affinity of the remaining three sites (Carroll et al., 1991). Binding of ryanodine to all three low affinity sites leads to channel closure. In a second version of the ISM, a sequential process is proposed wherein binding of ryanodine to each site successively lowers the affinity of the remaining unbound sites, leading to four classes of ryanodine binding sites having different K_D values (Pessah and Zimanyi, 1991). Binding of ryanodine to each site is also proposed to alter successively the conductance of the RyR channel to lower and lower fractional values of the fully conducting state. Ultimately, this leads to stabilization of a closed state of the channel, when all four sites have bound ryanodine.

In the DSM, two distinct classes of binding sites that differ in their affinity for ryanodine exist in each RyR tetramer at a stoichiometry of 1:1. As in the ISM, binding of ryanodine to the high affinity site requires the RyR to assume a conformation associated with an open state of the channel and stabilizes the protein in a conformation exhibiting a fractional conductance. Ryanodine binding to the low affinity site results in channel inhibition. Because the two types of ryanodine binding sites have distinct properties, it is not necessary to have negatively cooperative interactions between sites in the DSM to produce low affinity binding. An intersite interaction that is part of this model is the binding of ryanodine to the low affinity site that leads to a decrease in the rate of dissociation of ryanodine bound to the high affinity site (McGrew et al., 1989; Wang et al., 1993). The nature of this effect, e.g., whether it (a) involves steric interactions and a physical trapping of ryanodine bound to the high affinity site or (b) allosteric interactions between different regions of the RyR, remains to be determined.

A key difference between the DSM and ISM is that, in the DSM, the same physical sites do not subserve both high and low affinity ryanodine binding. In both the DSM and ISM, high and low affinity ryanodine binding is affected by conformational changes in the RyR, and, in both models, these conformational changes are a function of the activation state of the channel. In addition in the ISM, conformational changes producing a negatively cooperative effect are also a function of the extent of ryanodine binding. In comparing the DSM and ISM, it should be considered that determinations of the properties of binding sites with K_D values in the micromolar range using filtration assays can have a degree of uncertainty.

Additional experimentation is required to establish the number and the identity of the ryanodine binding sites. One approach that is being taken to resolve this issue is to identify the binding sites within the context of the primary sequence of the RyR. In this regard, significant advances have been made recently, and two lines of evidence indicate that both ryanodine binding site(s) may be localized to the C-terminal 76 kDa of the receptor. In an elegant series of studies, Callaway et al. (1994) took advantage of the ability of ryanodine binding to a low affinity site(s) to stabilize ryanodine bound to the high affinity site. Binding to the latter sites was maintained following proteolysis with trypsin to yield a 76-kDa fragment that was derived from the carboxy terminus of the RyR monomer. Independently, Witcher et al. (1994) used 10-O-(3-[4-azidobenzamido]propanoyl)ryanodine (84B), a photolabile cross-linking derivative of ryanodine, to covalently bind tritiated ligand to the RyR under conditions that resulted in high affinity binding. Again, a 76-kDa fragment derived from the C-terminus of the receptor was cross-linked. There can be some uncertainty associated with the approach used in the latter study, as regions not involved in ligand binding may be cross-linked. However, the agreement reached by both of the above complementary approaches indicate strongly that at least a significant part of the high affinity and presumably also the low affinity ryanodine binding sites are localized as indicated by these studies. Further utilization of such approaches to the site(s) responsible for low affinity ryanodine binding will permit a more precise determination of the physical relationship existing between the high and low affinity binding sites. For example, the studies by Callaway et al. (1994) suggest that at least part of the low affinity ryanodine binding site is present in the C-terminal 76 kDa fragment of the RyR, because high concentrations of ryanodine still were able to slow the dissociation of ryanodine bound to the high affinity site in this fragment.

A pharmacological approach to identifying the number and nature of the ryanodine binding sites could be taken if ligands specific for either the high or low affinity site(s) were available. For example, in contrast to ryanodine, which both increases and decreases SR Ca²⁺ permeability at nanomolar and micromolar concentrations, respectively, the ryanoid designated as the ester A (65) (Ruest et al., 1985) only causes an increase in the Ca²⁺ permeability of skeletal muscle SR membranes at concentrations up to 3 mM (figs. 1-3, and data not shown) (Sutko et al., 1990). The ester A binds to the high affinity ryanodine binding site with a K_D = 110 nM (Welch et al., 1994). If this ryanoid is shown not to bind to the low affinity site, e.g., by using [³H]ester A, the selective nature of the effects of this agent on SR Ca²⁺ permeability would suggest one of two possibilities. The first possibility is that the high and low affinity binding sites are distinct entities, as proposed in the DSM. In this case, ryanodine should still bind to the low affinity sites in the presence of the ester A. A second possibility is that the binding of the ester A does not lead to the conformational change necessary to induce the negative cooperativity required to convert high affinity to low affinity sites, as described in the ISM. In this case, the binding of ryanodine to both the high and low affinity sites should be attenuated by pretreating the RyRs with the ester A. Ryanodine binding to the high affinity site would be reduced due to direct competition between ryanodine and the ester A. The number of low affinity sites available to bind ryanodine would be decreased by the inability of ester A binding to the high affinity site to produce the conformation change necessary to create these sites.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   The effects of ryanodine and the ester A on the passive efflux of Ca2+ from skeletal muscle terminal cisternae vesicles. Membrane protein (5 mg/ml) was suspended in a solution containing 0.1 M KCl, 10 mm Pipes/Tris (pH 6.8) and 0.1 mM 45CaCl2 and incubated for 2 h at 37°C in the absence or presence of the concentrations of ryanodine and the ester A indicated along the abscissa. Ca2+ efflux occurring during 1-sec intervals was measured at 25°C by diluting 5 µl of the vesicle suspension into 500 µl of a solution containing 0.1 M KCl, 10 mM Pipes/Tris (pH 6.8). Ca2+ efflux was terminated by the rapid addition of 5 ml of an ice-cold solution containing 0.3 M sucrose, 10 µM ruthenium red, 0.5 mM HgCl2, 0.5 mM LaCl3 and 10 mM Pipes/Tris (pH 6.8). The vesicles were trapped on Whatman GF/A filters and washed with two additional 5-ml aliquots of the terminating solution. The 45Ca retained on the filters was measured using liquid scintillation counting techniques. The quantity of 45Ca present in the vesicles after the efflux period was expressed as a percentage of that present before the initiation of the efflux. The difference between the values obtained for the preparations treated with ryanodine and ester A and for nontreated vesicles are presented in this figure. Control vesicles retained 61.45 ± 1.48% of their initial Ca2+ content after the 1-sec efflux period, and the initial vesicular content of Ca2+ was not affected by either ryanodine or the ester A. Values more negative and more positive than the control values are indicative of enhanced and inhibited effluxes, respectively. Similar results were obtained when ryanodine or the ester A were present in the efflux medium. The effects of coadministration of ryanodine and the ester A were assessed 20 and 120 min after the simultaneous addition of a 300 µM concentration of each agent. The values shown are means ±standard error for the number of preparations indicated within the bars. This figure is taken from Sutko et al. (1990) with the permission of the Plenum Publishing Corporation.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   The effects of ryanodine and the ester A on the passive influx of Ca2+ into skeletal muscle terminal cisternae vesicles. Membrane protein (5 mg/ml) was suspended in a solution containing 0.1 M KCl, 10 mm Pipes/Tris (pH 6.8) and was incubated for 2 h at 37°C in the absence and presence of ryanodine and the ester A at the concentrations shown along the abscissa. The quantity of Ca2+ entering the vesicles during a 1-sec period was determined at 25°C by mixing 46.3 µl of the protein suspension with 3.6 µl of 45CaCl2. The final concentration of Ca2+ was either 0.1 or 1.0 mM. The results obtained with either concentration were similar once normalized and have been combined. Ca2+ influx was terminated, and vesicular Ca2+ was measured as described for figure 1. The quantity of Ca2+ entering the vesicles during the 1-sec uptake interval is expressed as a percentage of the equilibrium vesicular Ca2+ content obtained after 20 min in the presence of the Ca2+ ionophore, ionomyocin (1 µM). Neither ryanodine nor the ester A affected the latter quantity. The values presented are means ± standard error for the number of preparations shown in each bar. This figure is taken from Sutko et al. (1990) with the permission of the Plenum Publishing Corporation.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   The effects of ryanodine (Ry) and the ester A (A) on ATP-dependent Ca2+ accumulation by skeletal muscle terminal cisternae membranes. Membrane protein (0.2 to 0.8 mg/ml) was added to a medium containing 50 mM histidine (pH 7.0), 3 mm MgCl2, 5 mM ATP, 3 mM K oxalate, 100 mM KCl and 38 µM 45 CaCl2; the resulting compound was incubated for 10 min at 37°C in the absence and presence of 300 µM concentrations of ryanodine or the ester A. Ca2+ uptake was terminated and vesicular Ca2+ measured as described for figure 1. The values shown have been corrected for Ca2+ binding occurring in the absence of ATP (which was unaffected by either ryanodine or the ester A) and represent means ± standard error for the number of preparations indicated in the bars. This figure is taken from Sutko et al. (1990) with the permission of the Plenum Publishing Corporation.

The potential for using a pharmacological approach, such as that described in the preceding paragraph, to investigate the properties of ryanodine binding sites is supported further by the finding that other ryanoids also differ in their relative abilities to increase and decrease the Ca²⁺ permeability of SR membranes. For example, Besch and coworkers (Humerickhouse et al., 1993, 1994; Gerzon et al., 1993; Bidasee et al., 1995) have found that the ratios of the concentrations required to increase and decrease the Ca²⁺ permeability of SR membranes vary between ryanoids. Moreover, Mitchell et al. (1996) found that the EC₅₀ concentration of a ryanoid required to cause half-maximal efflux of Ca²⁺ from SR vesicles is similar to the dissociation constants measured for high affinity binding in all cases tested. In contrast, the EC₅₀ concentration of ryanoids required to close the channel varied from 5 to 1000 times the high affinity dissociation constants. Recall that when mediated by the RyR, these two effects are thought to involve, respectively, binding to the high and low affinity ryanodine binding sites. Thus, these data suggest that these sites differ in their relative affinities for several ryanoids, a complexity that may be most easily explained by the existence of two distinct classes of binding sites. It has been observed also that exposure of RyR2 channels to one of these compounds, -alanyl ryanodine (84A), precludes inhibition of the channel by a subsequent exposure to high concentrations of ryanodine, a treatment that normally produces this effect (Tinker et al., 1996). This result indicates the complex nature of the interactions between the high and low affinity ryanodine binding sites that will require additional experiments to understand.

Consideration of the functional consequences of ryanodine binding to the RyRs raises the issue of the physiological relevance of the partial conductance states (substates) exhibited by these proteins when their activity is recorded in vitro. It is important to note that in several laboratories, substates are observed in the absence of ryanodine and, therefore, may represent normal conductance modes of the channel that are stabilized by ryanodine binding. In addition, substates are not routinely observed in every laboratory; consequently, the possibility exists that they may be induced by the conditions associated with either sample preparation and/or the experiment. An interesting observation in this regard is that the association of FKBP12 with RyR1 results in a predominance of channel openings to a full conductance level, whereas substates frequently are observed in the absence of the immunophilin (Brillantes et al., 1994). The extent to which different laboratories have studied RyRs without and with bound FKBP12 must be considered.

An important question concerns whether the substate conductances serve as a physiologically important mechanism for regulating the average quantity of Ca²⁺ released per unit time by a RyR. If this is the case, it will be important to define how switching between these conductance modes is regulated in vivo. The latter will require investigations of RyR conductances in situ, and in vitro under conditions known to mimic those existing in situ, where the regulatory state of the protein has been defined.

In summary, several questions exist concerning (a) the molecular and biophysical properties of the RyR channels and (b) the roles of these proteins in signal transduction systems in different cell types. These questions involve the intracellular distribution of functional RyR channels, the number and nature of ryanodine binding sites, and the manner in which the different RyR isoforms, particularly those that are co-expressed within the same cell, function to generate cellular Ca²⁺ signals. Resolution of these questions will benefit from the development of new pharmacological agents that have binding site-specific and RyR-isoform-specific channel actions and that either activate or inhibit RyR channel activity. In the next section, we consider the chemistry of ryanodine and related compounds and the progress that has been made in achieving selective modifications of the structure of these compounds.

	IV. Ryanoids: Ryanodine and Related Compounds

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Ryanodine (1) (fig. 4) is the complex polycyclic, polyhydroxylic diterpene (+)-ryanodol (2) esterified at C3 with pyrrole-2-carboxylic acid (see Jenden and Fairhurst, 1969; Jefferies and Casida, 1994, for review). The structure of ryanodine (1) was established by Wiesner and colleagues (Wiesner, 1972) more than 25 years ago in what remains a classic example of chemical structure elucidation using chemical degradation. The x-ray analysis of a p-bromobenzyl ether derivative (3) of ryanodol confirmed this structure with a minor change that the isopropyl and the hydroxyl groups at C2 had to be reversed (Srivastava and Przybylska, 1968) and are arranged as shown in structure 2.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Dehydration of ryanodine and ryanodol. Diterpenoids from Cinnamomi cortex.

Ryanodol (C₂₀H₃₂O₈) is a pentacyclic diterpene that has a ring skeleton consisting of 14 carbon atoms and a single oxygen atom. For uniformity, the numbering system of ryanodol will be used throughout this review. Four of the rings in ryanodol (A, B, C, and E) are carbocyclic, whereas the fifth ring (D) contains an oxygen atom. There is a methyl group, at C1, and an isopropyl group, at C2, attached to cyclopentane ring A, one tertiary methyl group at C5 in cyclopentane ring B, and a secondary equatorial methyl group at C9 in cyclohexane ring C. There are five tertiary hydroxyl groups at C2, C4, C6, C12, and C15. The hydroxyl at C15 is part of a hemiketal group formed by the tertiary hydroxyl group at C11 and the carbonyl group at C15. There are two secondary hydroxyl groups, one in ring A (-oriented) at C3 and one in ring C at C10 (-oriented and equatorial). Many diterpenoids from Cinnamomi cortex, known as cinnzeylanols (see 6) and cinncassiols (see 7), have a carbon framework closely related to that of ryanodol (2) (Nohara et al., 1981).

In acidic medium, ryanodine (1) dehydrates easily (Wiesner, 1972) (fig. 4) and gives anhydroryanodine (4), losing at the same time its typical biological activities (Pessah et al., 1985; Waterhouse et al., 1987; Jefferies et al., 1991, 1992b). The loss of water is accompanied by an important modification in the skeleton of the molecule. Anhydroryanodine (4) and anhydroryanodol (5) were important degradation products in the elucidation of the structure of ryanodine (Wiesner, 1972). As described below in this section, anhydroryanodol was a relay compound in the total synthesis of ryanodol (2) (as described in the next section and illustrated in fig. 8).

The ryanodine molecule can be viewed as having a hydrophilic face bearing five hydroxyl groups at C2, C4, C6, C12, and C10 and a lipophilic surface formed by the isopropyl group and the hydrogens attached to C14, C20, C7, C8, and C21. Both faces can be extended or shortened by a proper orientation of the pyrrole ester group at C3 (Jefferies et al., 1991, 1992b).

The total synthesis of (+)-ryanodol (2) was accomplished by Deslongchamps, Ruest, and colleagues in the late 1970s (Belanger et al., 1979; Deslongchamps et al., 1990) and remains an elegant and unique achievement in molecular construction. The strategies and methodologies involved in this synthesis have been described recently in detail (Deslongchamps et al., 1990). The starting materials for this synthesis are the activated diene 12 obtained in 11 steps from vanillin (10) (fig. 5) and the optically active dienophile 9 obtained in eight steps from S-(+)-carvone (8). It should be noted that vanillin has been isolated from extracts of Ryania speciosa Vahl (Ruest and Deslongchamps, unpublished observations). The synthetic fate of each of the carbon atoms in structures 9 and 12 has been shown by numbering them using the numbering system of ryanodol.

View larger version (33K): [in this window] [in a new window]   Fig. 5.   Total synthesis of ryanodol: Diels-Alder adducts from a dienophile and a diene obtained from carvone and vanillin, respectively.

The Diels-Alder reaction of spirolactone dienone 12 and enone 9 provided a mixture of four diastereoisomeric adducts13A and B, and 14A and Bin quantitative yield. Sequential treatments of the mixture in basic media (to give 15 endo-exo and 16 endo-exo, fig. 6), acidic media, and then basic media, led to the optically active pentacyclic aldehyde 17 that was stored as its protected form 19. Diastereoselective formation of the desired isomer 17 from the precedent mixture has been explained by steric hindrance between the isopropyl group and the 1,2-diol moiety that prevented the formation of the undesired diastereoisomer 18 during the last step, which involves the aldol condensation between C3 and the C4-carbonyl group.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Total synthesis of ryanodol: formation of the pentacyclic intermediate 17.

Baeyer-Villiger-retroepoxidation sequence on the pentacyclic olefin-ketone 19 (fig. 7) gave the olefin lactone 20. Ozonolysis of 20 in protic medium resulted in the formation of the desired aldol condensation product 21 that was transformed into the equatorial monomethylated compound 22. Subsequent reduction of ketone 22 led selectively to the equatorial alcohol 23. Protection of the hydroxycarbonate moiety of this compound as a methoxymethyl orthocarbonate group and reduction of the lactone function gave the triol 24 that was selectively oxidized to the hemiketal 25. The secondary hydroxyl in this intermediate was transformed to mesylate 26, which underwent a basic medium fragmentation to yield the nine-membered lactone 27 that was immediately hydrolyzed to carbonate 28. Selective epoxidation of the olefin 28 gave the epoxide 29, which furnished a six-membered lactone 30 after mild aqueous basic treatment. The triol lactone 30 was converted into the primary p-nitrobenzoate derivative 31, which was oxidized to the ketone 32. Sequential reduction of this compound and acetylation of the resulting alcohol 33 furnished the equatorial acetate derivative 34.

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Total synthesis of ryanodol: preparation of rings B, C and D.

Modifications of ring A of compound 34 led to the substituted cyclopentenol moiety of anhydroryanodol (5); compound 34 (fig. 8) was first converted to a mixture of enol ethers (35) that was oxidized by ozone to cyclopentanone 36. Transformation of ketone 36 to its enol acetate 37 was followed by a basic treatment that yielded the desired enone 38. Selective reduction of the C3 carbonyl group gave the endoallylic alcohol 39. Removal of the carbonate and acetate groups under mild basic aqueous conditions gave a mixture (3:1) of anhydroryanodol (5, C15-O-C11 bridging) and its regioisomer (40, C15-O-C3 bridging) previously named epianhydroryanodol (Belanger et al., 1979; Deslongchamps et al., 1990). Selective epoxidation of either isomer, 5 or 40, or of a mixture of both compounds, yielded the anticipated -epoxide in the form of its C3-lactone 41, the most stable isomer in that medium. Basic aqueous treatment of 41 led to epoxide 42 of anhydroryanodol, that on treatment with lithium in ammonia gave (+)-ryanodol (2). This last sequence implies an attack by a dianion, or a radical anion (see 43, fig. 9) previously formed at C15 by the reduction of the lactone carbonyl onto the carbon atom at position 1, which opened the oxiran moiety to give the C2-hydroxyl group with the desired stereochemistry.

View larger version (30K): [in this window] [in a new window]   Fig. 8.   Total synthesis of ryanodol: preparation of ring A.

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Total synthesis of ryanodol: preparation of rings A and E.

Attempts to obtain native ryanodine (1) from ryanodol (2), attempts that require esterification of the latter compound at position 3 with 2-pyrrole carboxylic acid, have been unsuccessful. Models show that selective access to the hindered -hydroxyl group at C3 is very difficult and may require severe esterification conditions that would not be tolerated by the fragile ryanodol structure (Belanger et al., 1979; Deslongchamps et al., 1990).

It has been shown recently (Ruest and Deslongchamps, 1993) that esterification (Neises and Steglich, 1978) of (+)-ryanodol by pyrrole-2-carboxylic acid yields 10-O-pyrrolecarbonylryanodol (44), a compound that retains significant binding to the ryanodine receptor and is proving useful as the starting point for a series of compounds that will be used to test the regions of the ryanodine binding site that interact with the pyrrole-2-carboxylic acid moiety (Welch et al., 1996a) (see fig. 17, compounds 2A through 2J, and section IV.I.).

The total synthesis of 3-epiryanodine (50) (fig. 10) starting from either anhydroryanodine (4) or anhydroryanodol (5) has been achieved recently (Ruest and Deslongchamps, 1993). Treatment of the starting material with lithium in ammonia gave 2,3-dideoxy-²-ryanodol (45), which yielded 1,2-epoxy-3-epianhydroryanodol (C15-O-C11 bridging) (48) upon oxidation with an excess of peracid. The unisolated 2,3-epoxyryanodol (46) was unstable in this medium and suffered the well known cleavage to the anhydro series, forming unisolated 3-epianhydroryanodol (47) that was readily epoxidized. Treatment of this epoxide with lithium in ammonia resulted in 3-epiryanodol (49) in a good yield. Subsequent acylation (Neises and Steglich, 1978) of this compound with pyrrole-2-carboxylic acid gave 3-epiryanodine (50) with a satisfactory yield. The 3-epi isomer of naturally occurring ryanodine binds to the ryanodine receptor and provides the starting point for forming derivatives at the C3 position (fig. 17, compounds 49A through 49K) (see section IV.I.2.).