Ryanodine Receptor

The toxic plant alkaloid ryanodine was first purified and characterized from the powdered stem wood and roots of Ryania speciosa Vahl by Rogers and coworkers in 1948. The alkaloid produces profound rigidity in skeletal muscle.^{[17] [19] [20]} Isolation of 9,21-dehydroryanodine from Ryania facilitated the synthesis of radiolabeled ryanodine ([³ H]ryanodine) and permitted direct studies aimed at understanding the mechanism of this muscle poison. The availability of [³ H]ryanodine led to identification of the RYR receptor, which is synonymous with the junctional foot protein and possesses Ca²⁺ -release channel activity. RYRs bind [³ H]ryanodine with selectivity, and the high-affinity binding interaction is sensitive to the conformational state of the channel (see Fig. 29-2 ). High-affinity and low-affinity ryanodine binding sites appear to be located within a 76-kd tryptic fragment from the carboxyl terminus of RYR1 of rabbit skeletal muscle ( Fig. 29-3 ). MacLennan's group^[19] reports that the hydrophobic segments within residues 3985–4362 are thought to form the M₁ through M₄ transmembrane domains, enabling RYR1 to span the SR membrane four times. The M₁ through M₄ domains of RYR1 have high sequence homology to the analogous domains of inositol triphosphate receptors, suggesting a possible role in forming the sarcoplasmic/endoplasmic reticulum Ca²⁺ channel pore.

Skeletal (RYR1), cardiac (RYR2), and brain (RYR3) isoforms are encoded by three genes located on human chromosomes 19q13.1, 1q42.1-q43, and 15q14-q15, respectively. Based on sedimentation analysis and channel reconstitution studies in bilayer lipid membranes, each functional RYR consists of four identical subunits. Multiple isoforms of RYR are coexpressed in many cell types. Coexpression of different RYR isoforms in HEK293 cells has revealed that RYR2 is capable of physically interacting with RYR3 and RYR1, but that RYR1 does not interact with RYR3.^[20] Whether formation of mixed oligomeric RYRs extends beyond heterologous expression models such as HEK293 cells to mammalian tissue in situ has not been determined.

Complementary DNA (cDNA) sequence analysis reveals that each RYR protomer is composed of 5032 to 5037, 4968 to 4976, and 4872 amino residues with calculated molecular masses of 564 to 565, 565, and 552 kd for the RYR1, RYR2, and RYR3 isoforms, respectively. Typically, a sequence homology of 66% to 70% is observed between any two conspecific isoforms. RYR receptors are also highly conserved in the same tissue among different species (>95% sequence homology of RYR1 found in mammalian skeletal muscle of human, pig, and rabbit). The tetrameric organization of the RYR1 from skeletal muscle has been corroborated by electron microscopy of cryosections of purified RYR1 protein. Three-dimensional reconstruction of RYR1 cryosections has revealed the quatrefoil appearance of each homo-oligomer, with four radial channels on the cytoplasmic face that may converge into a single, common transmembrane pore on the luminal face. Evidence of direct coupling of α_1S -DHPR and RYR1 has been demonstrated by expressing chimeric α_1S/C -DHPR cDNAs in dysgenic myotubes that lack constitutive expression of α_1S -DHPR. Such studies have provided compelling evidence that the cytoplasmic region between repeats II and III (i.e., cytosolic II-III loop) contains a stretch of 46 amino acids (L720 to Q765) that is essential for engaging bidirectional signaling with RYR1, even in the presence of drastic alterations of sequence surrounding residues L720 to L765.^{[19] [21] [22]}

In addition to α_1S DHPR, RYR1 has been shown to interact with and be modulated by several intracellular accessory proteins. Calmodulin interacts directly with RYR1 of skeletal muscle, with a stoichiometry of two to three calmodulin molecules per subunit. The calmodulin sites with greatest affinity have been localized to the foot region of RYR1 (see Fig 29-2 ). Through a mechanism independent of kinase activity, calmodulin enhances channel activity at low cytoplasmic Ca²⁺ , whereas it inhibits channel activity at optimal Ca²⁺ (10 to 100 nM). Calsequestrin, the major Ca²⁺ binding protein within the SR lumen, links indirectly to a luminal domain of RYR1. The conformational change in RYR1 also conveys information to the SR lumen through calsequestrin and may be essential in regulating the Ca²⁺ release process. Functional interactions between RYR1 and calsequestrin may play an important role in regulating excitability of the Ca²⁺ channel in response to different filling states of SR. Triadin, a 95-kd, highly basic glycoprotein, was initially suggested to couple RYR1 and the α₁ -subunit of DHPR; however, amino acid analysis of triadin indicates only a single pass through the SR membrane, which contradicts its hypothesized role in coupling. The extremely high density of basic residues in the luminal terminus of triadin may be critical in interacting with the acidic moiety of RYR1. The linkage between RYR1 and triadin is thought to provide an anchorage site for calsequestrin within the SR lumen.

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FKBP12, the major T-cell immunophilin, tightly associates with RYR1 in skeletal muscle with a stoichiometry of four molecules per channel oligomer. The site on RYR1 that recognizes FKBP12 is distinct from that which binds calmodulin (see Fig. 29-2 ). Binding of FKBP12 appears to stabilize the closed conformation of the Ca²⁺ channel complex and its full conductance transitions. The immunosuppressant FK506 promotes dissociation of FKBP12 from RYR1 by competing with a common binding site essential for protein-protein interaction of the heterocomplex. The resulting FKBP12-deficient channel conducts current with multiple subconductance states. In the presence of channel activators such as Ca²⁺ and caffeine, activity of the FKBP12-deficient channel is further enhanced by increasing the mean open time and open probability. Association of FKBP12 to RYR1 may promote cooperativity among subunits. Dissociation of FKBP12 with FK506 increases maximal binding capacity of [³ H]ryanodine with lowered binding affinity, suggesting loss of negative allosteric interaction between high-affinity and low-affinity [³ H]ryanodine binding sites. The association of FKBP12 with the RYR1 complex may be involved in promoting cooperativity between neighboring channels. The "coupled gating" behavior of multiple channels has been reported in measurements with multiple channels reconstituted in membrane lipid bilayer by use of recombinant RYR1 (coexpressed with FKBP12) and native SR. Introduction of FK506 dissociates FKBP12 from the recombinant RYR1 complex and eliminates the coupled gating behavior of multiple channels. The cooperativity between neighboring RYR1 channels may contribute significantly to the robust release of Ca²⁺ from SR during EC coupling.

Homer proteins form an adapter system that regulates coupling of group 1 metabotropic glutamate receptors with intracellular inositol triphosphate receptors and is modified by neuronal activity. Homer proteins physically associate with RYR1 and regulate gating responses to Ca²⁺ , depolarization, and caffeine. The EVH1 domain appears to mediate the actions of Homer on RYR1 function.^[23] Dyspedic myotubes expressing RYR1 with a point mutation of a putative Homer-binding domain exhibit significantly reduced amplitude in their responses to potassium ion (K⁺ ) depolarization compared with cells expressing wild-type protein. Homer therefore appears to be a direct modulator of increased Ca²⁺ release and EC coupling in skeletal myotubes.

Two novel proteins (60 kd and 90 kd), whose function is unknown, are also associated with the RYR1 complex. One possesses kinase activity, and the other is the substrate of this kinase. Another 150/160-kd protein also is associated with RYR1. Phosphorylation of the 150/160-kd protein by casein II kinase inhibits RYR1 channel activity.