FKBP binding characteristics of cardiac microsomes from diverse vertebrates

FK506 binding protein (FKBP) is a cytosolic receptor for the immunosuppressive drug FK-506. The common isoform, FKBP12, was found to be associated with the calcium release channel (ryanodine receptor 1) of different species of vertebrate skeletal muscle, whereas 12.6, a novel FKBP isoform was found to be associated with canine cardiac ryanodine receptor (ryanodine receptor 2). Until recently, canine cardiac sarcoplasmic reticulum was considered to be the prototype for studying heart RyR2 and its interactions with FKBP. In this study, cardiac microsomes were isolated from diverse vertebrates: human, rabbit, rat, mice, dog, chicken, frog, and fish and were analyzed for their ability to bind or exchange with FKBP isoforms 12 and 12.6. Our studies indicate that RyR2 from seven out of the eight animals contain both FKBP12 and 12.6. Dog is the exception. It can now be concluded that the association of FKBP isoforms with RyR2 is widely conserved in the hearts of different species of vertebrates.     

Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line

Of the three known ryanodine receptor (RyR) isoforms expressed in muscle, RyR1 and RyR2 have well-defined roles in contraction. However, studies on mammalian RyR3 have been difficult because of low expression levels relative to RyR1 or RyR2. Using the herpes simplex virus 1 (HSV-1) helper-free amplicon system, we expressed either RyR1 or RyR3 in 1B5 RyR-deficient myotubes. Western blot analysis revealed that RyR1- or RyR3-transduced cells expressed the appropriate RyR isoform of the correct molecular mass. Although RyR1 channels exhibited the expected unitary conductance for Cs(+) in bilayer lipid membranes, 74 of 88 RyR3 channels exhibited pronounced subconductance behavior. Western blot analysis with an FKBP12/12.6-selective antibody reveals that differences in gating behavior exhibited by RyR1 and RyR3 may be, in part, the result of lower affinity of RyR3 for FKBP12. In calcium imaging studies, RyR1 restored skeletal-type excitation-contraction coupling, whereas RyR3 did not. Although RyR3-expressing myotubes were more sensitive to caffeine than those expressing RyR1, they were much less sensitive to 4-chloro-m-cresol (CMC). In RyR1-expressing cells, regenerative calcium oscillations were observed in response to caffeine and CMC but were never seen in RyR3-expressing 1B5 cells. In [(3)H]ryanodine binding studies, only RyR1 exhibited sensitivity to CMC, but both RyR isoforms responded to caffeine. These functional differences between RyR1 and RyR3 expressed in a mammalian muscle context may reflect differences in association with accessory proteins, especially FKBP12, as well as structural differences in modulator binding sites.     

Calumenin, a multiple EF-hands Ca2+-binding protein, interacts with ryanodine receptor-1 in rabbit skeletal sarcoplasmic reticulum

Calumenin is a multiple EF-hand Ca2+-binding protein located in endo/sarcoplasmic reticulum of mammalian tissues. In the present study, we cloned two rabbit calumenin isoforms (rabbit calumenin-1 and -2, GenBank Accession Nos. SY225335 and AY225336, respectively) by RT-PCR. Both isoforms contain a 19 aa N-terminal signal sequence, 6 EF-hand domains, and a C-terminal ER/SR retrieval signal, HDEF. Both calumenin isoforms exist in rabbit cardiac and skeletal muscles, but calumenin-2 is the main isoform in skeletal muscle. Presence of calumenin in rabbit sarcoplasmic reticulum (SR) was identified by Western blot analysis. GST-pull down and co-immunoprecipitation experiments showed that ryanodine receptor 1 (RyR1) interacted with calumenin-2 in millimolar Ca2+ concentration range. Experiments of gradual EF-hand deletions suggest that the second EF-hand domain is essential for calumenin binding to RyR1. Adenovirus-mediated overexpression of calumenin-2 in C2C12 myotubes led to increased caffeine-induced Ca2+ release, but decreased depolarization-induced Ca2+ release. Taken together, we propose that calumenin-2 in the SR lumen can directly regulate the RyR1 activity in Ca2+-dependent manner.     

RYR1 and RYR3 have different roles in the assembly of calcium release units of skeletal muscle

Calcium release units (CRUs) are junctions between the sarcoplasmic reticulum (SR) and exterior membranes that mediates excitation contraction (e-c) coupling in muscle cells. In skeletal muscle CRUs contain two isoforms of the sarcoplasmic reticulum Ca(2+)release channel: ryanodine receptors type 1 and type 3 (RyR1 and RyR3). 1B5s are a mouse skeletal muscle cell line that carries a null mutation for RyR1 and does not express either RyR1 or RyR3. These cells develop dyspedic SR/exterior membrane junctions (i.e., dyspedic calcium release units, dCRUs) that contain dihydropyridine receptors (DHPRs) and triadin, two essential components of CRUs, but no RyRs (or feet). Lack of RyRs in turn affects the disposition of DHPRs, which is normally dictated by a linkage to RyR subunits. In the dCRUs of 1B5 cells, DHPRs are neither grouped into tetrads nor aligned in two orthogonal directions. We have explored the structural role of RyR3 in the assembly of CRUs in 1B5 cells independently expressing either RyR1 or RyR3. Either isoform colocalizes with DHPRs and triadin at the cell periphery. Electron microscopy shows that expression of either isoform results in CRUs containing arrays of feet, indicating the ability of both isoforms to be targeted to dCRUs and to assemble in ordered arrays in the absence of the other. However, a significant difference between RyR1- and RyR3-rescued junctions is revealed by freeze fracture. While cells transfected with RyR1 show restoration of DHPR tetrads and DHPR orthogonal alignment indicative of a link to RyRs, those transfected with RyR3 do not. This indicates that RyR3 fails to link to DHPRs in a specific manner. This morphological evidence supports the hypothesis that activation of RyR3 in skeletal muscle cells must be indirect and provides the basis for failure of e-c coupling in muscle cells containing RyR3 but lacking RyR1 (see the accompanying report, ).     

Type 3 and type 1 ryanodine receptors are localized in triads of the same mammalian skeletal muscle fibers.

The type 3 ryanodine receptor (RyR3) is a ubiquitous calcium release channel that has recently been found in mammalian skeletal muscles. However, in contrast to the skeletal muscle isoform (RyR1), neither the subcellular distribution nor the physiological role of RyR3 are known. Here, we used isoform-specific antibodies to localize RyR3 in muscles of normal and RyR knockout mice. In normal hind limb and diaphragm muscles of young mice, RyR3 was expressed in all fibers where it was codistributed with RyR1 and with the skeletal muscle dihydropyridine receptor. This distribution pattern indicates that RyR3 is localized in the triadic junctions between the transverse tubules and the sarcoplasmic reticulum. During development, RyR3 expression declined rapidly in some fibers whereas other fibers maintained expression of RyR3 into adulthood. Comparing the distribution of RyR3-containing fibers with that of known fiber types did not show a direct correlation. Targeted deletion of the RyR1 or RyR3 gene resulted in the expected loss of the targeted isoform, but had no adverse effects on the expression and localization of the respective other RyR isoform. The localization of RyR3 in skeletal muscle triads, together with RyR1, is consistent with an accessory function of RyR3 in skeletal muscle excitation-contraction coupling.     

Comparison of properties of Ca2+ release channels between rabbit and frog skeletal muscles

Biochemical investigation of Ca2+ release channel proteins has been carried out mainly with rabbit skeletal muscles, while frog skeletal muscles have been preferentially used for physiological investigation of Ca2+ release. In this review, we compared the properties of ryanodine receptors (RyR), Ca2+ release channel protein, in skeletal muscles between rabbit and frog. While the Ryr1 isoform is the main RyR of rabbit skeletal muscles, two isoforms, - and -RyR which are homologous to Ryr1 and Ryr3 isoforms in mammals, respectively, coexist as a homotetramer in a similar amount in frog skeletal muscles. The two isoforms in an isotonic medium show very similar property in [3H]ryanodine binding activity which is parallel to Ca2+-induced Ca2+ release (CICR) activity, and make independent contributions to the activities of the sarcoplasmic reticulum. CICR and [3H]ryanodine binding activities of rabbit and frog are qualitatively similar in stimulation by Ca2+, adenine nucleotide and caffeine, however, they showed the following quantitative differences. First, rabbit RyR showed higher Ca2+ affinity than the frog. Second, rabbit RyR showed higher activity in the presence of Ca2+ alone with less stimulation by adenine nucleotide than the frog. Third, rabbit RyR displayed less enhancement of [3H]ryanodine binding by caffeine in spite of having a similar magnitude of Ca2+ sensitization than the frog, which may explain the occasional difficulty by researchers to demonstrate caffeine contracture with mammalian skeletal muscles. Finally, but not least, rabbit RyR still showed marked inhibition of [3H]ryanodine binding in the presence of high Ca2+ concentrations in the 1 M NaCl medium, while frog RyR showed disinhibition. Other matters relevant to Ca2+ release were also discussed.     

Ryanodine receptors in muscarinic receptor-mediated bronchoconstriction

Ryanodine receptors (RyRs), intracellular calcium release channels essential for skeletal and cardiac muscle contraction, are also expressed in various types of smooth muscle cells. In particular, recent studies have suggested that in airway smooth muscle cells (ASMCs) provoked by spasmogens, stored calcium release by the cardiac isoform of RyR (RyR2) contributes to the calcium response that leads to airway constriction (bronchoconstriction). Here we report that mouse ASMCs also express the skeletal muscle and brain isoforms of RyRs (RyR1 and RyR3, respectively). In these cells, RyR1 is localized to the periphery near the cell membrane, whereas RyR3 is more centrally localized. Moreover, RyR1 and/or RyR3 in mouse airway smooth muscle also appear to mediate bronchoconstriction caused by the muscarinic receptor agonist carbachol. Inhibiting all RyR isoforms with > or = 200 microM ryanodine attenuated the graded carbachol-induced contractile responses of mouse bronchial rings and calcium responses of ASMCs throughout the range of carbachol used (50 nM to > or = 3 microM). In contrast, inhibiting only RyR1 and RyR3 with 25 microM dantrolene attenuated these responses caused by high (>500 nM) but not by low concentrations of carbachol. These data suggest that, as the stimulation of muscarinic receptor in the airway smooth muscle increases, RyR1 and/or RyR3 also mediate the calcium response and thus bronchoconstriction. Our findings provide new insights into the complex calcium signaling in ASMCs and suggest that RyRs are potential therapeutic targets in bronchospastic disorders such as asthma.     

Expression levels of RyR1 and RyR3 control resting free Ca2+ in skeletal muscle

To better understand the role of the transient expression of ryanodine receptor (RyR) type 3 (RyR3) on Ca²⁺ homeostasis during the development of skeletal muscle, we have analyzed the effect of expression levels of RyR3 and RyR1 on the overall physiology of cultured myotubes and muscle fibers. Dyspedic myotubes were infected with RyR1 or RyR3 containing virions at 0.2, 0.4, 1.0, and 4.0 moieties of infection (MOI), and analysis of their pattern of expression, caffeine sensitivity, and resting free Ca²⁺ concentration ([Ca²⁺]_r) was performed. Although increased MOI resulted in increased expression of each receptor isoform, it did not significantly affect the immunopattern of RyRs or the expression levels of calsequestrin, triadin, or FKBP-12. Interestingly, myotubes expressing RyR3 always had significantly higher [Ca²⁺]_r and lower caffeine EC₅₀ than did cells expressing RyR1. Although some of the increased sensitivity of RyR3 to caffeine could be attributed to the higher [Ca²⁺]_r in RyR3-expressing cells, studies of [³H]ryanodine binding demonstrated intrinsic differences in caffeine sensitivity between RyR1 and RyR3. Tibialis anterior (TA) muscle fibers at different stages of postnatal development exhibited a transient increase in [Ca²⁺]_r coordinately with their level of RyR3 expression. Similarly, adult soleus fibers, which also express RyR3, had higher [Ca²⁺]_r than did adult TA fibers, which exclusively express RyR1. These data show that in skeletal muscle, RyR3 increases [Ca²⁺]_r more than RyR1 does at any expression level. These data suggest that the coexpression of RyR1 and RyR3 at different levels may constitute a novel mechanism by which to regulate [Ca²⁺]_r in skeletal muscle. ryanodine receptor; calcium release; ryanodine binding; muscle fibers     

Functional properties of the ryanodine receptor type 3 (RyR3) Ca2+ release channel.

Single-channel analysis of sarcoplasmic reticulum vesicles prepared from diaphragm muscle, which contains both RyR1 and RyR3 isoforms, revealed the presence of two functionally distinct ryanodine receptor calcium release channels. In addition to channels with properties typical of RyR1 channels, a second population of ryanodine-sensitive channels with properties distinct from those of RyR1 channels was observed. The novel channels displayed close-to-zero open-probability at nanomolar Ca2+ concentrations in the presence of 1 mM ATP, but were shifted to the open conformation by increasing Ca2+ to micromolar levels and were not inhibited at higher Ca2+ concentrations. These novel channels were sensitive to the stimulatory effects of cyclic adenosine 5'-diphosphoribose (cADPR). Detection of this second population of RyR channels in lipid bilayers was always associated with the presence of the RyR3 isoform in muscle preparations used for single-channel measurements and was abrogated by the knockout of the RyR3 gene in mice. Based on the above, we associated the novel population of channels with the RyR3 isoform of Ca2+ release channels. The functional properties of the RyR3 channels are in agreement with a potential qualitative contribution of this channel to Ca2+ release in skeletal muscle and in other tissues.     

Cloning and characterization of fiber type-specific ryanodine receptor isoforms in skeletal muscles of fish

We have cloned agroup of cDNAs that encodes the skeletal ryanodine receptor isoform(RyR1) of fish from a blue marlin extraocular muscle library. The cDNAsencode a protein of 5,081 amino acids with a calculated molecular massof 576,302 Da. The deduced amino acid sequence shows strong sequenceidentity to previously characterized RyR1 isoforms. An RNA probederived from a clone of the full-length marlin RyR1 isoform hybridizesto RNA preparations from extraocular muscle and slow-twitch skeletalmuscle but not to RNA preparations from fast-twitch skeletal or cardiacmuscle. We have also isolated a partial RyR clone from marlin andtoadfish fast-twitch muscles that shares 80% sequence identity withthe corresponding region of the full-length RyR1 isoform, and a RNAprobe derived from this clone hybridizes to RNA preparations fromfast-twitch muscle but not to slow-twitch muscle preparations. Westernblot analysis of slow-twitch muscles in fish indicates the presence ofonly a single high-molecular-mass RyR proteincorresponding to RyR1. [³H]ryanodine bindingassays revealed the fish slow-twitch muscle RyR1 had a greatersensitivity for Ca²⁺ than thefast-twitch muscle RyR1. The results indicate that, in fish muscle,fiber type-specific RyR1 isoforms are expressed and the two proteinsare physiologically distinct.

     

Mutational analysis of putative calcium binding motifs within the skeletal ryanodine receptor isoform, RyR1

The functional relevance of putative Ca(2+) binding motifs previously identified with Ca(2+) overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca(2+)-imaging techniques, [(3)H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [(3)H]ryanodine binding measurements, both Ca(2+) activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [(3)H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca(2+) concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3-5) did not affect functional responses in 1B5 myotubes except that the EC(50) for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [(3)H]ryanodine binding experiments, the Ca(2+)-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.     

Calcium release from sarcoplasmic reticulum is facilitated in human myotubes derived from carriers of the ryanodine receptor type 1 mutations Ile2182Phe and Gly2375Ala

Malignant hyperthermia (MH) is caused by increased calcium release from sarcoplasmic reticulum, triggered by volatile anesthetics or depolarizing muscle relaxants. Numerous mutations associated with MH have been detected in the skeletal muscle type ryanodine receptor gene (RyR1), but so far facilitated calcium release has only been demonstrated for a few of them. This is a prerequisite for confirming the causative role of an RyR1 mutation for MH. Calcium release from sarcoplasmic reticulum induced by 4-chloro-m-cresol (4CmC), caffeine, and halothane was determined in human myotubes by calcium imaging. The RyR1 Ile2182Phe mutation and the RyR1 Gly2375Ala mutation have been identified in individuals susceptible to MH. In myotubes of individuals carrying the RyR1 Ile2182Phe or the RyR1 Gly2375Ala mutation, the EC(50) for caffeine and halothane was reduced; in the Ile2182Phe myotubes, the EC(50) for 4CmC was also reduced, all consistent with facilitated calcium release from the sarcoplasmic reticulum. From these data we conclude that both mutations are pathogenic for MH.     

Type 1 and type 3 ryanodine receptors generate different Ca(2+) release event activity in both intact and permeabilized myotubes.

In this investigation we use a "dyspedic" myogenic cell line, which does not express any ryanodine receptor (RyR) isoform, to examine the local Ca(2+) release behavior of RyR3 and RyR1 in a homologous cellular system. Expression of RyR3 restored caffeine-sensitive, global Ca(2+) release and causes the appearance of relatively frequent, spontaneous, spatially localized elevations of [Ca(2+)], as well as occasional spontaneous, propagating Ca(2+) release, in both intact and saponin-permeabilized myotubes. Intact myotubes expressing RyR3 did not, however, respond to K(+) depolarization. Expression of RyR1 restored depolarization-induced global Ca(2+) release in intact myotubes and caffeine-induced global release in both intact and permeabilized myotubes. Both intact and permeabilized RyR1-expressing myotubes exhibited relatively infrequent spontaneous Ca(2+) release events. In intact myotubes, the frequency of occurrence and properties of these RyR1-induced events were not altered by partial K(+) depolarization or by application of nifedipine, suggesting that these RyR1 events are independent of the voltage sensor. The events seen in RyR1-expressing myotubes were spatially more extensive than those seen in RyR3-expressing myotubes; however, when analysis was limited to spatially restricted "Ca(2+) spark"-like events, events in RyR3-expressing myotubes were larger in amplitude and duration compared with those in RyR1. Thus, in this skeletal muscle context, differences exist in the spatiotemporal properties and frequency of occurrence of spontaneous release events generated by RyR1 and RyR3. These differences underscore functional differences between the Ca(2+) release behavior of RyR1 and RyR3 in this homologous expression system.     

Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy

Using cryo-electron microscopy and single particle image processing techniques, we present the first three-dimensional reconstructions of isoform 3 of the ryanodine receptor/calcium release channel (RyR3). Reconstructions were carried out on images obtained from a purified, detergent-solubilized receptor for two different buffer conditions, which were expected to favor open and closed functional states of the channel. As for the heart (RyR2) and skeletal muscle (RyR1) receptor isoforms, RyR3 is a homotetrameric complex comprising two main components, a multidomain cytoplasmic assembly and a smaller ( approximately 20% of the total mass) transmembrane region. Although the isoforms show structural similarities, consistent with the approximately 70% overall sequence identity of the isoforms, detailed comparisons of RyR3 with RyR1 showed one region of highly significant difference between them. This difference indicated additional mass present in RyR1, and it likely corresponds to a region of the RyR1 sequence (residues 1303-1406, known as diversity region 2) that is absent from RyR3. The reconstructions of RyR3 determined under "open" and "closed" conditions were similar to each other in overall architecture. A difference map computed between the two reconstructions reveals subtle changes in conformation at several widely dispersed locations in the receptor, the most prominent of which is a approximately 4 degrees rotation of the transmembrane region with respect to the cytoplasmic assembly.     

Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro

Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95- kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential- induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.     

Excitation-contraction coupling in airway smooth muscle

Excitation-contraction (EC) coupling in striated muscles is mediated by the cardiac or skeletal muscle isoform of voltage-dependent L-type Ca(2+) channel (Ca(v)1.2 and Ca(v)1.1, respectively) that senses a depolarization of the cell membrane, and in response, activates its corresponding isoform of intracellular Ca(2+) release channel/ryanodine receptor (RyR) to release stored Ca(2+), thereby initiating muscle contraction. Specifically, in cardiac muscle following cell membrane depolarization, Ca(v)1.2 activates cardiac RyR (RyR2) through an influx of extracellular Ca(2+). In contrast, in skeletal muscle, Ca(v)1.1 activates skeletal muscle RyR (RyR1) through a direct physical coupling that negates the need for extracellular Ca(2+). Since airway smooth muscle (ASM) expresses Ca(v)1.2 and all three RyR isoforms, we examined whether a cardiac muscle type of EC coupling also mediates contraction in this tissue. We found that the sustained contractions of rat ASM preparations induced by depolarization with KCl were indeed partially reversed ( approximately 40%) by 200 mum ryanodine, thus indicating a functional coupling of L-type channels and RyRs in ASM. However, KCl still caused transient ASM contractions and stored Ca(2+) release in cultured ASM cells without extracellular Ca(2+). Further analyses of rat ASM indicated that this tissue expresses as many as four L-type channel isoforms, including Ca(v)1.1. Moreover, Ca(v)1.1 and RyR1 in rat ASM cells have a similar distribution near the cell membrane in rat ASM cells and thus may be directly coupled as in skeletal muscle. Collectively, our data implicate that EC-coupling mechanisms in striated muscles may also broadly transduce diverse smooth muscle functions.     

兔膈肌骨骼肌型DHPRα1亚单位和RyRs的mRNA与蛋白表达测定

本文测定了兔膈肌钙释放单位中骨骼肌型ＤＨＰＲα１－亚单位和ＲｙＲｓ的ｍＲＮＡ与蛋白表达水平,探讨了兔膈肌钙释放单位的结构组成特征。采用ＲＴ－ＰＣＲ、原位杂交和免疫组织化学技术,分别测定兔膈肌骨骼肌型ＤＨＰＲα１－亚单位和ＲｙＲ１、ＲｙＲ２及ＲｙＲ３的ｍＲＮＡ与蛋白表达。结果显示,兔膈肌可见较高水平的骨骼肌型α１－亚单位和ＲｙＲ１ｍＲＮＡ与蛋白表达;较低水平的ＲｙＲ３ ｍＲＮＡ与蛋白表达。表明兔膈肌     

Ca2+ release induced by cyclic ADP ribose in mice lacking type 3 ryanodine receptor.

The action of cyclic-ADP-ribose was studied on calcium release from sarcoplasmic reticulum of skeletal muscles of neonatal and adult wild-type and RyR3-deficient mice. cADPR increased calcium efflux from microsomes, enhanced caffeine-induced calcium release, and, in 20% of the tests, triggered calcium release in single muscle fibers. These responses occurred only in the diaphragm of adult RyR3-deficient mice. cADPR action was abolished by ryanodine, ruthenium red, and 8-brome-cADPR. These results strongly favor a specific action of cADPR on RyR1. The responsiveness of RyR1 appears in adult muscles when RyR3 is lacking.     

Three-dimensional visualization of FKBP12.6 binding to an open conformation of cardiac ryanodine receptor

The cardiac isoform of the ryanodine receptor (RyR2) from dog binds predominantly a 12.6-kDa isoform of the FK506-binding protein (FKBP12.6), whereas RyR2 from other species binds both FKBP12.6 and the closely related isoform FKBP12. The role played by FKBP12.6 in modulating calcium release by RyR2 is unclear at present. We have used cryoelectron microscopy and three-dimensional (3D) reconstruction techniques to determine the binding position of FKBP12.6 on the surface of canine RyR2. Buffer conditions that should favor the "open" state of RyR2 were used. Quantitative comparison of 3D reconstructions of RyR2 in the presence and absence of FKBP12.6 reveals that FKBP12.6 binds along the sides of the square-shaped cytoplasmic region of the receptor, adjacent to domain 9, which forms part of the four clamp (corner-forming) structures. The location of the FKBP12.6 binding site on "open" RyR2 appears similar, but slightly displaced (by 1-2 nm) from that found previously for FKBP12 binding to the skeletal muscle ryanodine receptor that was in the buffer that favors the "closed" state. The conformation of RyR2 containing bound FKBP12.6 differs considerably from that depleted of FKBP12.6, particularly in the transmembrane region and in the clamp structures. The x-ray structure of FKBP12.6 was docked into the region of the 3D reconstruction that is attributable to bound FKBP12.6, to show the relative orientations of amino acid residues (Gln-31, Asn-32, Phe-59) that have been implicated as being critical in interactions with RyR2. A thorough understanding of the structural basis of RyR2-FKBP12.6 interaction should aid in understanding the roles that have been proposed for FKBP12.6 in heart failure and in certain forms of sudden cardiac death.