Ryanodine receptors (RyRs) are intracellular calcium release channels that participate in controlling cytosolic calcium levels. At variance with the probably ubiquitous inositol 1,4,5-trisphosphate-operated calcium channels (1,4,5-trisphosphate receptors), RyRs have been mainly regarded as the calcium release channels controlling skeletal and cardiac muscle contraction. Increasing evidence has recently suggested that RyRs may be more widely expressed, but this has never been extensively examined. Therefore, we cloned three cDNAs corresponding to murine RyR homologues to carry a comprehensive analysis of their expression in murine tissues. Here, we report that the three genes are expressed in almost all tissues analyzed, where tissue-specific patterns of expression were observed. In the uterus and vas deferens, expression of RyR3 was localized to the smooth muscle component of these organs. In the testis, expression of RyR1 and RyR3 was detected in germ cells. RyR mRNAs were also detected in in vitro-cultured cell lines. RyR1, RyR2, and RyR3 mRNA were detected in the cerebrum and in the cerebellum. In situ analysis revealed a cell type-specific pattern of expression in the different regions of the central nervous system. The differential expression of the three ryanodine receptor genes in the central nervous system was also confirmed using specific antibodies against the respective proteins. This widespread pattern of expression suggests that RyRs may participate in the regulation of intracellular calcium homeostasis in a range of cells wider than previously recognized.

Emerging evidence indicates that epileptic encephalopathies are genetically highly heterogeneous, underscoring the need for large cohorts of well-characterized individuals to further define the genetic landscape. Through a collaboration between two consortia (EuroEPINOMICS and Epi4K/EPGP), we analyzed exome-sequencing data of 356 trios with the "classical" epileptic encephalopathies, infantile spasms and Lennox Gastaut syndrome, including 264 trios previously analyzed by the Epi4K/EPGP consortium. In this expanded cohort, we find 429 de novo mutations, including de novo mutations in DNM1 in five individuals and de novo mutations in GABBR2, FASN, and RYR3 in two individuals each. Unlike previous studies, this cohort is sufficiently large to show a significant excess of de novo mutations in epileptic encephalopathy probands compared to the general population using a likelihood analysis (p = 8.2 × 10(-4)), supporting a prominent role for de novo mutations in epileptic encephalopathies. We bring statistical evidence that mutations in DNM1 cause epileptic encephalopathy, find suggestive evidence for a role of three additional genes, and show that at least 12% of analyzed individuals have an identifiable causal de novo mutation. Strikingly, 75% of mutations in these probands are predicted to disrupt a protein involved in regulating synaptic transmission, and there is a significant enrichment of de novo mutations in genes in this pathway in the entire cohort as well. These findings emphasize an important role for synaptic dysregulation in epileptic encephalopathies, above and beyond that caused by ion channel dysfunction.

Calcium (Ca(2+)) is a highly versatile second messenger that controls vascular smooth muscle cell (VSMC) contraction, proliferation, and migration. By means of Ca(2+) permeable channels, Ca(2+) pumps and channels conducting other ions such as potassium and chloride, VSMC keep intracellular Ca(2+) levels under tight control. In healthy quiescent contractile VSMC, two important components of the Ca(2+) signaling pathways that regulate VSMC contraction are the plasma membrane voltage-operated Ca(2+) channel of the high voltage-activated type (L-type) and the sarcoplasmic reticulum Ca(2+) release channel, Ryanodine Receptor (RyR). Injury to the vessel wall is accompanied by VSMC phenotype switch from a contractile quiescent to a proliferative motile phenotype (synthetic phenotype) and by alteration of many components of VSMC Ca(2+) signaling pathways. Specifically, this switch that culminates in a VSMC phenotype reminiscent of a non-excitable cell is characterized by loss of L-type channels expression and increased expression of the low voltage-activated (T-type) Ca(2+) channels and the canonical transient receptor potential (TRPC) channels. The expression levels of intracellular Ca(2+) release channels, pumps and Ca(2+)-activated proteins are also altered: the proliferative VSMC lose the RyR3 and the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase isoform 2a pump and reciprocally regulate isoforms of the ca(2+)/calmodulin-dependent protein kinase II. This review focuses on the changes in expression of Ca(2+) signaling proteins associated with VSMC proliferation both in vitro and in vivo. The physiological implications of the altered expression of these Ca(2+) signaling molecules, their contribution to VSMC dysfunction during vascular disease and their potential as targets for drug therapy will be discussed.

Ryanodine receptors are intracellular Ca2+ channels that have been known for more than a decade to have a role in releasing Ca2+ from the sarcoplasmic reticulum to regulate contraction in skeletal and cardiac muscle fibres. Vincenzo Sorrentino and Pompeo Volpe review some recent developments: the ryanodine receptor channels have now been found to be expressed in the central nervous system, and the cloning of a third ryanodine receptor gene (RYR3) has revealed that this new isoform is widely expressed in several tissues and cells. In consequence, the view of ryanodine receptors as Ca2+ channels of muscle cells is rapidly changing, and these channels seem set to take a more central position on the stage of intracellular Ca2+ signalling.

Alzheimer's disease (AD) is an incurable neurodegenerative disorder clinically characterized by progressive cognitive impairment. A prominent pathologic hallmark in the AD brain is the abnormal accumulation of the amyloid-β 1-42 peptide (Aβ), but the exact pathways mediating Aβ neurotoxicity remain enigmatic. Endoplasmic reticulum (ER) stress is induced during AD, and has been indirectly implicated as a mediator of Aβ neurotoxicity. We report here that Aβ activates the ER stress response factor X-box binding protein 1 (XBP1) in transgenic flies and in mammalian cultured neurons, yielding its active form, the transcription factor XBP1s. XBP1s shows neuroprotective activity in two different AD models, flies expressing Aβ and mammalian cultured neurons treated with Aβ oligomers. Trying to identify the mechanisms mediating XBP1s neuroprotection, we found that in PC12 cells treated with Aβ oligomers, XBP1s prevents the accumulation of free calcium (Ca(2+)) in the cytosol. This protective activity can be mediated by the downregulation of a specific isoform of the ryanodine Ca(2+) channel, RyR3. In support of this observation, a mutation in the only ryanodine receptor (RyR) in flies also suppresses Aβ neurotoxicity, indicating the conserved mechanisms between the two AD models. These results underscore the functional relevance of XBP1s in Aβ toxicity, and uncover the potential of XBP1 and RyR as targets for AD therapeutics.

Ryanodine receptors (RyRs) are large, high conductance Ca2+ channels that control the level of intracellular Ca2+ by releasing Ca2+ from an intracellular compartment, the sarco/endoplasmic reticulum. Mammalian tissues express 3 closely related ryanodine receptors (RyRs) known as skeletal muscle (RyR1), cardiac muscle (RyR2) and brain (RyR3). The RyRs are isolated as 30S protein complexes comprised of four 560 kDa RyR2 subunits and four 12.6 kDa FK506 binding protein (FKBP12.6) subunits. Multiple endogenous effector molecules and posttranslational modifications regulate the RyRs. This chapter reviews the regulation of the mammalian RyRs by endogenous effector molecules.

As an inhibitor of Ca(2+) release through ryanodine receptor (RYR) channels, the skeletal muscle relaxant dantrolene has proven to be both a valuable experimental probe of intracellular Ca(2+) signaling and a lifesaving treatment for the pharmacogenetic disorder malignant hyperthermia. However, the molecular basis and specificity of the actions of dantrolene on RYR channels have remained in question. Here we utilize [(3)H]ryanodine binding to further investigate the actions of dantrolene on the three mammalian RYR isoforms. The inhibition of the pig skeletal muscle RYR1 by dantrolene (10 microm) was associated with a 3-fold increase in the K(d) of [(3)H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles such that dantrolene effectively reversed the 3-fold decrease in the K(d) for [(3)H]ryanodine binding resulting from the malignant hyperthermia RYR1 Arg(615) ->> Cys mutation. Dantrolene inhibition of the RYR1 was dependent on the presence of the adenine nucleotide and calmodulin and reflected a selective decrease in the apparent affinity of RYR1 activation sites for Ca(2+) relative to Mg(2+). In contrast to the RYR1 isoform, the cardiac RYR2 isoform was unaffected by dantrolene, both in native cardiac SR vesicles and when heterologously expressed in HEK-293 cells. By comparison, the RYR3 isoform expressed in HEK-293 cells was significantly inhibited by dantrolene, and the extent of RYR3 inhibition was similar to that displayed by the RYR1 in native SR vesicles. Our results thus indicate that both the RYR1 and the RYR3, but not the RYR2, may be targets for dantrolene inhibition in vivo.

Calcium (Ca(2+)) release from intracellular stores controls numerous cellular processes, including cardiac and skeletal muscle contraction, synaptic transmission and metabolism. The ryanodine receptors (RyRs: RyR1, RyR2, RyR3) and inositol 1,4,5-trisphosphate receptors (IP3Rs: IP3R1, IP3R2, IP3R3) are the major Ca(2+) release channels (CRCs) on the endo/sarcoplasmic reticulum (ER/SR). RyRs and IP3Rs comprise macromolecular signaling complexes that include modulatory proteins which regulate channel activity in response to extracellular signals resulting in intracellular Ca(2+) release. Here we focus on the roles of CRCs in heart, skeletal muscle, brain, metabolism, and aging.

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.

Intracellular Ca2+-release channels on the sarcoplasmic reticulum of striated muscle [ryanodine receptors (RyRs)] and on the endoplasmic reticulum of almost all types of cells [inositol 1,4,5-trisphosphate receptors (IP3Rs)] comprise a unique family of molecules that are structurally and functionally distinct from all other known ion channels. These channels play crucial roles in Ca2+-mediated signaling that triggers excitation-contraction coupling, T-lymphocyte activation, fertilization, and many other cellular functions. Three forms of RyR have been identified: RyR1, expressed predominantly in skeletal muscle; RyR2, expressed predominantly in cardiac muscle; and RyR3, expressed in specialized muscles and nonmuscle tissues including the brain. RyR channels are tetramers composed of four subunits each with a molecular mass of approximately 560,000 Da. The tetrameric structures of RyR1 and RyR2 are stabilized by a channel-associated protein known as the FK506 binding protein (FKBP). FKBP is the cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin that inhibit the prolyl isomerase activity of FKBP and can dissociate FKBP from RyRs. Rapamycin and FK506 increase the sensitivity of RyRs to agonists such as caffeine and could be a cause of cardiac dysfunction associated with high-dose immunosuppressant therapy by promoting leakage of Ca2+ from the sarcoplasmic reticulum. The role of prolyl isomerase activity of FKBP in regulating RyR function remains uncertain, and several models have been proposed that could explain how the channel is modulated by its association with FKBP. Three forms of IP3Rs (types 1, 2 and 3) have been characterized by cDNA cloning. Most cells have at least one form of IP3R, and many express all three types. Like RyRs, the IP3R channels are tetramers composed of four subunits (approximately 300,000 Da each). IP3R1 function is regulated by at least two major cellular signaling pathways: the second messenger IP3 activates the channel, and phosphorylation by nonreceptor protein tyrosine kinases (e.g., Fyn) increase its open probability. During end-stage human heart failure, RyR2 mRNA and protein are downregulated, whereas IP3R1 is upregulated, suggesting that altered Ca2+-release channel levels may contribute to defects in Ca2+ homeostasis. Cells that are deficient in IP3R1 exhibit defective T cell-receptor signaling and thus cannot be activated by T cell-receptor stimulation. IP3R1-deficient cells are also resistant to induced apoptosis. Thus RyRs and IP3Rs play critical roles in fundamental and diverse signaling phenomena that include excitation-contraction coupling, T-cell activation, and programmed cell death.

The skeletal isoform of Ca2+ release channel, RyR1, plays a central role in activation of skeletal muscle contraction. Another isoform, RyR3, has been observed recently in some mammalian skeletal muscles, but whether it participates in regulating skeletal muscle contraction is not known. The expression of RyR3 in skeletal muscles was studied in mice from late fetal stages to adult life. RyR3 was found to be expressed widely in murine skeletal muscles during the post-natal phase of muscle development, but was not detectable in muscles of adult mice, with the exception of the diaphragm and soleus muscles. RyR3 knockout mice were generated, and it was shown that skeletal muscle contraction in these mice was impaired during the first weeks after birth. In skeletal muscles isolated from newborn RyR3(-/- )mice, but not in those from adult mice, the twitch elicited by electrical stimulation and the contracture induced by caffeine were strongly depressed. These results provide the first evidence that RyR3 has a physiological role in excitation-contraction coupling of neonatal skeletal muscles. The disproportion between the low amount of RyR3 and the large impact of the RyR3 knockout suggests that this isoform contributes to the amplification of Ca2+ released by the existing population of ryanodine receptors (RyR1).

We have evaluated and provided evidence that the ryanodine receptor 3 gene (RYR3), which encodes a large protein that forms a calcium channel, is important for the growth, morphology, and migration of breast cancer cells. A putative binding site for microRNA-367 (miR-367) exists in the 3'UTR of RYR3, and a genetic variant, rs1044129 A→G, is present in this binding region. We confirmed that miR-367 regulates the expression of a reporter gene driven by the RYR3 3'UTR and that the regulation was affected by the RYR3 genotype. A thermodynamic model based on base pairing and the secondary structure of the RYR3 mRNA and miR-367 miRNA showed that miR-367 had a higher binding affinity for the A genotype than for the G genotype. The rs1044129 SNP was genotyped in 1,532 breast cancer cases and 1,600 healthy Chinese women. The results showed that compared with the AA genotype, G was a risk genotype for breast cancer development and was also associated with breast cancer calcification and poor survival. Thus, rs1044129 is a unique SNP that resides in a miRNA-gene regulatory loop that affects breast cancer risk, calcification, and survival.

Recent findings on the ryanodine receptor of vertebrates, a Ca-release channel protein for the caffeine- and ryanodine-sensitive Ca pools, are reviewed in this article. Three distinct genes, i.e., ryr1, ryr2, and ryr3, express different isoforms in specific locations: Ryr1 in skeletal muscle and Purkinje cells of cerebellum; Ryr2 in cardiac muscle and brain, especially cerebellum; Ryr3 in skeletal muscle of nonmammalian vertebrates, the corpus striatum, and limbic cortex of brain, smooth muscles, and the other cells in vertebrates. While only one isoform (Ryr1) is expressed in mammalian skeletal muscles, two isoforms (alpha- and beta-isoforms expressed by ryr1 and ryr3, respectively) are found in nonmammalian vertebrate skeletal muscles. Although the coexistence of two isoforms may merely be related to differentiation and specialization, the biological significance remains to be clarified. Ryanodine receptors in vertebrate skeletal muscles are believed to mediate two different modes of Ca release: Ca(2+)-induced Ca release and action potential-induced Ca release. All results obtained so far with any isoform of ryanodine receptor are related to Ca(2+)-induced Ca release and show very similar characteristics. Ca(2+)-induced Ca release, however, cannot be the underlying mechanism of Ca release on skeletal muscle activation. Susceptibility of the ryanodine receptor's ryanodine-binding activity to modification by physical factors, such as osmolality of the medium, might be related to action potential-induced Ca release. A hypothesis of molecular interaction in view of the plunger model of action potential-induced Ca release is discussed, suggesting that the model could be compatible with Ryr1 and Ryr3, but incompatible with Ryr2. The functional relevance of ryanodine receptor isoforms, especially Ryr3, in brain also remains to be clarified. Among ryr1 gene-related diseases, malignant hyperthermia was the first to be identified; however, there is still the possibility of involvement of the other genes. Central core disease has been added to the list recently. A molecular approach for the diagnosis and treatment of diseases is now in progress.

Activation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs) and ryanodine receptors (RyRs) can lead to the release of Ca(2+) from intracellular stores and propagating Ca(2+) waves. Previous studies of these proteins in neurons have focused on their distribution in adult tissue, whereas, recent functional studies have examined neural tissue extracted from prenatal and young postnatal animals. In this study we examined the distribution of InsP(3)R isotypes 1-3 and RyR isotypes 1-3 in rat hippocampus during postnatal maturation, with a focus on InsP(3)R1 because it is most prominent in the hippocampus. InsP(3)R1 was observed in pyramidal cells and granule cells, InsP(3)R2 immunoreactivity was observed in perivascular astrocytes and endothelial cells, and InsP(3)R3 immunoreactivity was detected in axon terminals located in stratum pyramidale of CA1 and microvessels in stratum radiatum. RyR1 immunolabeling was enriched in CA1, RyR2 was most intense in CA3 and the dentate gyrus, and RyR3 immunolabeling was detected in all subfields of the hippocampus, but was most intense in stratum lacunosum-moleculare. During maturation from 2 to 10 weeks of age there was a shift in InsP(3)R1 immunoreactivity from a high density in the proximal apical dendrites to a uniform distribution along the dendrites. Independent of age, InsP(3)R1 immunoreactivity was observed to form clusters within the primary apical dendrite and at dendritic bifurcations of pyramidal neurons. As CA1 pyramidal neurons matured, InsP(3)R1 was often co-localized with the Ca(2+) binding protein calbindin D-28k. In contrast, InsP(3)R1 immunolabel was never co-localized with calbindin D-28k immunopositive interneurons located outside of stratum pyramidale or with parvalbumin, typically found in hippocampal basket cells, suggesting that InsP(3)R1s do not play a role in internal Ca(2+) release in these interneurons. These findings should help to interpret past functional studies and inform future studies examining the characteristics and consequences of InsP(3)R-mediated internal Ca(2+) release and Ca(2+) waves in hippocampal neurons.

To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca(2+) release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca(2+) release. Murine primary cultures were confocally imaged for Ca(2+) detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca(2+) sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice (mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca(2+) saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 microM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca(2+) saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca(2+)] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca(2+)] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca(2+) release channels and/or their susceptibility to Ca(2+)-induced activation, thereby suppressing the production of Ca(2+) sparks.

The aim of the study was to identify ion channel transcripts expressed in the sinoatrial node (SAN), the pacemaker of the heart. Functionally, the SAN can be divided into central and peripheral regions (center is adapted for pacemaking only, whereas periphery is adapted to protect center and drive atrial muscle as well as pacemaking) and the aim was to study expression in both regions. In rabbit tissue, the abundance of 30 transcripts (including transcripts for connexin, Na(+), Ca(2+), hyperpolarization-activated cation and K(+) channels, and related Ca(2+) handling proteins) was measured using quantitative PCR and the distribution of selected transcripts was visualized using in situ hybridization. Quantification of individual transcripts (quantitative PCR) showed that there are significant differences in the abundance of 63% of the transcripts studied between the SAN and atrial muscle, and cluster analysis showed that the transcript profile of the SAN is significantly different from that of atrial muscle. There are apparent isoform switches on moving from atrial muscle to the SAN center: RYR2 to RYR3, Na(v)1.5 to Na(v)1.1, Ca(v)1.2 to Ca(v)1.3 and K(v)1.4 to K(v)4.2. The transcript profile of the SAN periphery is intermediate between that of the SAN center and atrial muscle. For example, Na(v)1.5 messenger RNA is expressed in the SAN periphery (as it is in atrial muscle), but not in the SAN center, and this is probably related to the need of the SAN periphery to drive the surrounding atrial muscle.

Ryanodine receptors (RyR) amplify activity-dependent calcium influx via calcium-induced calcium release. Calcium signals trigger postsynaptic pathways in hippocampal neurons that underlie synaptic plasticity, learning, and memory. Recent evidence supports a role of the RyR2 and RyR3 isoforms in these processes. Along with calcium signals, brain-derived neurotrophic factor (BDNF) is a key signaling molecule for hippocampal synaptic plasticity and spatial memory. Upon binding to specific TrkB receptors, BDNF initiates complex signaling pathways that modify synaptic structure and function. Here, we show that BDNF-induced remodeling of hippocampal dendritic spines required functional RyR. Additionally, incubation with BDNF enhanced the expression of RyR2, RyR3, and PKMζ, an atypical protein kinase C isoform with key roles in hippocampal memory consolidation. Consistent with their increased RyR protein content, BDNF-treated neurons generated larger RyR-mediated calcium signals than controls. Selective inhibition of RyR-mediated calcium release with inhibitory ryanodine concentrations prevented the PKMζ, RyR2, and RyR3 protein content enhancement induced by BDNF. Intrahippocampal injection of BDNF or training rats in a spatial memory task enhanced PKMζ, RyR2, RyR3, and BDNF hippocampal protein content, while injection of ryanodine at concentrations that stimulate RyR-mediated calcium release improved spatial memory learning and enhanced memory consolidation. We propose that RyR-generated calcium signals are key features of the complex neuronal plasticity processes induced by BDNF, which include increased expression of RyR2, RyR3, and PKMζ and the spine remodeling required for spatial memory formation.

To investigate the channel properties of the mammalian type 3 ryanodine receptor (RyR3), we have cloned the RyR3 cDNA from rabbit uterus by reverse transcriptase-polymerase chain reaction and expressed the cDNA in HEK293 cells. Immunoblotting studies showed that the cloned RyR3 was indistinguishable from the native mammalian RyR3 in molecular size and immunoreactivity. Ca2+ release measurements using the fluorescence Ca2+ indicator fluo 3 revealed that the cloned RyR3 functioned as a caffeine- and ryanodine-sensitive Ca2+ release channel in HEK293 cells. Functional properties of the cloned RyR3 were further characterized by using single channel recordings in lipid bilayers. The cloned RyR3 channel exhibited a K+ conductance of 777 picosiemens in 250 mM KCl and a Ca2+ conductance of 137 picosiemens in 250 mM CaCl2 and displayed a pCa2+/pK+ ratio of 6.3 and an open time constant of about 1.16 ms. The response of the cloned RyR3 to cytoplasmic Ca2+ concentrations was biphasic. The channel was activated by Ca2+ at about 100 nM and inactivated at about 10 mM. Ca2+ alone was able to activate the cloned RyR3 fully. Calmodulin activated the cloned RyR3 at low Ca2+ concentrations but inhibited the channel at high Ca2+ concentrations. The cloned RyR3 was activated by ATP, caffeine, and perchlorate, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Cyclic ADP-ribose did not seem to affect single channel activity of the cloned RyR3. The most prominent differences of the cloned RyR3 from the rabbit skeletal muscle ryanodine receptor were in the gating kinetics, extent of maximal activation by Ca2+, and sensitivity to Ca2+ inactivation. Results of the present study provide initial insights into the single channel properties of the mammalian RyR3.

To evaluate the role in synaptic plasticity of ryanodine receptor type 3 (RyR3), which is normally enriched in hippocampal area CA1, we generated RyR3-deficient mice. Mutant mice exhibited facilitated CA1 long-term potentiation (LTP) induced by short tetanus (100 Hz, 100 ms) stimulation. Unlike LTP in wild-type mice, this LTP was not blocked bythe NMDA receptor antagonist D-AP5 but was partially dependent on L-type voltage-dependent Ca2+ channels (VDCCs) and metabotropic glutamate receptors (mGluRs). Long-term depression (LTD) was not induced in RyR3-deficient mice. RyR3-deficient mice also exhibited improved spatial learning on a Morris water maze task. These results suggest that in wild-type mice, in contrast to the excitatory role of Ca2+ influx, RyR3-mediated intracellular Ca2+ ([Ca2+]i) release from endoplasmic reticulum (ER) may inhibit hippocampal LTP and spatial learning.

A key event in skeletal muscle activation is the rapid release of Ca(2+) from the sarcoplasmic reticulum (SR), the Ca(2+) storage organelle in the muscle cell. The surface membrane/transverse tubules and the SR form functional units (calcium release units containing one or two couplons or junctions), where the voltage-sensing dihydropyridine receptor of the surface membrane interacts with the SR Ca(2+) release channel [ryanodine receptor (RyR)] and depolarization of the cell membrane is converted into Ca(2+) release from the SR. Although RyR1 is the most important isoform in skeletal muscle, some muscles also express high levels of RyR3, an isoform with a wide tissue distribution. The cytoplasmic domains of RyRs are visible in the electron microscope as periodically disposed feet. We find that, in muscles containing only RyR1, feet are exclusively located over the junctional SR surface facing the surface membrane/transverse tubule. In muscles containing RyR1 as well as RyR3, additional feet are located in lateral parajunctional regions immediately adjacent to junctional SR. Biochemical content of RyR3 and content of parajunctional feet are highly correlated in different muscles and the disposition of parajunctional versus junctional feet are notably different. On the basis of these two observations, we postulate that RyR3s are restricted to the parajunctional region, and thus their activation must be indirect and derivative during excitation-contraction coupling.

Intracellular Ca(2+) levels control both contraction and relaxation in vascular smooth muscle cells (VSMCs). Ca(2+)-dependent relaxation is mediated by discretely localized Ca(2+) release events through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). These local increases in Ca(2+) concentration, termed sparks, stimulate nearby Ca(2+)-activated K(+) (BK) channels causing BK currents (spontaneous transient outward currents or STOCs). STOCs are hyperpolarizing currents that oppose vasoconstriction. Several RyR isoforms are coexpressed in VSMCs; however, their role in Ca(2+) spark generation is unknown. To provide molecular information on RyR cluster function and assembly, we examined Ca(2+) sparks and STOCs in RyR3-deficient freshly isolated myocytes of resistance-sized cerebral arteries from knockout mice and compared them to Ca(2+) sparks in cells from wild-type mice. We used RT-PCR to identify RyR1, RyR2, and RyR3 mRNA in cerebral arteries. Ca(2+) sparks in RyR3-deficient cells were similar in peak amplitude (measured as F/F(0)), width at half-maximal amplitude, and duration compared with wild-type cell Ca(2+) sparks. However, the frequency of STOCs (between -60 mV and -20 mV) was significantly higher in RyR3-deficient cells than in wild-type cells. Ca(2+) sparks and STOCs in both RyR3-deficient and wild-type cells were inhibited by ryanodine (10 micromol/L), external Ca(2+) removal, and depletion of SR Ca(2+) stores by caffeine (1 mmol/L). Isolated, pressurized cerebral arteries of RyR3-deficient mice developed reduced myogenic tone. Our results suggest that RyR3 is part of the SR Ca(2+) spark release unit and plays a specific molecular role in the regulation of STOCs frequency in mouse cerebral artery VSMCs after decreased arterial tone.

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl- currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3-/-) mice, hypoxia-induced, but not submaximal noradrenaline-induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RyR3-/- mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline-induced Ca2+ and contractile responses in PASMCs.

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, ).

While the roles of subtypes 1 and 2 of the ryanodine receptors (RYRs) have been studied in cellular systems expressing specifically one or the other of these subtypes (i.e. skeletal and cardiac muscle), the function of these receptors has not been evaluated in smooth muscles. We have previously reported RYR-mediated elementary (Ca(2+) sparks) and global Ca(2+) responses in rat portal vein myocytes. Here, we investigated the respective roles of all three RYR subtypes expressed in these cells as revealed by reverse transcriptase-polymerase chain reaction. Antisense oligonucleotides targeting each one of the three RYR subtypes were shown to specifically inhibit the expression of the corresponding mRNA and protein without affecting the other RYR subtypes. Confocal Ca(2+) measurements revealed that depolarization-induced Ca(2+) sparks and global Ca(2+) responses were blocked when either RYR1 or RYR2 expression was suppressed. Caffeine-induced Ca(2+) responses were partly inhibited by the same antisense oligonucleotides. Neither the corresponding scrambled oligonucleotides nor the antisense oligonucleotides targeting RYR3 affected depolarization- or caffeine-induced Ca(2+) responses. Our results show that, in vascular myocytes, the two RYR1 and RYR2 subtypes are required for Ca(2+) release during Ca(2+) sparks and global Ca(2+) responses, evoked by activation of voltage-gated Ca(2+) channels.