Expression and functional characterization of the cardiac muscle ryanodine receptor Ca(2+) release channel in Chinese hamster ovary cells.

To study the function and regulation of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel, we expressed the RyR2 proteins in a Chinese hamster ovary (CHO) cell line, and assayed its function by single channel current recording and confocal imaging of intracellular Ca(2+) ([Ca(2+)](i)). The 16-kb cDNA encoding the full-length RyR2 was introduced into CHO cells using lipofectAmine and electroporation methods. Incorporation of microsomal membrane vesicles isolated from these transfected cells into lipid bilayer membrane resulted in single Ca(2+) release channel activities similar to those of the native Ca(2+) release channels from rabbit cardiac muscle SR membranes, both in terms of gating kinetics, conductance, and ryanodine modification. The expressed RyR2 channels were found to exhibit more frequent transitions to subconductance states than the native RyR2 channels and RyR1 expressed in CHO cells. Caffeine, an exogenous activator of RyR, induced release of [Ca(2+)](i) from these cells. Confocal imaging of cells expressing RyR2 did not detect spontaneous or caffeine-induced local Ca(2+) release events (i.e., "Ca(2+) sparks") typically seen in cardiac muscle. Our data show that the RyR2 expressed in CHO cells forms functional Ca(2+) release channels. Furthermore, the lack of localized Ca(2+) release events in these cells suggests that Ca(2+) sparks observed in cardiac muscle may involve cooperative gating of a group of Ca(2+) release channels and/or their interaction with muscle-specific proteins.     

Evidence for a role of C-terminus in Ca(2+) inactivation of skeletal muscle Ca(2+) release channel (ryanodine receptor).

Six chimeras of the skeletal muscle (RyR1) and cardiac muscle (RyR2) Ca(2+) release channels (ryanodine receptors) previously used to identify RyR1 dihydropyridine receptor interactions [Nakai et al. (1998) J. Biol. Chem. 273, 13403] were expressed in HEK293 cells to assess their Ca(2+) dependence in [(3)H]ryanodine binding and single channel measurements. The results indicate that the C-terminal one-fourth has a major role in Ca(2+) activation and inactivation of RyR1. Further, our results show that replacement of RyR1 regions with corresponding RyR2 regions can result in loss and/or reduction of [(3)H]ryanodine binding affinity while maintaining channel activity.     

Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating

The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.     

Single channel properties of heterotetrameric mutant RyR1 ion channels linked to core myopathies

Skeletal muscle excitation-contraction coupling involves activation of homotetrameric ryanodine receptor ion channels (RyR1s), resulting in the rapid release of Ca(2+) from the sarcoplasmic reticulum. Previous work has shown that Ca(2+) release is impaired by mutations in RyR1 linked to Central Core Disease and Multiple Minicore Disease. We studied the consequences of these mutations on RyR1 function, following their expression in human embryonic kidney 293 cells and incorporation in lipid bilayers. RyR1-G4898E, -G4898R, and -DeltaV4926/I4927 mutants in the C-terminal pore region of RyR1 and N-terminal RyR1-R110W/L486V mutant all showed negligible Ca(2+) permeation and loss of Ca(2+)-dependent channel activity but maintained reduced K(+) conductances. Co-expression of wild type and mutant RyR1s resulted in Ca(2+)-dependent channel activities that exhibited intermediate Ca(2+) selectivities compared with K(+), which suggested the presence of tetrameric RyR1 complexes composed of wild type and mutant subunits. The number of wild-type subunits to maintain a functional heterotetrameric channel differed among the four RyR1 mutants. The results indicate that homozygous RyR1 mutations associated with core myopathies abolish or greatly reduce sarcoplasmic reticulum Ca(2+) release during excitation-contraction coupling. They further suggest that in individuals, expressing wild type and mutant alleles, a substantial portion of RyR1 channels is able to release Ca(2+) from sarcoplasmic reticulum.     

A ryanodine fluorescent derivative reveals the presence of high-affinity ryanodine binding sites in the Golgi complex of rat sympathetic neurons, with possible functional roles in intracellular Ca(2+) signaling

The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca(2+) release channels. Although these channels are present in a variety of cell types, their functional role in nerve cells is still puzzling. Here, a monosubstituted fluorescent Ry analogue, B-FL-X Ry, was used to reveal the distribution of RyRs in cultured rat sympathetic neurons. B-FL-X Ry competitively inhibited the binding of [3H]Ry to rabbit skeletal muscle SR membranes, with an IC(50) of 150 nM, compared to 7 nM of unlabeled Ry. Binding of B-FL-X Ry to the cytoplasm of sympathetic neurons is saturable, reversible and of high affinity. The pharmacology of B-FL-X Ry showed marked differences with unlabeled Ry, which are partially explained by its lower affinity: (1) use-dependent reversible inhibition of caffeine-induced intracellular Ca(2+) release; (2) diminished voltage-gated Ca(2+) influx, due to a positive shift in the activation of voltage gated Ca(2+) currents. B-FL-X Ry-stained sympathetic neurons, viewed under confocal microscopy, showed conspicuous labeling of crescent-shaped structures pertaining to the Golgi complex, a conclusion supported by experiments showing co-localization with Golgi-specific fluorescent probes and the breaking up of crescent-shaped staining after treatment with drugs that disassemble Golgi complex. The presence of RyRs to the Golgi could be confirmed with specific anti-RyR(2) antibodies, but evidence of caffeine-induced Ca(2+) release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca(2+) signal was detected close to this organelle. In spite of that, short-term incubation with brefeldin A (BFA) suppressed the fast component of caffeine-induced Ca(2+) release, and the Ca(2+) release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca(2+) homeostasis and signaling in nerve cells, could be relevant to reports involving derangement of the Golgi complex as a probable cause of some forms of progressive neuronal degeneration, such as Alzheimer's disease and amyotrophic lateral sclerosis.     

RyR3 amplifies RyR1-mediated Ca(2+)-induced Ca(2+) release in neonatal mammalian skeletal muscle

The neonatal mammalian skeletal muscle contains both type 1 and type 3 ryanodine receptors (RyR1 and RyR3) located in the sarcoplasmic reticulum membrane. An allosteric interaction between RyR1 and dihydropyridine receptors located in the plasma membrane mediates voltage-induced Ca(2+) release (VICR) from the sarcoplasmic reticulum. RyR3, which disappears in adult muscle, is not involved in VICR, and the role of the transiently expressed RyR3 remains elusive. Here we demonstrate that RyR1 participates in both VICR and Ca(2+)-induced Ca(2+) release (CICR) and that RyR3 amplifies RyR1-mediated CICR in neonatal skeletal muscle. Confocal measurements of intracellular Ca(2+) in primary cultured mouse skeletal myotubes reveal active sites of Ca(2+) release caused by peripheral coupling between dihydropyridine receptors and RyR1. In myotubes lacking RyR3, the peripheral VICR component is unaffected, and RyR1s alone are able to support inward CICR propagation in most cells at an average speed of approximately 190 microm/s. With the co-presence of RyR1 and RyR3 in wild-type cells, unmitigated radial CICR propagates at 2,440 microm/s. Because neonatal skeletal muscle lacks a well developed transverse tubule system, the RyR3 reinforcement of CICR seems to ensure a robust, uniform, and synchronous activation of Ca(2+) release throughout the cell body. Such functional interplay between RyR1 and RyR3 can serve important roles in Ca(2+) signaling of cell differentiation and muscle contraction.     

Mice null for calsequestrin 1 exhibit deficits in functional performance and sarcoplasmic reticulum calcium handling

In skeletal muscle, the release of calcium (Ca(2+)) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca(2+) release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca(2+) buffering as well as its potential for modulating RyR1, the L-type Ca(2+) channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca(2+)]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca(2+) content and SR Ca(2+) release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca(2+) release with single action potentials and a collapse of the Ca(2+) release with repetitive trains. Under voltage clamp, SR Ca(2+) release flux and total SR Ca(2+) release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca(2+) release flux appears to be solely due to elimination of the slowly decaying component of SR Ca(2+) release, whereas the rapidly decaying component of SR Ca(2+) release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca(2+)] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca(2+)](free) in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca(2+) buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca(2+) release.     

The role of calmodulin for inositol 1,4,5-trisphosphate receptor function

Intracellular calcium release is a fundamental signaling mechanism in all eukaryotic cells. The ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP(3)R) are intracellular calcium release channels. Both channels can be regulated by calcium and calmodulin (CaM). In this review we will first discuss the role of calcium as an activator and inactivator of the IP(3)R, concluding that calcium is the most important regulator of the IP(3)R. In the second part we will further focus on the role of CaM as modulator of the IP(3)R, using results of the voltage-dependent Ca(2+) channels and the RyR as reference material. Here we conclude that despite the fact that different CaM-binding sites have been characterized, their function for the IP(3)R remains elusive. In the third part we will discuss the possible functional role of CaM in IP(3)-induced Ca(2+) release (IICR) by direct and indirect mechanisms. Special attention will be given to the Ca(2+)-binding proteins (CaBPs) that were shown to activate the IP(3)R in the absence of IP(3).     

Store-operated Ca2+ entry in porcine airway smooth muscle

Ca(2+) influx triggered by depletion of sarcoplasmic reticulum (SR) Ca(2+) stores [mediated via store-operated Ca(2+) channels (SOCC)] was characterized in enzymatically dissociated porcine airway smooth muscle (ASM) cells. When SR Ca(2+) was depleted by either 5 microM cyclopiazonic acid or 5 mM caffeine in the absence of extracellular Ca(2+), subsequent introduction of extracellular Ca(2+) further elevated [Ca(2+)](i). SOCC was insensitive to 1 microM nifedipine- or KCl-induced changes in membrane potential. However, preexposure of cells to 100 nM-1 mM La(3+) or Ni(2+) inhibited SOCC. Exposure to ACh increased Ca(2+) influx both in the presence and absence of a depleted SR. Inhibition of inositol 1,4,5-trisphosphate (IP)-induced SR Ca(2+) release by 20 microM xestospongin D inhibited SOCC, whereas ACh-induced IP(3) production by 5 microM U-73122 had no effect. Inhibition of Ca(2+) release through ryanodine receptors (RyR) by 100 microM ryanodine also prevented Ca(2+) influx via SOCC. Qualitatively similar characteristics of SOCC-mediated Ca(2+) influx were observed with cyclopiazonic acid- vs. caffeine-induced SR Ca(2+) depletion. These data demonstrate that a Ni(2+)/La(3+)-sensitive Ca(2+) influx via SOCC in porcine ASM cells involves SR Ca(2+) release through both IP(3) and RyR channels. Additional regulation of Ca(2+) influx by agonist may be related to a receptor-operated, noncapacitative mechanism.     

Store-operated Ca2+ influx causes Ca2+ release from the intracellular Ca2+ channels that is required for T cell activation

The precise control of many T cell functions relies on cytosolic Ca(2+) dynamics that is shaped by the Ca(2+) release from the intracellular store and extracellular Ca(2+) influx. The Ca(2+) influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca(2+) concentration ([Ca(2+)](i)) necessary for T cell activation, whereas the role of intracellular Ca(2+) release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca(2+)](i) elevation observed upon activation of the store-operated Ca(2+) entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca(2+) release channels were activated in parallel with SOCE and contributed to global [Ca(2+)](i) elevation. Pretreating cells with (-)-xestospongin C (10 microM) or ryanodine (400 microM), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca(2+)](i) elevation evoked by the passive store depletion or TCR ligation. Although the Ca(2+) release from the IP3R can be activated by TCR stimulation, the Ca(2+) release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca(2+)](i) signaling in T cells, that is SOCE-dependent Ca(2+) release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca(2+)-dependent functions of T lymphocytes.     

Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor

Recent studies have suggested a central role for Ca(2+) in the signaling pathway of apoptosis and certain anti-apoptotic effects of Bcl-2 family of proteins have been attributed to changes in intracellular Ca(2+) homeostasis. Here we report that depletion of Ca(2+) from endoplasmic reticulum (ER) leads to apoptosis in Chinese hamster ovary cells. Stable expression of ryanodine receptor (RyR) in these cells enables rapid and reversible changes of both cytosolic Ca(2+) and ER Ca(2+) content via activation of the RyR/Ca(2+) release channel by caffeine and ryanodine. Sustained depletion of the ER Ca(2+) store leads to apoptosis in Chinese hamster ovary cells, whereas co-expression of Bcl-xL and RyR in these cells prevents apoptotic cell death but not necrotic cell death. The anti-apoptotic effect of Bcl-xL does not correlate with changes in either the Ca(2+) release process from the ER or the capacitative Ca(2+) entry through the plasma membrane. The data suggest that Bcl-xL likely prevents apoptosis of cells at a stage downstream of ER Ca(2+) release and capacitative Ca(2+) entry.     

Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.     

Ryanodine receptor activation by Ca v 1.2 is involved in dendritic cell major histocompatibility complex class II surface expression

Dendritic cells express the skeletal muscle ryanodine receptor (RyR1), yet little is known concerning its physiological role and activation mechanism. Here we show that dendritic cells also express the Ca(v)1.2 subunit of the L-type Ca(2+) channel and that release of intracellular Ca(2+) via RyR1 depends on the presence of extracellular Ca(2+) and is sensitive to ryanodine and nifedipine. Interestingly, RyR1 activation causes a very rapid increase in expression of major histocompatibility complex II molecules on the surface of dendritic cells, an effect that is also observed upon incubation of mouse BM12 dendritic cells with transgenic T cells whose T cell receptor is specific for the I-A(bm12) protein. Based on the present results, we suggest that activation of the RyR1 signaling cascade may be important in the early stages of infection, providing the immune system with a rapid mechanism to initiate an early response, facilitating the presentation of antigens to T cells by dendritic cells before their full maturation.     

Role of FKBP12.6 in cADPR-induced activation of reconstituted ryanodine receptors from arterial smooth muscle

cADP ribose (cADPR) serves as second messenger to activate the ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) and mobilize intracellular Ca(2+) in vascular smooth muscle cells. However, the mechanisms mediating the effect of cADPR remain unknown. The present study was designed to determine whether FK-506 binding protein 12.6 (FKBP12.6), an accessory protein of the RyRs, plays a role in cADPR-induced activation of the RyRs. A 12.6-kDa protein was detected in bovine coronary arterial smooth muscle (BCASM) and cultured CASM cells by being immunoblotted with an antibody against FKBP12, which also reacted with FKBP12.6. With the use of planar lipid bilayer clamping techniques, FK-506 (0.01-10 microM) significantly increased the open probability (NP(O)) of reconstituted RyR/Ca(2+) release channels from the SR of CASM. This FK-506-induced activation of RyR/Ca(2+) release channels was abolished by pretreatment with anti-FKBP12 antibody. The RyRs activator cADPR (0.1-10 microM) markedly increased the activity of RyR/Ca(2+) release channels. In the presence of FK-506, cADPR did not further increase the NP(O) of RyR/Ca(2+) release channels. Addition of anti-FKBP12 antibody also completely blocked cADPR-induced activation of these channels, and removal of FKBP12.6 by preincubation with FK-506 and subsequent gradient centrifugation abolished cADPR-induced increase in the NP(O) of RyR/Ca(2+) release channels. We conclude that FKBP12.6 plays a critical role in mediating cADPR-induced activation of RyR/Ca(2+) release channels from the SR of BCASM.     

Calmodulin oxidation and methionine to glutamine substitutions reveal methionine residues critical for functional interaction with ryanodine receptor-1

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.     

Evidence for a role of the lumenal M3-M4 loop in skeletal muscle Ca(2+) release channel (ryanodine receptor) activity and conductance

We tested the hypothesis that part of the lumenal amino acid segment between the two most C-terminal membrane segments of the skeletal muscle ryanodine receptor (RyR1) is important for channel activity and conductance. Eleven mutants were generated and expressed in HEK293 cells focusing on amino acid residue I4897 homologous to the selectivity filter of K(+) channels and six other residues in the M3-M4 lumenal loop. Mutations of amino acids not absolutely conserved in RyRs and IP(3)Rs (D4903A and D4907A) showed cellular Ca(2+) release in response to caffeine, Ca(2+)-dependent [(3)H]ryanodine binding, and single-channel K(+) and Ca(2+) conductances not significantly different from wild-type RyR1. Mutants with an I4897 to A, L, or V or D4917 to A substitution showed a decreased single-channel conductance, loss of high-affinity [(3)H]ryanodine binding and regulation by Ca(2+), and an altered caffeine-induced Ca(2+) release in intact cells. Mutant channels with amino acid residue substitutions that are identical in the RyR and IP(3)R families (D4899A, D4899R, and R4913E) exhibited a decreased K(+) conductance and showed a loss of high-affinity [(3)H]ryanodine binding and loss of single-channel pharmacology but maintained their response to caffeine in a cellular assay. Two mutations (G4894A and D4899N) were able to maintain pharmacological regulation both in intact cells and in vitro but had lower single-channel K(+) and Ca(2+) conductances than the wild-type channel. The results support the hypothesis that amino acid residues in the lumenal loop region between the two most C-terminal membrane segments constitute a part of the ion-conducting pore of RyR1.     

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca(2+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](rest) that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](rest) in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca(2+) entry at rest.     

Molecular genetics of ryanodine receptors Ca2+-release channels

The family of ryanodine receptor (RyR) genes encodes three highly related Ca(2+)-release channels: RyR1, RyR2 and RyR3. RyRs are known as the Ca(2+)-release channels that participate to the mechanism of excitation-contraction coupling in striated muscles, but they are also expressed in many other cell types. Actually, in several cells two or three RyR isoforms can be co-expressed and interactive feedbacks among them may be important for generation of intracellular Ca(2+) signals and regulation of specific cellular functions. Important developments have been obtained in understanding the biochemical complexity underlying the process of Ca(2+) release through RyRs. The 3-D structure of these large molecules has been obtained and some regulatory regions have been mapped within these 3-D reconstructions. Recent studies have clarified the role of protein kinases and phosphatases that, by physically interacting with RyRs, appear to play a role in the regulation of these Ca(2+)-release channels. These and other recent advancements in understanding RyR biology will be the object of this review.