Calmodulin kinase is a molecular switch for cardiac excitation-contraction coupling

Signaling between cell membrane-bound L-type Ca(2+) channels (LTCC) and ryanodine receptor Ca(2+) release channels (RyR) on sarcoplasmic reticulum (SR) stores grades excitation-contraction coupling (ECC) in striated muscle. A physical connection regulates LTCC and RyR in skeletal muscle, but the molecular mechanism for coordinating LTCC and RyR in cardiomyocytes, where this physical link is absent, is unknown. Calmodulin kinase (CaMK) has characteristics suitable for an ECC coordinating molecule: it is activated by Ca(2+)/calmodulin, it regulates LTCC and RyR, and it is enriched in the vicinity of LTCC and RyR. Intact cardiomyocytes were studied under conditions where CaMK activity could be controlled independently of intracellular Ca(2+) by using an engineered Ca(2+)-independent form of CaMK and a highly specific CaMK inhibitory peptide. CaMK reciprocally enhanced L-type Ca(2+) current and reduced release of Ca(2+) from the SR while increasing SR Ca(2+) content. These findings support the hypothesis that CaMK is required to functionally couple LTCC and RyR during cardiac ECC.     

Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor

Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca(2+) release in skeletal muscle and a potentially fatal rise in perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation-contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and Ca(V)1.1, the principal subunit of the L-type Ca(2+) channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca(2+) entry. The Ca(V)1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all Ca(V) channels [Carpenter D, et al. (2009) BMC Med Genet 10:104-115.]. To define the functional consequences of this mutation, we expressed it in dysgenic (Ca(V)1.1 null) myotubes. Unlike previously described MH-linked mutations in Ca(V)1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca(2+) release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in Ca(V)1.1 R174W-expressing myotubes, resting myoplasmic Ca(2+) levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type Ca(V)1.1. Our results indicate that Ca(V)1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca(2+) leak from the SR, and that perturbation of Ca(V)1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers.     

Orthograde dihydropyridine receptor signal regulates ryanodine receptor passive leak

The skeletal muscle dihydropyridine receptor (DHPR) and ryanodine receptor (RyR1) are known to engage a form of conformation coupling essential for muscle contraction in response to depolarization, referred to as excitation-contraction coupling. Here we use WT and Ca(V)1.1 null (dysgenic) myotubes to provide evidence for an unexplored RyR1-DHPR interaction that regulates the transition of the RyR1 between gating and leak states. Using double-barreled Ca(2+)-selective microelectrodes, we demonstrate that the lack of Ca(V)1.1 expression was associated with an increased myoplasmic resting [Ca(2+)] ([Ca(2+)](rest)), increased resting sarcolemmal Ca(2+) entry, and decreased sarcoplasmic reticulum (SR) Ca(2+) loading. Pharmacological control of the RyR1 leak state, using bastadin 5, reverted the three parameters to WT levels. The fact that Ca(2+) sparks are not more frequent in dysgenic than in WT myotubes adds support to the hypothesis that the leak state is a conformation distinct from gating RyR1s. We conclude from these data that this orthograde DHPR-to-RyR1 signal inhibits the transition of gated RyR1s into the leak state. Further, it suggests that the DHPR-uncoupled RyR1 population in WT muscle has a higher propensity to be in the leak conformation. RyR1 leak functions are to keep [Ca(2+)](rest) and the SR Ca(2+) content in the physiological range and thus maintain normal intracellular Ca(2+) homeostasis.     

Conformational activation of Ca2+ entry by depolarization of skeletal myotubes

Store-operated Ca(2+) entry (SOCE) occurs in diverse cell types in response to depletion of Ca(2+) within the endoplasmic/sarcoplasmic reticulum and functions both to refill these stores and to shape cytoplasmic Ca(2+) transients. Here we report that in addition to conventional SOCE, skeletal myotubes display a physiological mechanism that we term excitation-coupled Ca(2+) entry (ECCE). ECCE is rapidly initiated by membrane depolarization. Like excitation-contraction coupling, ECCE is absent in both dyspedic myotubes that lack the skeletal muscle-type ryanodine receptor 1 and dysgenic myotubes that lack the dihydropyridine receptor (DHPR), and is independent of the DHPR l-type Ca(2+) current. Unlike classic SOCE, ECCE does not depend on sarcoplasmic reticulum Ca(2+) release. Indeed, ECCE produces a large Ca(2+) entry in response to physiological stimuli that do not produce substantial store depletion and depends on interactions among three different Ca(2+) channels: the DHPR, ryanodine receptor 1, and a Ca(2+) entry channel with properties corresponding to those of store-operated Ca(2+) channels. ECCE may provide a fundamental means to rapidly maintain Ca(2+) stores and control important aspects of Ca(2+) signaling in both muscle and nonmuscle cells.     

Postulated role of inter-domain interaction within the ryanodine receptor in Ca(2+) channel regulation

Key steps of excitation-contraction (E-C) coupling are (1) binding of the activator portion of the dihydropyridine (DHP) receptor (in skeletal muscle) or binding of the Ca(2+) entered through the DHP receptor (in cardiac muscle) to the ryanodine receptor (RyR), (2) a global protein conformational change of the RyR, and (3) opening of the RyR Ca(2+) channel, leading to muscle contraction. The conformational change (step 2) plays a major role in the Ca(2+) channel regulation, and a number of "regulatory domains" must be involved in this process. We postulate that the interaction among these regulatory domains is the central mechanism for the conformation-mediated control of the Ca(2+) channel. In this review, we summarize the recent data supporting this concept.     

Evidence for a Ca(2+)-gated ryanodine-sensitive Ca2+ release channel in visceral smooth muscle.

Although a role for the ryanodine receptor (RyR) in Ca2+ signaling in smooth muscle has been inferred, direct information on the biochemical and functional properties of the receptor has been largely lacking. Studies were thus carried out to purify and characterize the RyR in stomach smooth muscle cells from the toad Bufo marinus. Intracellular Ca2+ measurements with the Ca(2+)-sensitive fluorescent indicator fura-2 under voltage clamp indicated the presence of a caffeine- and ryanodine-sensitive internal store for Ca2+ in these cells. The (CHAPS)-solubilized, [3H]ryanodine-labeled RyR of toad smooth muscle was partially purified from microsomal membranes by rate density centrifugation as a 30-S protein complex. SDS/PAGE indicated the comigration of a high molecular weight polypeptide with the peak attributed to 30-S RyR, which had a mobility similar to the cardiac RyR and on immunoblots cross-reacted with a monoclonal antibody to the canine cardiac RyR. Following planar lipid bilayer reconstitution of 30-S stomach muscle RyR fractions, single-channel currents (830 pS with 250 mM K+ as the permeant ion) were observed that were activated by Ca2+ and modified by ryanodine. In vesicle-45Ca2+ efflux measurements, the toad channel was activated to a greater extent at 100-1000 microM than 1-10 microM Ca2+. These results suggest that toad stomach muscle contains a ryanodine-sensitive Ca2+ release channel with properties similar but not identical to those of the mammalian skeletal and cardiac Ca(2+)-release channels.     

An Ryr1I4895T mutation abolishes Ca2+ release channel function and delays development in homozygous offspring of a mutant mouse line

A heterozygous Ile4898 to Thr (I4898T) mutation in the human type 1 ryanodine receptor/Ca(2+) release channel (RyR1) leads to a severe form of central core disease. We created a mouse line in which the corresponding Ryr1(I4895T) mutation was introduced by using a "knockin" protocol. The heterozygote does not exhibit an overt disease phenotype, but homozygous (IT/IT) mice are paralyzed and die perinatally, apparently because of asphyxia. Histological analysis shows that IT/IT mice have greatly reduced and amorphous skeletal muscle. Myotubes are small, nuclei remain central, myofibrils are disarranged, and no cross striation is obvious. Many areas indicate probable degeneration, with shortened myotubes containing central stacks of pyknotic nuclei. Other manifestations of a delay in completion of late stages of embryogenesis include growth retardation and marked delay in ossification, dermatogenesis, and cardiovascular development. Electron microscopy of IT/IT muscle demonstrates appropriate targeting and positioning of RyR1 at triad junctions and a normal organization of dihydropyridine receptor (DHPR) complexes into RyR1-associated tetrads. Functional studies carried out in cultured IT/IT myotubes show that ligand-induced and DHPR-activated RyR1 Ca(2+) release is absent, although retrograde enhancement of DHPR Ca(2+) conductance is retained. IT/IT mice, in which RyR1-mediated Ca(2+) release is abolished without altering the formation of the junctional DHPR-RyR1 macromolecular complex, provide a valuable model for elucidation of the role of RyR1-mediated Ca(2+) signaling in mammalian embryogenesis.     

Excitation--contraction uncoupling by a human central core disease mutation in the ryanodine receptor

Central core disease (CCD) is a human congenital myopathy characterized by fetal hypotonia and proximal muscle weakness that is linked to mutations in the gene encoding the type-1 ryanodine receptor (RyR1). CCD is thought to arise from Ca(2+)-induced damage stemming from mutant RyR1 proteins forming "leaky" sarcoplasmic reticulum (SR) Ca(2+) release channels. A novel mutation in the C-terminal region of RyR1 (I4898T) accounts for an unusually severe and highly penetrant form of CCD in humans [Lynch, P. J., Tong, J., Lehane, M., Mallet, A., Giblin, L., Heffron, J. J., Vaughan, P., Zafra, G., MacLennan, D. H. & McCarthy, T. V. (1999) Proc. Natl. Acad. Sci. USA 96, 4164--4169]. We expressed in skeletal myotubes derived from RyR1-knockout (dyspedic) mice the analogous mutation engineered into a rabbit RyR1 cDNA (I4897T). Here we show that homozygous expression of I4897T in dyspedic myotubes results in a complete uncoupling of sarcolemmal excitation from voltage-gated SR Ca(2+) release without significantly altering resting cytosolic Ca(2+) levels, SR Ca(2+) content, or RyR1-mediated enhancement of dihydropyridine receptor (DHPR) channel activity. Coexpression of both I4897T and wild-type RyR1 resulted in a 60% reduction in voltage-gated SR Ca(2+) release, again without altering resting cytosolic Ca(2+) levels, SR Ca(2+) content, or DHPR channel activity. These findings indicate that muscle weakness suffered by individuals possessing the I4898T mutation involves a functional uncoupling of sarcolemmal excitation from SR Ca(2+) release, rather than the expression of overactive or leaky SR Ca(2+) release channels.     

Putative roles of type 3 ryanodine receptor isoforms (RyR3)

Ca(2+)-release from the sarcoplasmic or endoplasmic reticulum, the intracellular Ca(2+) store, is mediated by the ryanodine receptor (RyR) and/or the inositol trisphosphate receptor (IP3R). While IP3R is a ligand(IP3)-operated channel, RyR can be gated by a ligand (Ca(2+)) and/or mechanical coupling with the voltage sensor. There are three genetically distinct isoforms among RyR in mammals: RyR1-3. RyR1, the primary isoform in the skeletal muscle, can be gated by direct or indirect coupling with the conformation change of the alpha 1S subunit of dihydropyridine receptor (DHPR) on the T-tubules (transversely invaginated sarcolemma) upon depolarization of skeletal muscles or by the increased cytoplasmic Ca(2+) (Ca(2+)-induced Ca(2+) release, CICR). RyR2, the primary isoform in the cardiac ventricular muscle (and, in a lesser amount, the brain), can be gated by Ca(2+) which flows in through DHPR, especially the alpha1C subunit on depolarization. RyR3 is distributed ubiquitously in various tissues and may be coexpressed with RyR1 and RyR2. RyR3 is considered to be similar to RyR2 in the respect that it can be activated by Ca(2+), in view of the lack of available evidence to show the activation by the alpha1S subunit. Therefore, it is anticipated that RyR3 might take part through CICR in Ca(2+) signaling in smooth muscle and other non-muscle cells. To address the possible involvement of the CICR mechanism in the Ca(2+) signal transduction, it is critical to assess the effect of Mg(2+) on the CICR activity and the cytoplasmic concentration of Mg(2+). In this brief review, our discussion focuses on the effects of Ca(2+) and Mg(2+) on the activity of RyR3.     

Interaction between the dihydropyridine receptor Ca2+ channel beta-subunit and ryanodine receptor type 1 strengthens excitation-contraction coupling

Previous studies have shown that the skeletal dihydropyridine receptor (DHPR) pore subunit Ca(V)1.1 (alpha1S) physically interacts with ryanodine receptor type 1 (RyR1), and a molecular signal is transmitted from alpha1S to RyR1 to trigger excitation-contraction (EC) coupling. We show that the beta-subunit of the skeletal DHPR also binds RyR1 and participates in this signaling process. A novel binding site for the DHPR beta1a-subunit was mapped to the M(3201) to W(3661) region of RyR1. In vitro binding experiments showed that the strength of the interaction is controlled by K(3495)KKRR_ _R(3502), a cluster of positively charged residues. Phenotypic expression of skeletal-type EC coupling by RyR1 with mutations in the K(3495)KKRR_ _R(3502) cluster was evaluated in dyspedic myotubes. The results indicated that charge neutralization or deletion severely depressed the magnitude of RyR1-mediated Ca(2+) transients coupled to voltage-dependent activation of the DHPR. Meantime the Ca(2+) content of the sarcoplasmic reticulum was not affected, and the amplitude and activation kinetics of the DHPR Ca(2+) currents were slightly affected. The data show that the DHPR beta-subunit, like alpha1S, interacts directly with RyR1 and is critical for the generation of high-speed Ca(2+) signals coupled to membrane depolarization. These findings indicate that EC coupling in skeletal muscle involves the interplay of at least two subunits of the DHPR, namely alpha1S and beta1a, interacting with possibly different domains of RyR1.     

Bidirectional signaling between calcium channels of skeletal muscle requires multiple direct and indirect interactions

We have defined regions of the skeletal muscle ryanodine receptor (RyR1) essential for bidirectional signaling with dihydropyridine receptors (DHPRs) and for the organization of DHPR into tetrad arrays by expressing RyR1-RyR3 chimerae in dyspedic myotubes. RyR1-RyR3 constructs bearing RyR1 residues 1-1681 restored wild-type DHPR tetrad arrays and, in part, skeletal-type excitation-contraction (EC) coupling (orthograde signaling) but failed to enhance DHPR Ca(2+) currents (retrograde signaling) to WT RyR1 levels. Within this region, the D2 domain (amino acids 1272-1455), although ineffective on its own, dramatically enhanced the formation of tetrads and EC coupling rescue by constructs that otherwise are only partially effective. These findings suggest that the orthograde signal and DHPR tetrad formation require the contributions of numerous RyR regions. Surprisingly, we found that RyR3, although incapable of supporting EC coupling or tetrad formation, restored a significant level of Ca(2+) current, revealing a functional interaction with the skeletal muscle DHPR. Thus, our data support the hypotheses that (i) the structural/functional link between RyR1 and the skeletal muscle DHPR requires multiple interacting regions, (ii) the D2 domain of RyR1 plays a key role in stabilizing this interaction, and (iii) a form of retrograde signaling from RyR3 to the DHPR occurs in the absence of direct protein-protein interactions.     

Ca(2+) sparks operated by membrane depolarization require isoform 3 ryanodine receptor channels in skeletal muscle

Stimuli are translated to intracellular calcium signals via opening of inositol trisphosphate receptor and ryanodine receptor (RyR) channels of the sarcoplasmic reticulum or endoplasmic reticulum. In cardiac and skeletal muscle of amphibians the stimulus is depolarization of the transverse tubular membrane, transduced by voltage sensors at tubular-sarcoplasmic reticulum junctions, and the unit signal is the Ca(2+) spark, caused by concerted opening of multiple RyR channels. Mammalian muscles instead lose postnatally the ability to produce sparks, and they also lose RyR3, an isoform abundant in spark-producing skeletal muscles. What does it take for cells to respond to membrane depolarization with Ca(2+) sparks? To answer this question we made skeletal muscles of adult mice expressing exogenous RyR3, demonstrated as immunoreactivity at triad junctions. These muscles showed abundant sparks upon depolarization. Sparks produced thusly were found to amplify the response to depolarization in a manner characteristic of Ca(2+)-induced Ca(2+) release processes. The amplification was particularly effective in responses to brief depolarizations, as in action potentials. We also induced expression of exogenous RyR1 or yellow fluorescent protein-tagged RyR1 in muscles of adult mice. In these, tag fluorescence was present at triad junctions. RyR1-transfected muscle lacked voltage-operated sparks. Therefore, the voltage-operated sparks phenotype is specific to the RyR3 isoform. Because RyR3 does not contact voltage sensors, their opening was probably activated by Ca(2+), secondarily to Ca(2+) release through junctional RyR1. Physiologically voltage-controlled Ca(2+) sparks thus require a voltage sensor, a master junctional RyR1 channel that provides trigger Ca(2+), and a slave parajunctional RyR3 cohort.     

Expression and regulation of ryanodine receptor/calcium release channels

Intracellular calcium release channels on the endoplasmic or sarcoplasmic reticula (ryanodine receptors, RyR, and inositol 1,4,5-trisphosphate receptors, IP(3)R) comprise a unique family of molecules that are structurally and functionally distinct from all other known ion channels. These channels play crucial roles in many cellular signaling pathways including excitation-contraction coupling, oocyte fertilization, hormone secretion, neurotransmitter release, and T lymphocyte activation. Three forms of RyR have been identified: RyR1 expressed predominantly in skeletal muscle, RyR2 in cardiac muscle, and RyR3 in the brain. The tetrameric structures of RyR1 and RyR2 are stabilized by a channel-associated protein, FKBP12. The immunosuppressant drugs FK506 and rapamycin inhibit the prolyl isomerase activity of FKBP12 and could cause cardiac dysfunction by inducing a Ca(2+) leak from the sarcoplasmic reticulum. RyR2 is downregulated and IP(3)R is upregulated during severe end-stage heart failure secondary to dilated cardiomyopathies in humans, suggesting that these channels may contribute to abnormalities in Ca(2+) homeostasis.     

Dyssynchronous (non-uniform) Ca2+ release in myocytes from streptozotocin-induced diabetic rats

Using biochemical/pharmacological approaches, we previously showed that type 2 ryanodine receptors (RyR2) become dysfunctional in hearts of streptozotocin-induced type 1 diabetic rats. However, the functional consequence of this observation remains incompletely understood. Here we use laser confocal microscopy to investigate whether RyR2 dysfunction during diabetes alters evoked and spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR). After 7-8 weeks of diabetes, steady-state levels of RyR2 remain unchanged in hearts of male Sprague-Dawley rats, but the number of functional receptors decreased by >37%. Interestingly, residual functional RyR2 from diabetic rat hearts exhibited increased sensitivity to Ca(2+) activation (EC(50activation) decreased from 80 microM to 40 microM, peak Ca(2+) activation decreased from 425 microM to 160 microM). When field stimulated, intracellular Ca(2+) release in diabetic ventricular myocytes was dyssynchronous (non-uniform) and this was independent of L-type Ca(2+) currents. Time to peak Ca(2+) increased 3.7-fold. Diabetic myocytes also exhibited diastolic Ca(2+) release and 2-fold higher frequency of spontaneous Ca(2+) sparks, albeit at a lower amplitude. The amplitude of caffeine-releasable Ca(2+) was also lower in diabetic myocytes. RyR2 from diabetic rat hearts exhibited increased phosphorylation at Ser2809 and contained reduced levels of FKBP12.6 (calstablin2). Collectively, these data suggest that RyR2 becomes leaky during diabetes and this defect may be responsible to the reduced SR Ca(2+) load. Diastolic Ca(2+) release could also serve as a substrate for delayed after-depolarizations, contributing to the increased incidence of arrhythmias and sudden cardiac death in type 1 diabetes.     

Type 1 ryanodine receptor knock-in mutation causing central core disease of skeletal muscle also displays a neuronal phenotype

The type 1 ryanodine receptor (RyR1) is expressed widely in the brain, with high levels in the cerebellum, hippocampus, and hypothalamus. We have shown that L-type Ca(2+) channels in terminals of hypothalamic magnocellular neurons are coupled to RyRs, as they are in skeletal muscle, allowing voltage-induced Ca(2+) release (VICaR) from internal Ca(2+) stores without Ca(2+) influx. Here we demonstrate that RyR1 plays a role in VICaR in nerve terminals. Furthermore, in heterozygotes from the Ryr1(I4895T/WT) (IT/+) mouse line, carrying a knock-in mutation corresponding to one that causes a severe form of human central core disease, VICaR is absent, demonstrating that type 1 RyR mediates VICaR and that these mice have a neuronal phenotype. The absence of VICaR was shown in two ways: first, depolarization in the absence of Ca(2+) influx elicited Ca(2+)syntillas (scintilla, spark, in a nerve terminal, a SYNaptic structure) in WT, but not in mutant terminals; second, in the presence of extracellular Ca(2+), IT/+ terminals showed a twofold decrease in global Ca(2+) transients, with no change in plasmalemmal Ca(2+) current. From these studies we draw two conclusions: (i) RyR1 plays a role in VICaR in hypothalamic nerve terminals; and (ii) a neuronal alteration accompanies the myopathy in IT/+ mice, and, possibly in humans carrying the corresponding RyR1 mutation.     

Immunogold-labeled L-type calcium channels are clustered in the surface plasma membrane overlying junctional sarcoplasmic reticulum in guinea-pig myocytes-implications for excitation-contraction coupling in cardiac muscle

Ca(2+) release through ryanodine receptors, located in the membrane of the junctional sarcoplasmic reticulum (SR), initiates contraction of cardiac muscle. Ca(2+)influx through plasma membrane L-type Ca(2+)channels is thought to be an important trigger for opening ryanodine receptors ("Ca(2+)-induced Ca(2+)-release"). Optimal transmission of the transmembrane Ca(2+)influx signal to SR release is predicted to involve spatial juxtaposition of L-type Ca(2+)channels to the ryanodine receptors of the junctional SR. Although such spatial coupling has often been implicitly assumed, and data from immunofluorescence microscopy are consistent with its existence, the definitive demonstration of such a structural organization in mammalian tissue is lacking at the electron-microscopic level. To determine the spatial distribution of plasma membrane L-type Ca(2+)channels and their location in relation to underlying junctional SR, we applied two high-resolution immunogold-labeling techniques, label-fracture and cryothin-sectioning, combined with quantitative analysis, to guinea-pig ventricular myocytes. Label-fracture enabled visualization of colloidal gold-labeled L-type Ca(2+)channels in planar freeze-fracture electron-microscopic views of the plasma membrane. Mathematical analysis of the gold label distribution (by nearest-neighbor distance distribution and the radial distribution function) demonstrated genuine clustering of the labeled channels. Gold-labeled cryosections showed that labeled L-type Ca(2+)channels quantitatively predominated in domains of the plasma membrane overlying junctional SR. These findings provide an ultrastructural basis for functional coupling between L-type Ca(2+)channels and junctional SR and for excitation-contraction coupling in guinea-pig cardiac muscle.     

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca(2+) release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200-500 microM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, P(o) < 0.005) and brief (<250 micros) gating events and insensitivity to Ca(2+). Addition of ryanodine restores Ca(2+)-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.     

Peptide toxins as probes of ryanodine receptor structure and function

Toxins from scorpion venom are emerging as useful ligands for structure/function studies of ryanodine receptors (RyR), the sarcoplasmic reticulum Ca(2+) release channels that elevate intracellular Ca(2+) to elicit contraction of cardiac and skeletal muscle. Imperatoxin A (IpTx(a)), a 3.7 kDa peptide from the African scorpion P. imperator, is an agonist of RyRs which, similar to the alkaloid ryanodine, binds with high affinity to the RyR protein and induces the appearance of a long-lived subconductance state. Imperatoxin I (IpTx(i)), a 15 kDa heterodimeric protein from the same venom that displays phospholipase A(2) activity, inhibits RyRs without a physical interaction with the channel protein, by releasing free fatty acids into the incubation medium. IpTx(a) and IpTx(i) are the first of a group of peptide probes of RyRs with diverse mechanism of action which overcome some of the undesirable characteristics of ryanodine.     

Transmission of information from cardiac dihydropyridine receptor to ryanodine receptor: evidence from BayK 8644 effects on resting Ca(2+) sparks

Coupling between L-type Ca(2+) channels (dihydropyridine receptors, DHPRs) and ryanodine receptors (RyRs) plays a pivotal role in excitation-contraction (E-C) coupling in cardiac myocytes, and Ca(2+) influx is generally accepted as the trigger of sarcoplasmic reticulum (SR) Ca(2+) release. The L-type Ca(2+) channel agonist BayK 8644 (BayK) has also been reported to alter RyR gating via a functional linkage between DHPR and RyR, independent of Ca(2+) influx. Here, the effect of rapid BayK application on resting RyR gating in intact ferret ventricular myocytes was measured as Ca(2+) spark frequency (CaSpF) by confocal microscopy and fluo 3. BayK increased resting CaSpF by 401+/-15% within 10 seconds in Ca(2+)-free solution, and depolarization had no additional effect. The effect of BayK on CaSpF was dose-dependent, but even 50 nmol/L BayK induced a rapid 245+/-12% increase in CaSpF. Nifedipine (5 micromol/L) had no effect by itself on CaSpF, but it abolished the BayK effect (presumably by competitive inhibition at the DHPR). The nondihydropyridine Ca(2+) channel agonist FPL-64176 (1 micromol/L) did not alter CaSpF (despite rapid and potent enhancement of Ca(2+) current, I(Ca)). In striking contrast to the very rapid and depolarization-independent effect of BayK on CaSpF, BayK increased I(Ca) only slowly (tau=18 seconds), and the effect was greatly accelerated by depolarization. We conclude that in ferret ventricular myocytes, BayK effects on I(Ca) and CaSpF both require drug binding to the DHPR, but postreceptor pathways may diverge in transmission to the gating of the L-type Ca(2+) channel and RyR.     

Characterization of the sarcoplasmic reticulum k(+) and Ca(2+)-release channel-ryanodine receptor-in human atrial cells

Since the role of sarcoplasmic reticulum (SR) in the E-C coupling of mammalian atrial cells has long been a subject of debate, biochemical, electrophysiological and immunological assays were performed in order to define and compare the properties of the Ca(2+)-release channel-ryanodine receptor (RyR)-from atrial and ventricular tissues. Cardiac SR preparations from human, canine and ovine tissues were compared using [(3)H]ryanodine binding, channel reconstitution into planar lipid bilayers and Western blot analysis involving RyR antibodies. [(3)H]ryanodine binding assays revealed a K(d)value of; 2.5 n M for all investigated cardiac tissues. Bound [(3)H]ryanodine was Ca(2+)-dependent with similar EC(50)values of 0.43, 0.49 and 0.79 microM for human atrium, canine ventricle and ovine atrium, respectively. However the density of binding sites was 4.5 times lower in atrial than in ventricular tissues. Beyond the presence of selective K(+)channels (gamma=188 pS) recorded in the SR enriched fraction of human atrium, the activity of a large conducting (gamma=671 pS) cationic channel was also observed. The latter displayed typical characteristics of Ca(2+)-release channels which were activated by 10 microM free [Ca(2+)] and 2 m M ATP. Western blot analysis revealed the presence of the RyR2 isoform in atrial and ventricular samples whereas no immunoreactivity was detected with specific RyR1 and RyR3 antibodies. Our results, obtained at the molecular level, are consistent with the presence of functional SR in human atrial cells. The human atrial Ca(2+)-release channel displays binding and regulating properties typical of the RyR2 isoform.