Sarcoplasmic Reticulum Calcium Release Channel Research Articles

Effects of 50 Hz sinusoidal magnetic field on Ca2+ release channel ryanodine receptor of sarcoplasmic reticulum vesicles

To investigate the effects of sinusoidal magnetic field on isolated sarcoplasmic reticulum (SR) calcium release channel (RyR1) function. With the Ca2+ dynamic spectrum and isotope labeled methods, the Ca2+ release and [(3)H]-Ryanodine binding, the initial rates of NADH oxidation and the production of superoxide of SR exposed to 50 Hz sinusoidal magnetic field (MF) were investigated respectively. 0.4 mT, 50 Hz sinusoidal MF exposure for 30 min increased SR Ca2+ release initial rate about 35% from (10.82 +/- 0.89) pmol.mg(-1) pro.s(-1) to (14.69 +/- 1.21) pmol.mg(-1) pro.s(-1); and the [(3)H]-Ryanodine binding by about 15% from (2.13 +/- 0.05) pmol/mg pro to (2.45 +/- 0.07) pmol/mg pro, which regulated by 1 mmol/L NADH with 1 mmol/L NAD+. Meanwhile MF upregulated the rate of NADH oxidation by about 22% from (0.88 +/- 0.11) x 10(-4) FI/s to (1.07 +/- 0.13) x 10(-4) FI/s and upregulated the production of superoxide by about 32% from (0.99 +/- 0.09) x 10(-5) FI/s to (1.31 +/- 0.06) x 10(-5) FI/s. 0.4 mT sinusoidal MF increases the activity of RyR1 within the low redox potential environment, and promotes NADH oxidase activity and superoxide production.

Loss of T-tubules and other changes to surface topography in ventricular myocytes from failing human and rat heart

T-tubular invaginations of the sarcolemma of ventricular cardiomyocytes contain junctional structures functionally coupling L-type calcium channels to the sarcoplasmic reticulum calcium-release channels (the ryanodine receptors), and therefore their configuration controls the gain of calcium-induced calcium release (CICR). Studies primarily in rodent myocardium have shown the importance of T-tubular structures for calcium transient kinetics and have linked T-tubule disruption to delayed CICR. However, there is disagreement as to the nature of T-tubule changes in human heart failure. We studied isolated ventricular myocytes from patients with ischemic heart disease, idiopathic dilated cardiomyopathy, and hypertrophic obstructive cardiomyopathy and determined T-tubule structure with either the fluorescent membrane dye di-8-ANNEPs or the scanning ion conductance microscope (SICM). The SICM uses a scanning pipette to produce a topographic representation of the surface of the live cell by a non-optical method. We have also compared ventricular myocytes from a rat model of chronic heart failure after myocardial infarction. T-tubule loss, shown by both ANNEPs staining and SICM imaging, was pronounced in human myocytes from all etiologies of disease. SICM imaging showed additional changes in surface structure, with flattening and loss of Z-groove definition common to all etiologies. Rat myocytes from the chronic heart failure model also showed both T-tubule and Z-groove loss, as well as increased spark frequency and greater spark amplitude. This study confirms the loss of T-tubules as part of the phenotypic change in the failing human myocyte, but it also shows that this is part of a wider spectrum of alterations in surface morphology.

Calcium Handling in Human Embryonic Stem Cell-Derived Cardiomyocytes

The objective of the current study was to characterize calcium handling in developing human embryonic stem cell-derived cardiomyocytes (hESC-CMs). To this end, real-time polymerase chain reaction (PCR), immunocytochemistry, whole-cell voltage-clamp, and simultaneous patch-clamp/laser scanning confocal calcium imaging and surface membrane labeling with di-8-aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC-CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor-2, and inositol-1,4,5-trisphosphate (IP3) receptors. Store calcium function was manifested as action-potential-induced calcium transients. Time-to-target plots showed that these action-potential-initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC-CMs also exhibited local calcium events ("sparks") that were localized to the surface membrane. The presence of caffeine-sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early-stage (with the initiation of beating), intermediate-stage (10 days post-beating [dpb]), and late-stage (30-40 dpb) hESC-CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3-releasable calcium pool was also demonstrated in the hESC-CMs in experiments using caged-IP3 photolysis and antagonist application (2 microM 2-Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early-stage hESC-CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC-CMs both for developmental studies and for the development of future myocardial cell replacement strategies. Disclosure of potential conflicts of interest is found at the end of this article.

Congenital muscle disorders with cores: the ryanodine receptor calcium channel paradigm

Dysregulation of calcium signals because of defects of the skeletal muscle sarcoplasmic reticulum calcium release channel (ryanodine receptor; RyR1) is causative of several congenital muscle disorders including malignant hyperthermia (MH; MIM #145600), central core disease (CCD; MIM #11700), specific forms of multi-minicore disease (MmD; MIM # 255320) and centronuclear myopathy (CNM). Experimental data have shown that RYR1 mutations result mainly in four types of channel defects: one class of RYR1 mutations (MH) cause the channels to become hypersensitive to activation by electrical and pharmacological stimuli. The second class of RYR1 mutations (CCD) result in leaky channels leading to depletion of Ca(2+) from SR stores. A third class of RYR1 mutations linked to CCD causes excitation-contraction uncoupling, whereby activation of the voltage sensor Cav1.1 is unable to release calcium from the SR. The fourth class of mutations are unveiled by wild type allele silencing, and cause a decrease of mutant RyR1 channels expression on SR membranes. In this review, we discuss the classes of RYR1 mutations which have been associated with CCD, MmD and related neuromuscular phenotypes.

An increase of late sodium current induces delayed afterdepolarizations and sustained triggered activity in atrial myocytes

This study determined the role of a slowly inactivating component of sodium current (I(Na)), late I(Na), to induce delayed afterdepolarizations (DADs) and triggered activity. We hypothesized that an increase of late I(Na) may induce not only early afterdepolarizations (EADs), but also intracellular calcium overload and DADs. Guinea pig atrial myocytes were studied using the whole cell patch-clamp technique. Anemone toxin II (ATX-II) (5-10 nmol/l) was used to enhance late I(Na). Ranolazine (10 micromol/l) and TTX (2 micromol/l) were applied to block ATX-II-induced late I(Na). ATX-II prolonged action potential duration and induced EADs. In the continuous presence of ATX-II, following the appearance of EADs, both DADs and sustained triggered activity occurred. Triggered activity was abolished and DADs were reduced by either ranolazine or TTX. Consistent with induction of DADs, ATX-II induced the transient inward current (I(TI)). The amplitude of I(TI) was significantly reduced by ranolazine. ATX-II induced only EADs, but no DADs, in the presence of the sodium-calcium exchange inhibitor KB-R7943 or the sarcoplasmic reticulum calcium release channel inhibitor ryanodine, or when the calcium chelator EGTA or BAPTA was included in the pipette solution. In conclusion, an increase of late I(Na), in addition to inducing EADs, can cause cellular calcium overload and induce DADs and sustained triggered activity in atrial myocytes. The data reveal that an increase of late I(Na) is a novel mechanism for initiation of atrial arrhythmic activity.

Malignant hyperthermia susceptibility diagnosed with a family-specific ryanodine receptor gene type 1 mutation

Malignant hyperthermia (MH) is an autosomal dominant disorder of skeletal muscle calcium regulation, and the rate of calcium-induced calcium release (CICR), determined by using skinned fibers of skeletal muscle, has been employed as a diagnostic test for MH susceptibility in Japan. The ryanodine receptor (RYR1), encoding the major calcium-release channel in skeletal muscle sarcoplasmic reticulum, has been shown to be mutated in a number of MH pedigrees. We experienced the detection of accelerated CICR and/or an RYR1 mutation in a patient with an MH episode and his family. Accelerated CICR and an RYR1 mutation (c.14512C>G, p.L4838V) were found in the patient and his father. The MH-causative mutation (c.14512C>G, p.L4838V) was also found in his brother and his son (resulting in the diagnosis of MH without the CICR test), but the mutation was not found in his mother or two daughters. With the detection of the family-specific mutation in other family members, the diagnosis of MH was made without the invasive CICR test.

Sarcoplasmic reticulum calcium leak and cardiac arrhythmias

Ventricular arrhythmias deteriorating into sudden cardiac death are a major cause of mortality worldwide. The recent linkage of a genetic form of cardiac arrhythmia to mutations in the gene encoding RyR2 (ryanodine receptor 2) has uncovered an important role of this SR (sarcoplasmic reticulum) calcium release channel in triggering arrhythmias. Mutant RyR2 channels give rise to spontaneous release of calcium (Ca(2+)) from the SR during diastole, which enhances the probability of ventricular arrhythmias. Several molecular mechanisms have been proposed to explain the gain-of-function phenotype observed in mutant RyR2 channels. Despite considerable differences between the models discussed in the present review, each predicts spontaneous diastolic Ca(2+) leak from the SR due to incomplete closure of the RyR2 channel. Enhanced SR Ca(2+) leak is also observed in common structural diseases of the heart, such as heart failure. In heart failure, defective channel regulation in the absence of inherited mutations may also increase SR Ca(2+) leak and initiate cardiac arrhythmias. Therefore inhibition of diastolic Ca(2+) leak through SR Ca(2+) release channels has emerged as a new and promising therapeutic target for cardiac arrhythmias.

Malignant hyperthemia

Malignant hyperthermia (MH) was first recognised as a potentially fatal pharmacogenetic disorder nearly 50 years ago. The abnormality has been localised to skeletal muscle, specifically the mechanisms regulating myoplasmic calcium ion homeostasis. The gene principally implicated is RYR1, which encodes the skeletal muscle sarcoplasmic reticulum calcium release channel, but the genetics of MH are relatively complex, with more than 100 mutations described in RYR1 and with a significant minority of cases not involving this gene. This review describes how the pathophysiology of MH relates to the clinical features, which follow a logical pattern. Reasons for the reaction reaching an untreatable stage are proposed, emphasising the importance of early diagnosis and aggressive treatment using the latest guidelines. Early intervention in suspected cases, and the lack of specificity of any single clinical feature for MH, necessitates laboratory confirmation of the diagnosis using muscle biopsy specimens. These relatively invasive tests can also be used to screen family members of known cases, although the limited introduction of DNA testing has obviated the need for some individuals to have a muscle biopsy. Patients at risk of developing MH can receive general anaesthesia and guidelines for this are described.

Central core disease

Central core disease (CCD) is an inherited neuromuscular disorder characterised by central cores on muscle biopsy and clinical features of a congenital myopathy. Prevalence is unknown but the condition is probably more common than other congenital myopathies. CCD typically presents in infancy with hypotonia and motor developmental delay and is characterized by predominantly proximal weakness pronounced in the hip girdle; orthopaedic complications are common and malignant hyperthermia susceptibility (MHS) is a frequent complication. CCD and MHS are allelic conditions both due to (predominantly dominant) mutations in the skeletal muscle ryanodine receptor (RYR1) gene, encoding the principal skeletal muscle sarcoplasmic reticulum calcium release channel (RyR1). Altered excitability and/or changes in calcium homeostasis within muscle cells due to mutation-induced conformational changes of the RyR protein are considered the main pathogenetic mechanism(s). The diagnosis of CCD is based on the presence of suggestive clinical features and central cores on muscle biopsy; muscle MRI may show a characteristic pattern of selective muscle involvement and aid the diagnosis in cases with equivocal histopathological findings. Mutational analysis of the RYR1 gene may provide genetic confirmation of the diagnosis. Management is mainly supportive and has to anticipate susceptibility to potentially life-threatening reactions to general anaesthesia. Further evaluation of the underlying molecular mechanisms may provide the basis for future rational pharmacological treatment. In the majority of patients, weakness is static or only slowly progressive, with a favourable long-term outcome.

Lipopolysaccharide Activates Calcineurin in Ventricular Myocytes

We investigated whether lipopolysaccharide (LPS), a proximate cause of inflammation, activates calcineurin in cardiac myocytes and if calcineurin regulates apoptosis in this setting. Calcineurin regulates myocardial growth and hypertrophy, but its role in inflammation is unknown. Calcineurin has proapoptotic or antiapoptotic effects depending on the stimuli. Calcineurin activity was measured in left ventricular myocytes from adult Sprague Dawley rats. Cardiac apoptosis was measured by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling staining and caspase-3 activity after in vitro and in vivo exposure to LPS. Lipopolysaccharide increased calcineurin activity in myocytes over 1 to 24 h (t 1/2 = 4.8 h) with an EC(50) of 0.80 ng/ml LPS (p < 0.05, n = 4). The LPS (10 ng/ml) effects were mimicked by angiotensin II (Ang II) (100 nmol/l); both increased calcineurin activity and induced apoptosis without additive effects (p < 0.05, n = 5 to 9). Lipopolysaccharide and/or Ang II effects were prevented by 1 h pre-treatment with an Ang II type 1 receptor blocker (losartan, 1 micromol/l), calcineurin inhibitor (cyclosporin A, 0.5 micromol/l), calcium chelator (1,2-Bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester, 0.1 micromol/l), or by inhibiting sarcoplasmic reticulum (SR) calcium (Ca)-ATPase (thapsigargin, 1 micromol/l) or SR calcium release channel (ryanodine, 1 micromol/l). Left ventricular apoptosis increased from 4 to 24 h after LPS (1 mg/kg intravenously) in vivo, but not in rats pre-treated with cyclosporin A (20 mg/kg/day subcutaneously) for 3 days (p < 0.05, n = 5). In cardiac myocytes, LPS activates calcineurin in association with apoptosis by Ang II and SR calcium-dependent mechanisms. This expands the paradigm for cardiac calcineurin to be activated by low levels of LPS in inflammation and chronic conditions (e.g., infections, smoking, and heart failure).

SERCA2a, Phospholamban, Sarcolipin, and Ryanodine Receptors Gene Expression in Children with Congenital Heart Defects

In animal models of conotruncal heart defects, an abnormal calcium sensitivity of the contractile apparatus and a depressed L-type calcium current have been described. Sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) is a membrane protein that catalyzes the ATP-dependent transport of Ca(2+) from the cytosol to the SR. The activity of SERCA is inhibited by phospholamban (PLN) and sarcolipin (SLN), and all these proteins participate in maintaining the normal intracellular calcium handling. Ryanodine receptors (RyRs) are the major SR calcium-release channels required for excitation-contraction coupling in skeletal and cardiac muscle. Our objective was to evaluate SERCA2a (i.e., the SERCA cardiac isoform), PLN, SLN, and RyR2 (i.e., the RyR isoform enriched in the heart) gene expression in myocardial tissue of patients affected by tetralogy of Fallot (TOF), a conotruncal heart defect. The gene expression of target genes was assessed semiquantitatively by RT-PCR using the calsequestrin (CASQ, a housekeeping gene) RNA as internal standard in the atrial myocardium of 23 pediatric patients undergoing surgical correction of TOF, in 10 age-matched patients with ventricular septal defect (VSD) and in 13 age-matched children with atrial septal defect (ASD). We observed a significantly lower expression of PLN and SLN in TOF patients, while there was no difference between the expression of SERCA2a and RyR2 in TOF and VSD. These data suggest a complex mechanism aimed to enhance the intracellular Ca(2+) reserve in children affected by tetralogy of Fallot.

Exclusion of linkage of the RYR1, CACNA1S, and ATP2A1 genes to recurrent exertional rhabdomyolysis in Thoroughbreds

To determine whether there was genetic linkage between the recurrent exertional rhabdomyolysis (RER) trait in Thoroughbred horse pedigrees and DNA markers in genes (the sarcoplasmic reticulum calcium release channel [RYR1] gene, the sarcoplasmic reticulum calcium ATPase [ATP2A1] gene, and the transverse tubule dihydropyridine receptor-voltage sensor [CACNA1S] gene) that are important in myoplasmic calcium regulation. 34 horses in the University of Minnesota RER resource herd and 62 Thoroughbreds from 3 families of Thoroughbreds outside of the university in which RER-affected status was assigned after 2 or more episodes of ER had been observed. Microsatellite DNA markers from the RYR1, ATP2A1, and CACNA1S gene loci on equine chromosomes 10, 13, and 30 were identified. Genotypes were obtained for all horses in the 4 families affected by RER, and data were used to test for linkage of these 3 loci to the RER phenotype. Analysis of the RYR1, CACNA1S, and ATP2A1 microsatellites excluded a link between those markers and the RER trait. It is likely that the heritable alterations in muscle contractility that are characteristic of RER are caused by a gene that is not yet known to cause related muscle disease in other species.

Cardiomyopathies and sudden cardiac death caused by RyR2 mutations: Are the channels the beginning and the end?☆

See article by Milting et al. [3] (pages 496–505) in this issue. A basic goal of medicine is to cure diseases. Acquired diseases may sometimes be prevented by various methods of exercising, by the “healthy way” of living. Inherited diseases are different; their effective treatment (provided that there is any) requires the recognition of the disease in a sufficiently early stage of life. One – and probably the most promising – way to do so is to establish the link between disorders and genetic alterations. This can be achieved by mapping the mutations corresponding to a given disease, and in the past decade a large number of mutations affecting molecules involved in calcium handling of skeletal and cardiac muscles, including the ryanodine receptor, have been identified [1]. Regarding the cardiac sarcoplasmic reticulum (SR) calcium release channel (RyR2), one of the first published mutations was presented in the pioneering work of Laitinen et al. [2]. In that study, three different RyR2 mutations (P2328S, Q4201R, and V4653F) were associated with familiar polymorphic ventricular tachycardia (FPVT), and each of them was shown to fully co-segregate with the characteristic arrhythmic phenotype. The importance of this kind of knowledge cannot be overestimated, since it makes it possible to recognize a hidden inherited cardiac disorder that exhibits no visible sign of myocardial damage while threatening with close to 30% mortality due to sudden cardiac death (SCD) upon heavy exercise. The article by Milting et al. in this issue of Cardiovascular Research reports two new single nucleotide polymorphisms (G1885E and G1886S) … * Corresponding author. Tel.: +36 52 416634; fax: +36 52 432289. Email address: nanasi{at}phys.dote.hu

Sarcoplasmic reticulum: The dynamic calcium governor of muscle

The sarcoplasmic reticulum (SR) provides feedback control required to balance the processes of calcium storage, release, and reuptake in skeletal muscle. This balance is achieved through the concerted action of three major classes of SR calcium-regulatory proteins: (1) luminal calcium-binding proteins (calsequestrin, histidine-rich calcium-binding protein, junctate, and sarcalumenin) for calcium storage; (2) SR calcium release channels (type 1 ryanodine receptor or RyR1 and IP3 receptors) for calcium release; and (3) sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) pumps for calcium reuptake. Proper calcium storage, release, and reuptake are essential for normal skeletal muscle function. We review SR structure and function during normal skeletal muscle activity, the proteins that orchestrate calcium storage, release, and reuptake, and how phenotypically distinct muscle diseases (e.g., malignant hyperthermia, central core disease, and Brody disease) can result from subtle alterations in the activity of several key components of the SR calcium-regulatory machinery.

Sarcoplasmic Reticulum Calcium Release Channels in Ventricles of Older Adult Hamsters

Whether the density of sarcoplasmic reticulum (SR) calcium release channels/ryanodine receptors in the heart declines with age is not clear. We investigated age-related changes in the density of [3H]-ryanodine receptors in crude ventricular homogenates, which contained all ligand binding sites in heart and in isolated junctional SR membranes. Experiments utilized young (120 days) and older adult (300 days) hamsters. [3H]-ryanodine binding site density did not change with age in crude homogenate preparations, although total heart protein concentration increased significantly with age. In contrast, the density of [3H]-ryanodine binding sites decreased markedly in heavy SR membranes purified from older hearts. These results show that demonstration of age-related changes in cardiac ryanodine receptor density depends upon the preparation used. Furthermore, the increase in total ventricular protein with age suggests that normalization of data by membrane protein should be used with caution in studies of aging heart.

Alterations of myocardial ultrastructure and gene expression of calcium handling proteins in diabetic rat heart

To investigate the ultrastructure of myocardium and gene expression of calcium handling proteins in diabetic rat heart. Diabetes was induced in male Sprague-Dawley rats by a single injection of alloxanm (40 mg/kg ) and the rats in control group were injected with normal saline. At the end of 2, 4, 6 weeks after the induction of diabetes, the animals were sacrificed. The expression of calcium handling proteins was detected by reverse transcription-polymerase chain reaction (RT-PCR) and actin mRNA was used as internal standard. Heart tissue at the apex was obtained for light and electron microscope study. At the end of 4 and 6 weeks, cardiosomatic ratio of diabetic rats was higher than that of control. Electron microscopy revealed a spectrum of subcellular remodeling in myocardium which was characterized by damaged myofibrils and mitochondria, dilated and swollen sarcoplasmic reticulum. Expression of phospholamban mRNAs was significantly increased, but 1,4,5-trisphosphate inositol receptor type 2, ryanodine receptor type 2 mRNAs were significantly decreased compared with those in the age-matched control rats. In contrast, the expression of sarco/endoplasmic reticulum Ca(2+)-ATPase mRNAs was not affected. In diabetic rat heart, gene expression of calcium handling proteins was characterized by up-regulation of phospholamban and down-regulation of sarcoplasmic reticulum calcium release channel while electron microscopic analysis of myocardium revealed a spectrum of subcellular remodeling.

The Novel Skeletal Muscle Sarcoplasmic Reticulum JP-45 Protein: MOLECULAR CLONING, TISSUE DISTRIBUTION, DEVELOPMENTAL EXPRESSION, AND INTERACTION WITH α1.1 SUBUNIT OF THE VOLTAGE-GATED CALCIUM CHANNEL

JP-45 is a novel integral protein constituent of the skeletal muscle sarcoplasmic reticulum junctional face membrane. We identified its primary structure from a cDNA clone isolated from a mouse skeletal muscle cDNA library. Mouse skeletal muscle JP-45 displays over 86 and 50% identity with two hypothetical NCBI data base protein sequences from mouse tongue and human muscle, respectively. JP-45 is predicted to have a cytoplasmic domain, a single transmembrane segment followed by an intralumenal domain enriched in positively charged amino acids. Northern and Western blot analyses reveal that the protein is mainly expressed in skeletal muscle. The mRNA encoding JP-45 appears in 17-day-old mouse embryos; expression of the protein peaks during the second month of postnatal development and then decreases approximately 3-fold during aging. Double immunofluorescence of adult skeletal muscle fibers demonstrates that JP-45 co-localizes with the sarcoplasmic reticulum calcium release channel. Co-immunoprecipitation experiments with a monoclonal antibody against JP-45 show that JP-45 interacts with the alpha1.1 subunit voltage-gated calcium channel and calsequestrin. These results are consistent with the localization of JP-45 in the junctional sarcoplasmic reticulum and with its involvement in the molecular mechanism underlying skeletal muscle excitation-contraction coupling.

Clinical and functional effects of a deletion in a COOH-terminal lumenal loop of the skeletal muscle ryanodine receptor.

We have identified a patient affected by a relatively severe form of central core disease (CCD), carrying a heterozygous deletion (amino acids 4863-4869) in the pore-forming region of the sarcoplasmic reticulum calcium release channel. The functional effect of this deletion was investigated (i) in lymphoblastoid cells from the affected patient and her mother, who was also found to harbour the mutation and (ii) in HEK293 cells expressing recombinant mutant channels. Lymphoblastoid cells carrying the RYR1 deletion exhibit an 'unprompted' calcium release from intracellular stores, resulting in significantly smaller thapsigargin-sensitive intracellular Ca(2+) stores, compared with lymphoblastoid cells from control individuals. Blocking the RYR1 with dantrolene restored the intracellular calcium stores to levels similar to those found in control cells. Single channel and [(3)H]ryanodine binding measurements of heterologously expressed mutant channels revealed a reduced ion conductance and loss of ryanodine binding and regulation by Ca(2+). Heterologous expression of recombinant RYR1 peptides and analysis of their membrane topology demonstrate that the deleted amino acids are localized in the lumenal loop connecting membrane-spanning segments M8 and M10. We provide evidence that a deletion in the lumenal loop of RYR1 alters channel function and causes CCD.

Location of Ryanodine and Dihydropyridine Receptors in Frog Myocardium

Frog myocardium depends almost entirely on calcium entry from extracellular spaces for its beat-to-beat activation. Atrial myocardium additionally shows internal calcium release under certain conditions, but internal release in the ventricle is absent or very low. We have examined the content and distribution of the sarcoplasmic reticulum (SR) calcium release channels (ryanodine receptors, RyRs) and the surface membrane calcium channels (dihydropyridine receptors, DHPRs) in myocardium from the two atria and the ventricle of the frog heart using binding of radioactive ryanodine, immunolabeling of RyR and DHPR, and thin section and freeze-fracture electron microscopy. In cells from both types of chambers, the SR forms peripheral couplings and in both chambers peripheral couplings colocalize with clusters of DHPRs. However, although a low level of high affinity binding of ryanodine is detectable and RyRs are present in peripheral couplings of the atrium, the ventricle shows essentially no ryanodine binding and RyRs are not detectable either by electron microscopy or immunolabeling. The results are consistent with the lack of internal calcium release in the ventricle, and raise questions regarding the significance of DHPR at peripheral couplings in the absence of RyR. Interestingly, the free SR membrane in both heart chambers shows a low but equal density of intramembrane particles representing the Ca 2+ ATPase.

Caffeine analogs: effects on ryanodine-sensitive calcium-release channels and GABAA receptors.

1. Caffeine at 0.3-10 mM enhanced the binding of [3H]ryanodine to calcium-release channels of rabbit muscle sarcoplasmic reticulum. A variety of other xanthines were as efficacious as caffeine or nearly so, but none appeared markedly more potent. 2. Caffeine at 1 mM markedly inhibited binding of [3H]diazepam to GABAA receptors in rat cerebral cortical membranes. 3. Other xanthines also inhibited binding with certain dimethylpropargylxanthines being nearly fivefold more potent than caffeine. 4. Caffeine at 1 mM stimulated binding of [35S]TBPS to GABAA receptors as did certain other xanthines. 5. The dimethylpropargylxanthines had little effect. 1,3-Dipropyl-8-cyclopentylxanthine at 100 microM had no effect on [3H]diazepam binding, but markedly inhibited [35S]TBPS binding. 6. Structure-activity relationships for xanthines do differ for calcium-release channels and and for different sites on GABAA receptors, but no highly selective lead compounds were identified.