Regulation Of Ryanodine Receptor Research Articles

CYCLIC-AMP INCREASES THE SPEED OF CARBACHOL-INDUCED APICAL TO BASAL CALCIUM WAVES IN PANCREATIC ACINAR CELLS

Intra-acinar zymogen activation is an early and critical feature of acute pancreatitis. High sustained calcium elevations within the pancreatic acinar cell precede pathologic zymogen activation. However, the determinants of the calcium signal are not clear. We have previously shown that cAMP sensitizes the pancreatic acinar cell to zymogen activation. In this study, we examined the role of cAMP in shaping the dynamics of the calcium signal. We hypothesized that cAMP regulates intra-acinar calcium channel opening in response to calcium-evoking agonists. In these experiments, isolated acinar cells were loaded with the calcium dye fluo4 and imaged using a confocal laser scanning microscope. They were stimulated with the calcium-evoking agonist carbachol (1 uM) in the absence or presence of the cAMP analog 8-Br-cAMP (100 uM). We noted that the rise time after carbachol stimulation was reduced by 55% (p < 0.005) in the basal region of the cell and to a lesser degree in the apical region. In addition, the slope of the calcium rise was increased 2.8 and 1.9 fold in the basal and apical regions, respectively, with cAMP pretreatment (p < 0.01). Furthermore, the speed of the apical-to-basolateral calcium wave increased from 8 to 21 um/sec (p < 0.05). Neither the baseline calcium wave speed nor the cAMP-mediated increase was affected by calcium-free media with EGTA (2 mM). However, dantrolene (100 uM), an inhibitor of the intra-cellular calcium channel the ryanodine receptor, abrogated the cAMP-mediated increase in wave speed (p < 0.05). No differences were noted in the peak calcium response in the apical or basal regions. These data suggest that cAMP affects the speed of the apical to basal calcium wave. Further, the effects of cAMP on the acinar cell calcium wave appear to be dependent on ryanodine receptor opening. We conclude that cAMP appears to regulate ryanodine receptor calcium opening in pancreatic acinar cells.

Regulation of Ryanodine Receptors in the Heart

See related article, pages 819–829 In response to exercise or other stresses, catecholamines are released into the circulation and within the heart. Catecholaminergic stimulation of β1-adrenergic receptors (β1ARs) in the heart increases heart rate (chronotropy) and contractility (inotropy), resulting in increased cardiac output during acute stress. Concurrent stimulation of β2-ARs dilates blood vessels, which increase blood flow to exercising muscles, thereby matching increased cardiac output to metabolic demands of the organs. The inotropic mechanisms investigated by Valdivia and colleagues1 in the current issue of Circulation Research are essential for the acute stress-dependent increase of cardiac output. Each of the millions of muscle cells in the heart (cardiomyocytes) contribute to myocardial force development. Cardiomyocyte contraction is controlled by intracellular Ca2+ release through a process called excitation-contraction coupling (ECC) that involves the following steps: (1) an action potential (AP) depolarizes the cell membrane; (2) voltage-dependent plasma membrane L-type calcium channels (Cav1.2) opening results in a whole-cell inward Ca2+ current ( I Ca); (3) I Ca activates cardiac ryanodine receptor (RyR2)/Ca2+ release channels located on the junctional sarcoplasmic reticulum (jSR), a process referred to as Ca2+-induced Ca2+ release (CICR); (4) Ca2+ binds to troponin C (TnC) leading to cross-bridge formation between myosin and actin and contraction of the sarcomere. Cardiomyocyte relaxation is signaled by a return of intracellular [Ca2+] to resting levels attributable to the following major mechanisms: (1) Cav1.2 inactivation; (2) RyR2 inactivation; (3) Ca2+ reuptake into the SR by SERCA2a pumps; and (4) Na+/Ca2+ exchange extrusion of Ca2+ to the extracellular space. Under resting conditions, net SR Ca2+ release contributes approximately 66% of Ca2+ necessary for myofilament activation in large mammals including humans, and approximately 90% in rodents.2 Stimulation …

Ca 2+ Stores Regulate Ryanodine Receptor Ca 2+ Release Channels via Luminal and Cytosolic Ca 2+ Sites

The free [Ca 2+] in endoplasmic/sarcoplasmic reticulum Ca 2+ stores regulates excitability of Ca 2+ release by stimulating the Ca 2+ release channels. Just how the stored Ca 2+ regulates activation of these channels is still disputed. One proposal attributes luminal Ca 2+-activation to luminal facing regulatory sites, whereas another envisages Ca 2+ permeation to cytoplasmic sites. This study develops a unified model for luminal Ca 2+ activation for single cardiac ryanodine receptors (RyR 2) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR 2 involves three modes of action associated with Ca 2+ sensors in different parts of the molecule; a luminal activation site ( L-site, 60 μM affinity), a cytoplasmic activation site ( A-site, 0.9 μM affinity), and a novel cytoplasmic inactivation site ( I 2-site, 1.2 μM affinity). RyR activation by luminal Ca 2+ is demonstrated to occur by a multistep process dubbed luminal-triggered Ca 2+ feedthrough. Ca 2+ binding to the L-site initiates brief openings (1 ms duration at 1–10 s −1) allowing luminal Ca 2+ to access the A-site, producing up to 30-fold prolongation of openings. The model explains a broad data set, reconciles previous conflicting observations and provides a foundation for understanding the action of pharmacological agents, RyR-associated proteins, and RyR 2 mutations on a range of Ca 2+-mediated physiological and pathological processes.

Non-Thiol Reagents Regulate Ryanodine Receptor Function by Redox Interactions That Modify Reactive Thiols

The Ca(2+) release channel (CRC) from sarcoplasmic reticulum (SR) is rich in thiol groups, and their oxidation/- reduction by thiol reagents activates/inhibits the CRC. Most channel regulators are not thiol reagents, and the mechanism of their action is illusive. Here the authors show that many channel activators act as electron acceptors, while many channel inhibitors act as electron donors in free radical reactions. The channel activator, caffeine, and the CRC inhibitor, tetracaine, are shown to interact competitively, which suggests that there exists a common site(s) on the CRC, that integrates the donor/acceptor effects of ligands. Moreover, channel activators shift the redox potential of reactive thiols on the ryanodine receptor (RyR) to more negative values and decrease the number of reactive thiols, while channel inhibitors shift the redox potential to more positive values and increase the number of reactive thiols. These observations suggest that the non-thiol channel modulators shift the thiol-disulfide balance within CRC by transiently exchanging electrons with the Ca(2+) release protein.

The Cysteine-rich Secretory Protein Domain of Tpx-1 Is Related to Ion Channel Toxins and Regulates Ryanodine Receptor Ca2+ Signaling

The cysteine-rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract as well as in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionary diverse N-terminal domain and a characteristic cysteine-rich C-terminal domain, which we refer to as the Crisp domain. We presented the NMR solution structure of the Crisp domain of mouse Tpx-1, and we showed that it contains two subdomains, one of which has a similar fold to the ion channel regulators BgK and ShK. Furthermore, we have demonstrated for the first time that the ion channel regulatory activity of Crisp proteins is attributed to the Crisp domain. Specifically, the Tpx-1 Crisp domain inhibited cardiac ryanodine receptor (RyR) 2 with an IC(50) between 0.5 and 1.0 microM and activated the skeletal RyR1 with an AC(50) between 1 and 10 microM when added to the cytoplasmic domain of the receptor. This activity was nonvoltage-dependent and weakly voltage-dependent, respectively. Furthermore, the Tpx-1 Crisp domain activated both RyR forms at negative bilayer potentials and showed no effect at positive bilayer potentials when added to the luminal domain of the receptor. These data show that the Tpx-1 Crisp domain on its own can regulate ion channel activity and provide compelling evidence for a role for Tpx-1 in the regulation of Ca(2+) fluxes observed during sperm capacitation.

Regulation of Cardiac Sarcoplasmic Reticulum Ca Release by Luminal [Ca] and Altered Gating Assessed with a Mathematical Model

Cardiac excitation-contraction coupling is initialized by the release of Ca from the sarcoplasmic reticulum (SR) in response to a sudden increase in local cytosolic [Ca] ([Ca] i) within the junctional cleft. We have tested the hypothesis that functional ryanodine receptor (RyR) regulation plays a major role in the regulation of myocyte Ca. A mathematical model with unique characteristics was used to simulate Ca homeostasis. Specifically, the model was designed to accurately represent the SR [Ca]-dependence of release from a variety of experimentally produced data sets. The simulated data for altered RyR Ca sensitivity demonstrated a regulatory feedback loop that resulted in the same release at lower [Ca] SR. This suggests that the primary role of myocyte RyR regulation may be to decrease SR [Ca] without decreasing the size of the [Ca] i transient. The model results suggest that this action moderates the increased SR [Ca] observed with adrenergic stimulation and may keep the [Ca] SR below the threshold for delayed afterdepolarizations and arrhythmia. However, increased Ca affinity of the RyR increased the probability of delayed afterdepolarizations when heart failure was simulated. We conclude that RyR regulation may play a role in preventing arrhythmias in healthy myocytes but that the same regulation may have the opposite effect in chronic heart failure.

Regulation of Ryanodine Receptors by Calsequestrin: Effect of High Luminal Ca2+ and Phosphorylation

Calsequestrin, the major calcium sequestering protein in the sarcoplasmic reticulum of muscle, forms a quaternary complex with the ryanodine receptor calcium release channel and the intrinsic membrane proteins triadin and junctin. We have investigated the possibility that calsequestrin is a luminal calcium concentration sensor for the ryanodine receptor. We measured the luminal calcium concentration at which calsequestrin dissociates from the ryanodine receptor and the effect of calsequestrin on the response of the ryanodine receptor to changes in luminal calcium. We provide electrophysiological and biochemical evidence that: 1), luminal calcium concentration of >/=4 mM dissociates calsequestrin from junctional face membrane, whereas in the range of 1-3 mM calsequestrin remains attached; 2), the association with calsequestrin inhibits ryanodine receptor activity, but amplifies its response to changes in luminal calcium concentration; and 3), under physiological calcium conditions (1 mM), phosphorylation of calsequestrin does not alter its ability to inhibit native ryanodine receptor activity when the anchoring proteins triadin and junctin are present. These data suggest that the quaternary complex is intact in vivo, and provides further evidence that calsequestrin is involved in the sarcoplasmic reticulum calcium signaling pathway and has a role as a luminal calcium sensor for the ryanodine receptor.

Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains.

Ryanodine receptors (RyR) function as Ca(2+) channels that regulate Ca(2+) release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca(2+)-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca(2+) handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca(2+) phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or "I"-domain) which critically modulated intracellular Ca(2+) handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca(2+) release channels.

Removal of clustered positive charge from dihydropyridine receptor II–III loop peptide augments activation of ryanodine receptors

Peptides based on the skeletal muscle DHPR II–III loop have been shown to regulate ryanodine receptor channel activity. The N-terminal region of this cytoplasmic loop is predicted to adopt an α-helical conformation. We have selected a peptide sequence of 26 residues (Ala 667–Asp 692) as the minimum sequence to emulate the helical propensity of the corresponding protein sequence. The interaction of this control peptide with skeletal and cardiac RyR channels in planar lipid bilayers was then assessed and was found to lack isoform specificity. At low concentrations peptide A 667–D 692 increased RyR open probability, whilst at higher concentrations open probability was reduced. By replacing a region of clustered positive charge with a neutral sequence with the same predisposition to helicity, the inhibitory effect was ablated and activation was enhanced. This novel finding demonstrates that activation does not derive from the presence of positively charged residues adjacent in the primary structure and, although it may be mediated by the alignment of basic residues down one face of an amphipathic helix, not all of these residues are essential.

Homer/Vesl proteins and their roles in CNS neurons.

Since their initial discovery in 1997, Homer/Vesl proteins have become increasingly investigated as putative regulators of receptor and ion-channel function in the central nervous system. Within a relatively brief period, numerous research reports have described manifold effects of Homer proteins, including the modulation of the trafficking of type I metabotropic glutamate receptors (mGluRs), axonal pathfinding, mGluR coupling to calcium and potassium channels, agonist-independent mGluR activity, ryanodine receptor regulation, locomotor activity, and behavioral plasticity. This review summarizes our current knowledge on the induction, expression, and structure of the various forms of Homer proteins, as well as their roles in neuronal function. In addition, we provide an outlook on novel developments with regard to the involvement of Homer-1a in hippocampal synaptic function.

Dysregulated Ryanodine Receptors Mediate Cellular Toxicity: RESTORATION OF NORMAL PHENOTYPE BY FKBP12.6

Ca2+ homeostasis is a vital cellular control mechanism in which Ca2+ release from intracellular stores plays a central role. Ryanodine receptor (RyR)-mediated Ca2+ release is a key modulator of Ca2+ homeostasis, and the defective regulation of RyR is pathogenic. However, the molecular events underlying RyR-mediated pathology remain undefined. Cells stably expressing recombinant human RyR2 (Chinese hamster ovary cells, CHOhRyR2) had similar resting cytoplasmic Ca2+ levels ([Ca2+]c) to wild-type CHO cells (CHOWT) but exhibited increased cytoplasmic Ca2+ flux associated with decreased cell viability and proliferation. Intracellular Ca2+ flux increased with human RyR2 (hRyR2) expression levels and determined the extent of phenotypic modulation. Co-expression of FKBP12.6, but not FKBP12, or incubation of cells with ryanodine suppressed intracellular Ca2+ flux and restored normal cell viability and proliferation. Restoration of normal phenotype was independent of the status of resting [Ca2+]c or ER Ca2+ load. Heparin inhibition of endogenous inositol trisphosphate receptors (IP3R) had little effect on intracellular Ca2+ handling or viability. However, purinergic stimulation of endogenous IP3R resulted in apoptotic cell death mediated by hRyR2 suggesting functional interaction occurred between IP3R and hRyR2 Ca2+ release channels. These data demonstrate that defective regulation of RyR causes altered cellular phenotype via profound perturbations in intracellular Ca2+ signaling and highlight a key modulatory role of FKBP12.6 in hRyR2 Ca2+ channel function.

Zipping in the Regulators

Marx et al. show that the leucine/isoleucine zipper (LZ) protein motif found in several ion channels appears to mediate interaction of the channels with key regulatory proteins. They characterized the role of LZ motifs in ryanodine receptors (RyRs), which are ligand-controlled ion channels essential for proper excitation-contraction coupling in muscle. The activity of RyRs is regulated by phosphorylation, which modulates the channel properties of these receptors, and increased phosphorylation of RyR2 (the cardiac form of RyRs) is associated with heart failure. RyR2 has three LZ domains, each of which interacted with a distinct cellular regulator: LZ1 conferred binding of protein phosphatase 1 (PP1) through interaction with the PP1-targeting protein spinophilin, LZ2 conferred binding of PP2A through the PP2A-targeting protein PR130, and LZ3 mediated binding of the cyclic AMP-dependent protein kinase through the targeting protein mAKAP (also known as A-kinase anchoring protein). Each of the three targeting proteins also has LZ domains, which are required for specific interactions with LZ domains of RyR2. Disruption of the LZ-mediated interactions by incubation of receptor complexes with fusion proteins containing the LZ domains of RyR2 resulted in changes in RyR channel activity and altered regulation by phosphorylation. The identification of LZ-mediated interactions enabled Marks et al. to predict kinase and phosphatase interactions with RyR1 and, thus, may facilitate identification of components in other channel complexes and, thus, may expose other potential targets for therapeutic intervention. S. O. Marx, S. Reiken, Y. Hisamatsu, M. Gaburjakova, J. Gaburjakova, Y.-M. Yang, N. Rosemblit, A. R. Marks, Phosphorylation-dependent regulation of ryanodine receptors: A novel role for leucine/isoleucine zippers. J. Cell Biol. 153 , 699-708 (2001). [Abstract] [Full Text]

Autoimmunity against the ryanodine receptor in myasthenia gravis.

Some myasthenia gravis (MG) patients have antibodies against skeletal muscle antigens in addition to the acetylcholine receptor (AChR). A major antigen for these antibodies is the Ca2+ release channel of the sarcoplasmic reticulum the ryanodine receptor (RyR). These antibodies are found mainly in MG patients with a thymoma MG and correlate with severe MG symptoms. The antibodies recognize a region near the N-terminus on the RyR, which seems to be of importance for RyR regulation. The antibodies cause allosteric inhibition of RyR function in vitro, inhibiting Ca2+ release from sarcoplasmic reticulum.

Role of Ins(1,4,5)P3, cADP-ribose and nicotinic acid-adenine dinucleotide phosphate in Ca2+ signalling in mouse submandibular acinar cells.

cADP-ribose (cADPr) and nicotinic acid-adenine dinucleotide phosphate (NAADP) are two putative second messengers; they were first shown to stimulate Ca(2+) mobilization in sea urchin eggs. We have used the patch-clamp whole-cell technique to determine the role of cADPr and NAADP in relation to that of Ins(1,4,5)P(3) in mouse submandibular acinar cells by measuring agonist-evoked and second-messenger-evoked changes in Ca(2+)-dependent K(+) and Cl(-) currents. Both Ins(1,4,5)P(3) and cADPr were capable of reproducing the full range of responses normally seen in response to stimulation with acetylcholine (ACh). Low concentrations of agonist (10-20 nM ACh) or second messenger [1-10 microM Ins(1,4,5)P(3) or cADPr] elicited a sporadic transient activation of the Ca(2+)-dependent currents; mid-range concentrations [50-500 nM ACh, 50 microM Ins(1,4,5)P(3) or 50-100 microM cADPr] elicited high-frequency (approx. 2 Hz) trains of current spikes; and high concentrations [more than 500 nM ACh, more than 50 microM Ins(1,4,5)P(3) or more than 100 microM cADPr] gave rise to a sustained current response. The response to ACh was inhibited by antagonists of both the Ins(1,4,5)P(3) receptor [Ins(1,4,5)P(3)R] and the ryanodine receptor (RyR) but could be completely blocked only by an Ins(1,4,5)P(3)R antagonist (heparin). NAADP (50 nM to 100 microM) did not itself activate the Ca(2+)-dependent ion currents, nor did it inhibit the activation of these currents by ACh. These results show that, in these cells, both Ins(1,4,5)P(3)R and RyR are involved in the propagation of the Ca(2+) signal stimulated by ACh and that cADPr can function as an endogenous regulator of RyR. Furthermore, although NAADP might have a role in hormone-stimulated secretion in pancreatic acinar cells, it does not contribute to ACh-evoked secretion in submandibular acinar cells.

Role of Ins(1,4,5)P3, cADP-ribose and nicotinic acid–adenine dinucleotide phosphate in Ca2+ signalling in mouse submandibular acinar cells

cADP-ribose (cADPr) and nicotinic acid–adenine dinucleotide phosphate (NAADP) are two putative second messengers; they were first shown to stimulate Ca2+ mobilization in sea urchin eggs. We have used the patch-clamp whole-cell technique to determine the role of cADPr and NAADP in relation to that of Ins(1,4,5)P3 in mouse submandibular acinar cells by measuring agonist-evoked and second-messenger-evoked changes in Ca2+-dependent K+ and Cl- currents. Both Ins(1,4,5)P3 and cADPr were capable of reproducing the full range of responses normally seen in response to stimulation with acetylcholine (ACh). Low concentrations of agonist (10–20nM ACh) or second messenger [1–10µM Ins(1,4,5)P3 or cADPr] elicited a sporadic transient activation of the Ca2+-dependent currents; mid-range concentrations [50–500nM ACh, 50µM Ins(1,4,5)P3 or 50–100µM cADPr] elicited high-frequency (approx. 2Hz) trains of current spikes; and high concentrations [more than 500nM ACh, more than 50µM Ins(1,4,5)P3 or more than 100µM cADPr] gave rise to a sustained current response. The response to ACh was inhibited by antagonists of both the Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] and the ryanodine receptor (RyR) but could be completely blocked only by an Ins(1,4,5)P3R antagonist (heparin). NAADP (50nM to 100µM) did not itself activate the Ca2+-dependent ion currents, nor did it inhibit the activation of these currents by ACh. These results show that, in these cells, both Ins(1,4,5)P3R and RyR are involved in the propagation of the Ca2+ signal stimulated by ACh and that cADPr can function as an endogenous regulator of RyR. Furthermore, although NAADP might have a role in hormone-stimulated secretion in pancreatic acinar cells, it does not contribute to ACh-evoked secretion in submandibular acinar cells.

Calcium Binding to Calmodulin Leads to an N-terminal Shift in Its Binding Site on the Ryanodine Receptor

The skeletal muscle calcium release channel, ryanodine receptor, is activated by calcium-free calmodulin and inhibited by calcium-bound calmodulin. Previous biochemical studies from our laboratory have shown that calcium-free calmodulin and calcium bound calmodulin protect sites at amino acids 3630 and 3637 from trypsin cleavage (Moore, C. P., Rodney, G., Zhang, J. Z., Santacruz-Toloza, L., Strasburg, G., and Hamilton, S. L. (1999) Biochemistry 38, 8532-8537). We now demonstrate that both calcium-free calmodulin and calcium-bound calmodulin bind with nanomolar affinity to a synthetic peptide matching amino acids 3614-3643 of the ryanodine receptor. Deletion of the last nine amino acids (3635-3643) destroys the ability of the peptide to bind calcium-free calmodulin, but not calcium-bound calmodulin. We propose a novel mechanism for calmodulin's interaction with a target protein. Our data suggest that the binding sites for calcium-free calmodulin and calcium-bound calmodulin are overlapping and, when calcium binds to calmodulin, the calmodulin molecule shifts to a more N-terminal location on the ryanodine receptor converting it from an activator to an inhibitor of the channel. This region of the ryanodine receptor has previously been identified as a site of intersubunit contact, suggesting the possibility that calmodulin regulates ryanodine receptor activity by regulating subunit-subunit interactions.

Characterization of a calcium-regulation domain of the skeletal-muscle ryanodine receptor

A negatively charged region of the N-terminal portion of the skeletal ryanodine receptor (RyR), located between residues 1872–1923, is involved in Ca 2+-dependent regulation of the Ca2+-release channel. This region is divergent between the skeletal (RyR1) and cardiac (RyR2) isoforms of the channel, and is known as D3. Ca2+ exerts important regulatory functions on the RyR, being involved in both activation and inactivation functions of the channel, i.e. the effects occurring at micromolar and millimolar Ca2+ concentrations respectively. To characterize the role of D3 in the Ca2+-dependent regulation of the Ca2+-release channel, we studied the functional consequences of deleting the D3 region from RyR1 (∆D3-RyR1) using a heterologous expression system, [3H]ryanodine binding assays and single-channel recordings in lipid bilayers. Deletion of the D3 region selectively affected Ca2+-dependent regulation of RyR1, but did not alter [3H]ryanodine binding or the effect of other modulators on the RyR. Compared with full-length RyR1 (wt-RyR1), the Ca2+-dependence curve of ∆D3-RyR1 is broader, reflecting increased sensitivity to Ca2+ activation and decreased sensitivity to Ca2+ inactivation. In addition, ∆D3-RyR1 was more resistant to inhibition by Mg2+. Comparison of the effect of caffeine on wt-RyR1 and ∆D3-RyR1 suggested that D3 is an important region of RyR that participates in Ca2+-dependent activation and inactivation of the Ca2+-release channel.

Identification of a polymorphic ryanodine receptor gene from Heliothis virescens (Lepidoptera: Noctuidae)

cDNAs encoding the C–terminal 1172 amino acids of a ryanodine receptor (RyR) from the lepidopteran pest Heliothis virescens ( Hv–RyR) have been cloned and characterised. Sequence comparisons, organisational studies on corresponding genomic regions and a genetic segregation analysis provide evidence for two polymorphic alleles of the Hv–RyR locus. Comparison of the Hv–RyR C–terminal amino acid sequence with equivalent regions of other RyRs reveals a high level of overall amino acid homology (74% identity with D. melanogaster and between 47.9 and 50.1% with vertebrate isoforms). Homologies are however not uniformly distributed, though regions of high and low similarity are consistent with patterns in other RyR isoforms. The structural similarity of Hv–RyR with other RyRs is also indicated by comparison of hydropathy profiles and other previously described functional domains. Such results are consistent with this region of Hv–RyR containing the Ca 2+ channel itself and being intimately involved in RyR regulation. Potential uses of the cDNAs described in the discovery and development of novel ryanodine like insecticides are discussed.

How many cysteine residues regulate ryanodine receptor channel activity?

RyRs contain 80-100 cysteine residues per subunit, of which approximately 25% are free for covalent modification, while the remainder are either modified or form intraprotein disulfides. Oxidizing and nitrosylating reagents have several effects on single RyR channel activity, which depend on the type of modifying reagent, the isoform of the RyR, and ligands bound to the channel. We present evidence here for four major classes of functional cysteine residues associated with RyR channels, i.e., two classes with free -SH groups that either activate or inhibit channels when covalently modified and two classes, with endogenous modification, that either inhibit or activate. Single-channel characteristics provide evidence for four discrete responses within the first activating class, two responses within the second inhibiting class and two types of response within the third endogenously modified class. All but one of these changes in channel properties depend on residues located on the cytoplasmic or membrane-associated domains of the RyR; the remaining response is confined to the luminal domain. If it is assumed that each type of response depends on a separate subclass of cysteine residue and that each subclass contains a minimum of one cysteine per subunit, our results suggest that there are at least nine cysteine residues per subunit with functional connections to the gating mechanism of RyR channels. These cysteine residues may be selectively modified under physiological and pathological conditions to regulate Ca2+ release from the sarcoplasmic reticulum and contraction.

Localization and regulation of ryanodine receptor in bovine oocytes.

We have previously reported that injection of ryanodine receptor agonists into mature bovine oocytes induces intracellular calcium release, indicative of the existence of ryanodine receptors. In this experiment, further evidence of the ryanodine receptor localization, and developmental regulation in bovine oocytes is presented. The possible physiological significance is also suggested. Using a rabbit antibody against the rabbit cardiac muscle ryanodine receptor, the ryanodine receptor was observed uniformly localized in the periphery of mature bovine oocytes, while a weak and discontinuous signal was observed in the germinal vesicle intact stage of bovine oocytes. As oocytes progress to the metaphase I stage, the ryanodine receptor localization became more intense and continuous, yet not comparable to that observed in the metaphase II oocytes. These modifications correlate with the intracellular calcium responsiveness to ryanodine. A 200-microM injection of ryanodine induces a low intracellular calcium transient in germinal vesicle-stage bovine oocytes, while peaked intracellular calcium transients are subsequently observed in the metaphase II-stage oocytes. However, no significant changes in the amplitude of intracellular calcium transients induced by 250 nM inositol 1,4,5-trisphosphate and 10 microM ionomycin were observed in oocytes at a comparable stage. Fertilization induced a significant decrease in ryanodine receptor signal; similar changes were also observed in oocytes injected with 200 microM ryanodine or incubated with 10 microM ionomycin. However, no changes in ryanodine signal were observed in oocytes injected with vehicle medium. Furthermore, injection of either ryanodine or inositol 1,4,5-trisphosphate induced subsequent pronuclear formation and cleavage. These data indicate that the ryanodine receptor is closely regulated and associated with early cellular changes following fertilization; stimulation of this receptor results in the activation of bovine oocytes, and it is likely that this receptor may play a role at fertilization.