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At least 19 recordsLinked to original sources

PubMed2002

Role of the proposed pore-forming segment of the Ca2+ release channel (ryanodine receptor) in ryanodine interaction.

In earlier studies we showed that point mutations introduced into the proposed pore-forming segment, GVRAGGGIGD (amino acids 4820-4829), of the mouse cardiac ryanodine receptor reduced or abolished high affinity [3H]ryanodine binding. Here we investigate the effects of these mutations on the affinity and dissociation properties of [3H]ryanodine binding and on ryanodine modification of the ryanodine receptor channel at the single channel and whole cell levels. Scatchard analysis and dissociation studies reveal that mutation G4824A decreases the equilibrium dissociation constant (K(d)) and the dissociation rate constant (k(off)), whereas mutations G4828A and D4829A increase the K(d) and k(off) values. The effect of ryanodine on single G4828A and D4829A mutant channels is reversible on the time scale of single channel experiments, in contrast to the irreversible effect of ryanodine on single wild-type channels. Ryanodine alone is able to induce a large and sustained Ca2+ release in HEK293 cells transfected with the R4822A or G4825A mutant cDNA at the resting cytoplasmic Ca2+ but causes little or no Ca2+ release in cells transfected with the wild-type cDNA. Mutation G4826C diminishes the functional effect of ryanodine on Ca2+ release but spares caffeine-induced Ca2+ release in HEK293 cells. Co-expression of the wild-type and G4826C mutant proteins produces single channels that interact with ryanodine reversibly and display altered conductance and ryanodine response. These results are consistent with the view that the proposed pore-forming segment is a critical determinant of ryanodine interaction. A putative model of ryanodine-ryanodine receptor interaction is proposed.

Amino Acid Sequence↗

PubMed2001

Ryanodine sensitizes the cardiac Ca(2+) release channel (ryanodine receptor isoform 2) to Ca(2+) activation and dissociates as the channel is closed by Ca(2+) depletion.

In single-channel recordings, the rabbit cardiac Ca(2+) release channel (RyR2) is converted to a fully open subconductance state with about 50% of full conductance by micromolar concentrations of ryanodine. At +30 mV, corresponding to a luminal to cytoplasmic cation current, the probability of opening (P(o)) of ryanodine-modified channels was only marginally altered at pCa 10 (pCa = -log(10) Ca concentration). However, at -30 mV, the P(o) was highly sensitive to Ca(2+) added to the cis (cytoplasmic) side and, at pCa 10, was reduced to less than 0.27. The EC(50) value for channel opening was about pCa 8. No significant Ca(2+) inactivation was observed for ryanodine-modified channels at either -30 mV or +30 mV. The opening of unmodified Ca(2+) channels is Ca(2+) sensitive, with an EC(50) value of about pCa 6 (two orders of magnitude less sensitive than ryanodine-modified channels) and IC(50) values of pCa 2.2 at -30 mV and 2.5 at +30 mV. Mg(2+) decreased the P(o) of ryanodine-modified channels at low Ca(2+) concentrations at both -30 and +30 mV. Caffeine, ATP, and ruthenium red were modulators of the P(o) of ryanodine-modified channels. In a [(3)H]ryanodine binding assay, [(3)H]ryanodine dissociation from the high-affinity binding site was found to be Ca(2+) sensitive, with an IC(50) of pCa 7.1. High concentrations of unlabeled ryanodine prevented [(3)H]ryanodine dissociation, but ruthenium red accelerated dissociation. These results suggest that ryanodine sensitizes Ca(2+) activation of the Ca(2+) release channel and desensitizes Ca(2+) inactivation through an allosteric interaction. [(3)H]Ryanodine dissociates from the high-affinity site when the channel is closed by removal of Ca(2+), implying that high-affinity ryanodine and Ca(2+) binding sites are linked through either short- or long-range interactions, probably involving conformational changes.

Animals↗

PubMed2001

Ryanodine sensitizes the Ca(2+) release channel (ryanodine receptor) to Ca(2+) activation.

Ryanodine, a plant alkaloid, is one of the most widely used pharmacological probes for intracellular Ca(2+) signaling in a variety of muscle and non-muscle cells. Upon binding to the Ca(2+) release channel (ryanodine receptor), ryanodine causes two major changes in the channel: a reduction in single-channel conductance and a marked increase in open probability. The molecular mechanisms underlying these alterations are not well understood. In the present study, we investigated the gating behavior and Ca(2+) dependence of the wild type (wt) and a mutant cardiac ryanodine receptor (RyR2) after being modified by ryanodine. Single-channel studies revealed that the ryanodine-modified wt RyR2 channel was sensitive to inhibition by Mg(2+) and to activation by caffeine and ATP. In the presence of Mg(2+), the ryanodine-modified single wt RyR2 channel displayed a sigmoidal Ca(2+) dependence with an EC(50) value of 110 nm, whereas the ryanodine-unmodified single wt channel exhibited an EC(50) of 120 microm for Ca(2+) activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca(2+) activation by approximately 1,000-fold. Furthermore, ryanodine is able to restore Ca(2+) activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit approximately 1,000-fold reduction in Ca(2+) sensitivity to activation. The E3987A mutation, however, affects neither [(3)H]ryanodine binding to, nor the stimulatory and inhibitory effects of ryanodine on, the RyR2 channel. These results demonstrate that ryanodine does not "lock" the RyR channel into an open state as generally believed; rather, it sensitizes dramatically the channel to activation by Ca(2+).

Alanine↗

PubMed1991

Characterization of multiple [3H]ryanodine binding sites on the Ca2+ release channel of sarcoplasmic reticulum from skeletal and cardiac muscle: evidence for a sequential mechanism in ryanodine action.

Kinetic and equilibrium measurements of [3H]ryanodine binding to the Ca2+ release channel of rabbit skeletal and rat cardiac sarcoplasmic reticulum (SR) are examined to ascertain the nature of cooperative interactions among high and low affinity binding sites and to quantitate their distribution. Equilibrium studies reveal affinities of 1-4 nM for the highest affinity binding site and of 30-50 nM, 500-800 nM, and 2-4 microM for the lower affinity sites in both preparations, with Hill coefficients of significantly less than 1, and initial rates of association and dissociation increase with increasing concentrations of ryanodine. SR vesicles are actively loaded in the presence of pyrophosphate, and fluctuations in extravesicular Ca2+ are measured by the absorbance change of antipyrylazo III. The data demonstrate a biphasic, time- and concentration-dependent action of ryanodine on the release of Ca2+, with an initial activation and a subsequent inactivation phase. Kinetic analysis of the activation of Ca2+ release by ryanodine, in consonance with the binding data, demonstrates the existence of multiple binding sites for the alkaloid on the channel complex, with nanomolar to micromolar affinities. Based on the present findings obtained by receptor binding analysis and Ca2+ transport measurements, we suggest a model that describes four, most plausibly negatively cooperative, binding sites on the Ca2+ release channel. Occupation of ryanodine binding sites produces sequential activation followed by inactivation of the SR channel, revealing the strong possibility of an irreversible uncoupling of the native function of the receptor/channel complex by high concentrations of ryanodine. A model relating ryanodine receptor occupancy with SR Ca2+ release stresses two important new findings regarding the interaction of ryanodine with its receptor. First, ryanodine binds to four sites on the oligomeric channel complex with decreasing affinities, which can be best described by allosteric negative cooperativity. Second, binding of ryanodine to its receptor activates the Ca2+ release channel in a concentration-dependent and saturable manner in the range of 20 nM to 1 mM and produces a kinetically limited and sequential inactivation of the Ca2+ channel, with the concomitant attainment of full negative cooperativity. The results presented suggest that driving of the complex toward full negative cooperativity with high concentrations of ryanodine promotes a long-lived conformational state in which ryanodine is physically occluded and hindered from free diffusion from its binding site.

Animals↗

PubMed1994

Photoaffinity labeling of the ryanodine receptor/Ca2+ release channel with an azido derivative of ryanodine.

Ryanodine receptors/Ca2+ release channels play an important role in regulating the intracellular free calcium concentrations in both muscle and nonmuscle cells. Ryanodine, a neutral plant alkaloid, specifically binds to and modulates these Ca2+ release channels. In the work described here, we characterize the interaction of a tritium-labeled, photoactivable derivative of ryanodine (3H-labeled 10-O-[3-(4-azidobenzamido)propionyl]ryanodine ([3H]ABRy)) with the ryanodine receptor of skeletal, cardiac, and brain membranes. Scatchard analysis demonstrates that this ligand binds to a single class of high affinity sites in skeletal muscle triads. Furthermore, competition binding assays of [3H]ryanodine with skeletal, cardiac, and brain membranes in the presence of increasing concentrations of unlabeled ABRy illustrate that this azido derivative of ryanodine is able to specifically displace [3H]ryanodine from its binding site(s). Analysis of the effects of Ca2+, ATP, and KCl on [3H]ABRy binding in triad membranes shows a similar modulation of binding to that seen in these membranes with [3H]ryanodine. Photoaffinity labeling of triads with [3H]ABRy resulted in specific and covalent incorporation of [3H]ABRy into a 565-kDa protein that was shown to be the skeletal muscle ryanodine receptor. Digestion of the labeled ryanodine receptor revealed a [3H]ABRy-labeled 76-kDa tryptic fragment that was identified with an antibody directed against the COOH-terminal of the receptor. These results demonstrate that the 76-kDa COOH-terminal tryptic fragment contains the high affinity binding site for ryanodine.

Adenosine Triphosphate↗

PubMed1998

Structure-function relationships among ryanodine derivatives. Pyridyl ryanodine definitively separates activation potency from high affinity.

Ryanodine derivatives are differentially effective on the two limbs of the ryanodine concentration-effect curve. This study comparing ryanodine, ryanodol, and pyridyl ryanodine and nine C10Oeq esters of them focuses on structure-function relations underlying their differential effectiveness. Ryanodol and pyridyl ryanodine had significantly lower affinities than ryanodine, but their EC50act values (concentration of ryanoid that induces one-half of full efficacy), potencies, and efficacies were not diminished in like fashion. Ryanodine and ryanodol were partial agonists, whereas pyridyl ryanodine was a full agonist, having a diminished deactivation potency. C10Oeq esterifications enhanced affinities and efficacies of the base ryanoids. The C10-Oeq ester derivatives of ryanodine and pyridyl ryanodine, but not those of ryanodol, lost their capacity to deactivate RyR1s. Thus, affinity differences among ryanoids clearly do not predicate functional differences as regards activation of Ca2+ release channels. The pyrrole carboxylate on the C3 of ryanodine is dispensable to ryanoid activation of Ca2+ release channels. Ryanodol lacks this ring, but it nevertheless effects substantial activation. Moreover, its C10-Oeq esters display full efficacy. The increased ability of all the C10-Oeq derivatives to release Ca2+ from the vesicles strengthens their role in directly impeding deactivation of RyR1, perhaps by interaction with some component within the transmembrane ionic flux pathway.

Animals↗

PubMed2001

Changes in ryanodine receptor-mediated calcium release during skeletal muscle differentiation. II. Resolution of a caffeine-ryanodine paradox.

Our previous study demonstrated a disparity of action between two established pharmacological modulators of the same calcium (Ca2+) release channel, the ryanodine receptor (RyR). Specifically, we observed that caffeine sensitivity was elicited at earlier stages of development than that of ryanodine. In the present study, we offer a hypothesis to resolve this paradox. We provide evidence that ryanodine acts as a pure uncompetitive inhibitor of Ca2+ transport, with respect to Ca2+ itself. This explains why little ryanodine inhibition was observed at low Ca2+ concentrations, while maximal ryanodine inhibition was observed at saturating Ca2+ concentrations. In order to exclude the possibility of nonspecific ryanodine actions as an alternative explanation, we established the phenomenon of capacitative calcium entry (CCE) for L6 cells. Since it is known that CCE is inversely correlated with [Ca2+] of the ER/SR lumen, the extent of CCE is therefore an indirect measure of Ca2+ concentration within the SR. We also demonstrated the functional pathway for Ca2+ entry. Employing pharmacological inhibitors, we found that a T-type plasma membrane channel was predominant in the myoblasts, while an L-type channel was predominant in the adult myotubes. Our data using these inhibitors made nonspecific ryanodine actions an unlikely explanation of the disparity in action between ryanodine and caffeine. Moreover, we found no evidence that inositol trisphosphate, a proposed regulator of CCE for other cells, could influence CCE in L6 cells. We conclude that the disparity between caffeine and ryanodine can be explained by Ca2+ dependence of ryanodine action. This study may also offer an explanation of other studies showing unclear actions of ryanodine binding and action.

Animals↗

PubMed2003

Residue Gln4863 within a predicted transmembrane sequence of the Ca2+ release channel (ryanodine receptor) is critical for ryanodine interaction.

Despite the pivotal role of ryanodine in ryanodine receptor (RyR) research, the molecular basis of ryanodine-RyR interaction remains largely undefined. We investigated the role of the proposed transmembrane helix TM10 in ryanodine interaction and channel function. Each amino acid residue within the TM10 sequence, 4844IIFDITFFFFVIVILLAIIQGLII4867, of the mouse RyR2 was mutated to either alanine or glycine. Mutants were expressed in human embryonic kidney 293 cells, and their properties were assessed. Mutations D4847A, F4850A, F4851A, L4858A, L4859A, and I4866A severely curtailed the release of intracellular Ca2+ in human embryonic kidney 293 cells in response to extracellular caffeine and diminished [3H]ryanodine binding to cell lysates. Mutations F4846A, T4849A, I4855A, V4856A, and Q4863A eliminated or markedly reduced [3H]ryanodine binding, but cells expressing these mutants responded to extracellular caffeine by releasing stored Ca2+. Interestingly these two groups of mutants, each with similar properties, are largely located on opposite sides of the predicted TM10 helix. Single channel analyses revealed that mutation Q4863A dramatically altered the kinetics and apparent affinity of ryanodine interaction with single RyR2 channels and abolished the effect of ryanodol, an analogue of ryanodine, whereas the single channel conductance of the Q4863A mutant and its responses to caffeine, ATP, and Mg2+ were comparable to those of the wild type channels. Furthermore the effect of ryanodine on single Q4863A mutant channels was influenced by the transmembrane holding potential. Together these results suggest that the TM10 sequence and in particular the Q4863 residue constitute an important determinant of ryanodine interaction.

Amino Acid Sequence↗

PubMed2005

IP3-mediated Ca2+ increases do not involve the ryanodine receptor, but ryanodine receptor antagonists reduce IP3-mediated Ca2+ increases in guinea-pig colonic smooth muscle cells.

Smooth muscle responds to IP3-generating (sarcolemma acting) neurotransmitters and hormones by releasing Ca2+ from the sarcoplasmic reticulum (SR) via IP3 receptors (IP3Rs). This release may propagate as Ca2+ waves. The Ca2+ signal emanating from IP3 generation may be amplified by its activating further Ca2+ release from ryanodine receptors (RyRs) in the process of Ca2+-induced Ca2+ release (CICR). Evidence for this proposal has relied largely on the use of blocking drugs such as ryanodine, tetracaine and dantrolene, reportedly specific inhibitors of RyRs. Here we have examined whether or not Ca2+ released via IP3Rs subsequently activates RyRs. In addition, the specificity of the blocking agents has been assessed by determining the extent of their ability to block IP3-mediated Ca2+ release under conditions in which RyRs were not activated. IP3-evoked Ca2+ release and Ca2+ waves did not require or activate RyRs. However, the RyR blocking drugs inhibited IP3-mediated Ca2+ signals at concentrations thought to be selective for RyRs. In single colonic smooth muscle cells, voltage clamped in the whole cell configuration, carbachol (CCh) evoked propagating Ca2+ waves which were not inhibited by ryanodine when the sarcolemma potential was -70 mV. At -20 mV, at which potential the SR Ca2+ content was increased and RyRs activated, ryanodine inhibited the Ca2+ waves. Photolysed caged IP3 increased [Ca2+]c; ryanodine, by itself, did not reduce the IP3-evoked [Ca2+]c increase when the sarcolemma potential was maintained at -70 mV. However, after activation of RyRs by caffeine, in the continued presence of ryanodine, the IP3-evoked [Ca2+]c increase was inhibited. In other experiments, RyRs were activated (as evidenced by the occurrence of spontaneous transient outward currents) by depolarizing the sarcolemma to -20 mV and again ryanodine was effective in inhibiting IP3-evoked Ca2+ increase. Thus while ineffective by itself, ryanodine inhibited IP3-evoked Ca2+ increases, presumably by causing persistent opening of the channel and depleting the SR of Ca2+, after RyRs were activated. These experiments establish that IP3-evoked Ca2+ release and Ca2+ waves do not activate RyRs; had they done so ryanodine would have inhibited the Ca2+ increase. However, under conditions where ryanodine was ineffective against the IP3-evoked Ca2+ transient (i.e. when RyRs were not activated, e.g. at a membrane potential of -70 mV) tetracaine and dantrolene each blocked IP3-evoked Ca2+ increases. The results show that although IP3-mediated Ca2+ release does not activate RyRs, RyR blockers can inhibit IP3-mediated Ca2+ signals.

Animals↗

PubMed1997

Ryanodine receptor type III (Ry3R) identification in mouse parotid acini. Properties and modulation of [3H]ryanodine-binding sites.

Immunoblot analysis and [3H]ryanodine binding were used to characterize and identify ryanodine receptors (RyRs) in nonexcitable mouse parotid acini. Western analysis revealed ryanodine receptor type III (Ry3R) to be the only detectable isoform in parotid microsomal membranes. Binding of [3H]ryanodine to microsomal fractions was dependent on Ca2+, salt, pH, and temperature. At 23 degrees C, and in the presence of 0.5 M KCl and 100 microM Ca2+, [3H]ryanodine bound specifically to membranes with high affinity (Kd = 6 nM); maximum binding capacity (Bmax) was 275 fmol/mg protein. Mg2+ and ruthenium red inhibited [3H]ryanodine binding (IC50 = 1.4 mM and 0.5 microM, respectively). 4-Chloro-3-ethylphenol enhanced the binding of [3H]ryanodine 2.5-fold; whereas ATP and caffeine were much less efficacious toward activating Ry3R (56% and 18% maximal enhancement, respectively). Bastadin, a novel modulator of the 12-kDa FK506 binding protein.RyR complex, increased [3H]ryanodine binding 3-4-fold by enhancing Kd. The immunosuppressant FK506 enhanced [3H]ryanodine receptor occupancy at >100 microM and antagonized the action of bastadin, suggesting that an immunophilin modulates Ry3R in parotid acini. These results suggest that Ry3R may play an important role in Ca2+ homeostasis in mouse parotid acini.

Adenosine Triphosphate↗

PubMed1995

Regulation of mouse egg activation: presence of ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose on uninseminated and inseminated eggs.

Sperm-induced activation of mammalian eggs is associated with a transient increase in Ca2+ concentrations thought to be derived from inositol 1,4,5-trisphosphate-sensitive and -insensitive intracellular stores. Whereas the importance of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores has been evaluated, the identity and role of inositol 1,4,5-trisphosphate-insensitive stores are poorly understood. To explore the role of the ryanodine-sensitive Ca2+ store, we first used reverse transcription-polymerase chain reaction to identify transcripts of the ryanodine receptor in eggs and determined that transcripts for the type 2 and 3 receptor were present. Immunoprecipitation of radioiodinated egg extracts with an antibody that recognizes both type 2 and 3 receptors detected specifically a band of Mr = 520,000. Immunolocalization of the receptor(s) using laser-scanning confocal microscopy revealed that the receptor(s) was uniformly distributed in the cortex of the germinal vesicle-intact oocyte, but became asymmetrically localized to the cortex in a region apposed to the meiotic spindle in the metaphase II-arrested egg; this asymmetrical localization developed by metaphase I. The role of the ryanodine receptor in mouse egg activation was examined by determining the effects of microinjected ryanodine or cyclic ADP ribose on endpoints of egg activation in either uninseminated or inseminated eggs. Ryanodine induced the conversion of the zona pellucida glycoprotein ZP2 to its postfertilization form ZP2f in a biphasic concentration-dependent manner; nanomolar concentrations stimulated this conversion, whereas micromolar concentrations had no stimulatory effect. Cyclic ADP ribose also promoted the ZP2 conversion, but with a hyperbolic concentration dependence. Neither of these compounds induced cell cycle resumption. Inhibiting the inositol 1,4,5-trisphosphate-sensitive Ca2+ store did not inhibit the ryanodine-induced ZP2 conversion and, reciprocally, inhibiting the ryanodine-sensitive Ca2+ store did not inhibit the inositol 1,4,5-trisphosphate-induced ZP2 conversion. Last, treatment of eggs under conditions that would block the release of Ca2+ from the ryanodine-sensitive store had no effect on any event of egg activation following fertilization. Results of these experiments suggest that although ryanodine receptors are present and functional, release of Ca2+ from this store is not essential for sperm-induced egg activation.

Adenosine Diphosphate Ribose↗

PubMed2004

Comparative analysis of in vitro contracture tests with ryanodine and a combination of ryanodine with either halothane or caffeine: a comparative investigation in malignant hyperthermia.

BACKGROUND: The diagnosis of susceptibility to malignant hyperthermia (MH) is currently performed on muscle biopsies subjected to halothane-caffeine in vitro contracture tests (IVCTs). There is a consensus on our need to improve the diagnostic potential of IVCTs if we are to maximize the information available for research and diagnosis in MH. This study was designed as a pilot comparative study and we aimed at comparing the ryanodine test and new tests using a combination of ryanodine, halothane and caffeine. METHODS: One hundred and thirty-two subjects (52 MHS and 80 MHN) were included in this study and new IVCTs were performed in additional muscle biopsy specimens. The contracture time-course was compared considering the onset time of contracture (OT) and the time to reach a 10 mN contracture (10T). Cut-off values were determined using ROC analyses. RESULTS: For the ryanodine test, sensitivity and specificity calculated for OT were 84.6% and 90.4%, respectively, and were better than those obtained using 10T. Combined tests using either caffeine and ryanodine or halothane and ryanodine did provide higher sensitivities (from 85.3 to 93.9%). A better specificity was only observed for the IVC tests combining halothane (cumulated) and caffeine both with ryanodine (93.9% for both). The largest sensitivity was observed when halothane was used as a bolus and combined with ryanodine. The specificity was always larger with the combined tests as compared to the test using ryanodine alone (from 79.1 to 90.9%). This superiority was confirmed, at least in part, when comparing genetic investigations and the results of new tests in a subgroup of subjects. CONCLUSIONS: This pilot study showed a clear diagnostic potential for new IVC tests combining halothane, the triggering agent of MH, and ryanodine acting at the calcium release channel, and should be considered as a first step in the investigation of combined tests.

Anesthetics, Inhalation↗

PubMed2001

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.

Allosteric Regulation↗

PubMed2002

Caffeine and ryanodine demonstrate a role for the ryanodine receptor in the organ of Corti.

The hypothesis that the release of Ca(2+) from ryanodine receptor activated Ca(2+) stores in vivo can affect the function of the cochlea was tested by examining the effects of caffeine (1-10 mM) and ryanodine (1-333 microM), two drugs that release Ca(2+) from these intracellular stores. The drugs were infused into the perilymph compartment of the guinea pig cochlea while sound (10 kHz) evoked cochlear potentials and distortion product otoacoustic emissions (DPOAEs; 2f(1)-f(2)=8 kHz, f(2)=12 kHz) were monitored. Caffeine significantly suppressed the compound action potential of the auditory nerve (CAP) at low intensity (56 dB SPL; 3.3 and 10 mM) and high intensity (92 dB SPL; 10 mM), increased N1 latency at high and low intensity (3 and 10 mM) and suppressed low intensity summating potential (SP; 10 mM) without an effect on high intensity SP. Ryanodine significantly suppressed the CAP at low intensity (100 and 333 microM) and at high intensity (333 microM), increased N1 latency at low intensity (33, 100 and 333 microM) and at high intensity (333 microM) and suppressed low intensity SP (100 and 333 microM) and increased high intensity SP (333 microM). The cochlear microphonic (CM) evoked by 10 kHz tone bursts was not affected by caffeine at high or low intensity, and ryanodine had no effect on it at low intensity but decreased it at high intensity (10, 33, 100 and 333 microM). In contrast, caffeine (10 mM) and ryanodine (33 and 100 microM) significantly increased CM evoked by l kHz tone bursts and recorded from the round window. Caffeine (10 mM) and ryanodine (100 microM) reversibly suppressed the cubic DPOAEs evoked by low intensity primaries. Overall, low intensity evoked responses were more sensitive and were suppressed to a greater extent by both drugs. This is consistent with the hypothesis that release of Ca(2+) from ryanodine receptor Ca(2+) stores, possibly in outer hair cells and supporting cells, affects the function of the cochlear amplifier.

Action Potentials↗

PubMed1996

Ryanodine receptor/calcium release channel conformations as reflected in the different effects of propranolol on its ryanodine binding and channel activity.

1. Propranolol, a beta-blocker, inhibited or stimulated ryanodine binding to both the membrane-bound and purified ryanodine receptor (RyR) depending on the assay conditions. At high NaCl concentrations, propranolol increased the number of ryanodine-binding sites (Bmax) with no effect on the binding affinity. In the presence of 0.2 M NaCl, ryanodine binding was inhibited by propranolol. Half-maximal inhibition was obtained at 1.2 mM and complete inhibition at 2 mM propranolol. The inhibitory effect of propranolol obtained at low NaCl concentration was not restored by increasing the NaCl concentration to 1 M. 2. Modulators of the RyR that are known to alter its conformational states, such as adenine nucleotides, Ca2+ concentration and pH, modified the effect of propranolol on ryanodine binding. In the presence of propranolol and at low NaCl concentrations, ryanodine binding was inhibited and showed no Ca(2+)-, pH-, or time-dependence. 3. Propranolol immediately and completely blocked the channel opening of RyR reconstituted into a planar lipid bilayer. Propranolol-modified non-active channel was reactivated to a subconductive state (about 40% of the control conductance) by ATP. 4. Competition experiments between lidocaine (a stimulatory drug) or tetracaine (an inhibitory drug) and propranolol at 0.2 or 1.0 M NaCl, respectively, suggest the existence of different interaction sites for local anaesthetics and propranolol. 5. These results suggest that propranolol interacts directly with the RyR and modifies its ryanodine binding and single-channel activities. Propranolol effects are altered by the RyR conformational state, suggesting its possible use as a conformational probe for RyR.

Adrenergic beta-Antagonists↗

PubMed1998

Characterization of recombinant rabbit cardiac and skeletal muscle Ca2+ release channels (ryanodine receptors) with a novel [3H]ryanodine binding assay.

A rapid assay for high affinity [3H]ryanodine binding to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized recombinant or native Ca2+ release channel proteins (ryanodine receptor, RyR) was devised. The key to preservation of high affinity [3H]ryanodine binding sites in the presence of increasing concentrations of CHAPS was the addition of phosphatidylcholine. This assay was used to characterize the equilibrium and kinetic properties of [3H]ryanodine binding to recombinant skeletal (RyR1) and cardiac (RyR2) Ca2+ release channels and the effects on binding of physiological modulators including ATP, Ca2+, and Mg2+. Both RyR1 and RyR2 had a single high affinity ryanodine binding site and low affinity sites, but [3H]ryanodine binding to recombinant RyR2 was not sensitive to ATP activation or Ca2+ inactivation and was less sensitive to Mg2+ inhibition. The [3H]ryanodine binding assay was used to estimate the expression level of recombinant RyR2 and RyR1, and to show that RyR2 can be expressed at very high levels in HEK-293 cells. Analysis of the properties of recombinant RyR2 and RyR1 by measurement of intracellular Fura-2 fluorescence revealed that the different properties of RyR2 and RyR1 are retained in the recombinant expressed proteins.

Adenosine Triphosphate↗

PubMed1998

Interactions of a reversible ryanoid (21-amino-9alpha-hydroxy-ryanodine) with single sheep cardiac ryanodine receptor channels.

The binding of ryanodine to a high affinity site on the sarcoplasmic reticulum Ca2+-release channel results in a dramatic alteration in both gating and ion handling; the channel enters a high open probability, reduced-conductance state. Once bound, ryanodine does not dissociate from its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-amino-9alpha-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca2+-release channel. The interaction of 21-amino-9alpha-hydroxy-ryanodine with the channel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the interaction of this ryanoid with the channel is reversible under steady state conditions, with dwell times in the modified state lasting seconds. By monitoring the reversible interaction of this ryanoid with single channels under voltage clamp conditions, we have established a number of novel features of the ryanoid binding reaction. (a) Modification of channel function occurs when a single molecule of ryanoid binds to the channel protein. (b) The ryanoid has access to its binding site only from the cytosolic side of the channel and the site is available only when the channel is open. (c) The interaction of 21-amino-9alpha-hydroxy-ryanodine with its binding site is influenced strongly by transmembrane voltage. We suggest that this voltage dependence is derived from a voltage-driven conformational alteration of the channel protein that changes the affinity of the binding site, rather than the translocation of the ryanoid into the voltage drop across the channel.

Animals↗

PubMed1989

Cardiac ryanodine receptor is absent in type I slow skeletal muscle fibers: immunochemical and ryanodine binding studies.

Cardiac ryanodine receptor was purified from canine ventricle as a single polypeptide of Mr 400,000 by a stepwise sucrose density gradient centrifugation and heparin-Sepharose CL-4B column chromatography. The [3H]ryanodine binding capacity (Bmax) was 60-fold enriched from cardiac microsomes without a change in affinity for [3H]ryanodine. The purity of the final preparation was determined to be greater than 95% by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using this purified preparation as an antigen, we produced six monoclonal antibodies which immunoprecipitated the cardiac ryanodine receptor. Three of these antibodies recognized the cardiac receptor on immunoblot analysis. In contrast, no protein in the microsomes isolated from Type I (slow) or Type II (fast) skeletal muscles was recognized by these antibodies. The [3H]ryanodine binding to cardiac and skeletal muscle microsomes was dependent on free Ca2+ concentration. In skeletal muscle microsomes, the [3H]ryanodine binding was remarkably enhanced by the addition of ATP or KCl and inhibited by high free Ca2+, whereas it was less sensitive to these agents in cardiac microsomes. All of these results clearly demonstrate that the cardiac ryanodine receptor is different from the skeletal muscle receptors and is not present even in Type I (slow) skeletal muscle fibers, in which cardiac isoforms of some of the muscle proteins are constitutively expressed.

Alkaloids↗