Regulation of ryanodine receptors by dopamine D1 receptors during methamphetamine-induced place conditioning.

Little is known about ryanodine receptors (RyRs) related to the methamphetamine (METH)-induced place preference. The present study was designed to ascertain whether RyRs could play a role in the development of METH-induced place preference in the mouse. The METH-induced place preference was dose-dependently suppressed by dantrolene, a RyRs receptor antagonist. The levels of RyRs 1 and 2 in the frontal cortex and RyRs 1 in the limbic forebrain were significantly increased in METH-conditioned mice. This up-regulation of RyRs were not inhibited by nifedipine. Both the dopamine D(1) receptor antagonist SCH23390 and the dopamine D(2) receptor antagonist sulpiride inhibited the development of METH-induced place conditioning. In contrast, the increases of RyRs 1 and 2 in the frontal cortex and of RyRs 1 in the limbic forebrain were completely abolished by SCH23390, whereas sulpiride had no effect. These findings indicate that up-regulation of RyRs is regulated through the activation of dopamine D(1) receptors in the METH-conditioning.

Retina expresses a novel variant of the ryanodine receptor.

Calcium released from intracellular stores via the ryanodine receptor (RyR) mediates a variety of signalling processes. We previously showed that retina expresses the three known types of RyR, but retinal membrane preparations exhibit unique characteristics such as Ca2+-independent [3H]ryanodine-binding and inhibition by caffeine. We have heretofore suggested that the major retinal RyR isoform is novel. The present study aimed to identify this receptor isoform and to localize RyR in mammalian retina. Immunoblotting with specific and pan-antibodies showed that the major retinal RyR has a mobility similar to that of RyR2 or RyR3. Real-time PCR revealed that the major type is RyR2, and RT-PCR followed by sequencing showed a transcript that encodes a protein with approximately 99% identity to RyR2, yet lacking two regions of seven and 12 amino acids and including an additional insertion of eight amino acids. An antibody against RyR2 localized this type to somas and primary dendrites of most retinal neurons. An antibody against RyR1 localized RyR to most somas but also revealed staining in photoreceptor outer segments, concentrated on the disk membranes at their rim. The ryanodine-binding properties and the electrophoretic mobility of RyR from the outer segments were similar to those of the whole retinal preparation. The results thus identify a novel variant of RyR2 which can contribute to regulating photoreceptor Ca2+ concentrations. The restricted localization of the outer segment RyR to the disk rim suggests that its activation mechanism involves a coupling between retinal RyR and the cGMP-gated channel.

Postulated role of interdomain interaction between regions 1 and 2 within type 1 ryanodine receptor in the pathogenesis of porcine malignant hyperthermia.

We have demonstrated recently that CICR (Ca2+-induced Ca2+ release) activity of RyR1 (ryanodine receptor 1) is held to a low level in mammalian skeletal muscle ('suppression' of the channel) and that this is largely caused by the interdomain interaction within RyR1 [Murayama, Oba, Kobayashi, Ikemoto and Ogawa (2005) Am. J. Physiol. Cell Physiol. 288, C1222-C1230]. To test the hypothesis that aberration of this suppression mechanism is involved in the development of channel dysfunctions in MH (malignant hyperthermia), we investigated properties of the RyR1 channels from normal and MHS (MH-susceptible) pig skeletal muscles with an Arg615-->Cys mutation using [3H]ryanodine binding, single-channel recordings and SR (sarcoplasmic reticulum) Ca2+ release. The RyR1 channels from MHS muscle (RyR1MHS) showed enhanced CICR activity compared with those from the normal muscle (RyR1N), although there was little or no difference in the sensitivity to several ligands tested (Ca2+, Mg2+ and adenine nucleotide), nor in the FKBP12 (FK506-binding protein 12) regulation. DP4, a domain peptide matching the Leu2442-Pro2477 region of RyR1 which was reported to activate the Ca2+ channel by weakening the interdomain interaction, activated the RyR1N channel in a concentration-dependent manner, and the highest activity of the affected channel reached a level comparable with that of the RyR1MHS channel with no added peptide. The addition of DP4 to the RyR1MHS channel produced virtually no further effect on the channel activity. These results suggest that stimulation of the RyR1MHS channel caused by affected inter-domain interaction between regions 1 and 2 is an underlying mechanism for dysfunction of Ca2+ homoeostasis seen in the MH phenotype.

Cell adhesion molecules regulate Ca2+-mediated steering of growth cones via cyclic AMP and ryanodine receptor type 3.

Axonal growth cones migrate along the correct paths during development, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (CAMs). Asymmetric Ca2+ elevations in the growth cone cytosol induce both attractive and repulsive turning in response to the guidance cues (Zheng, J.Q. 2000. Nature. 403:89-93; Henley, J.R., K.H. Huang, D. Wang, and M.M. Poo. 2004. Neuron. 44:909-916). Here, we show that CAMs regulate the activity of ryanodine receptor type 3 (RyR3) via cAMP and protein kinase A in dorsal root ganglion neurons. The activated RyR3 mediates Ca2+-induced Ca2+ release (CICR) into the cytosol, leading to attractive turning of the growth cone. In contrast, the growth cone exhibits repulsion when Ca2+ signals are not accompanied by RyR3-mediated CICR. We also propose that the source of Ca2+ influx, rather than its amplitude or the baseline Ca2+ level, is the primary determinant of the turning direction. In this way, axon-guiding and CAM-derived signals are integrated by RyR3, which serves as a key regulator of growth cone navigation.

Type-3 ryanodine receptor involved in Ca2+-induced Ca2+ release and transmitter exocytosis at frog motor nerve terminals.

Ca(2+)-induced Ca2+ release (CICR) occurs in frog motor nerve terminals after ryanodine receptors (RyRs) are primed for activation by conditioning large Ca2+ entry. We studied which type of RyR exists, whether CICR occurs without conditioning Ca2+ entry and how RyRs are primed. Immunohistochemistry revealed the existence of RyR3 in motor nerve terminals and axons and both RyR1 and RyR3 in muscle fibers. A blocker of RyR, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) slightly decreased rises in intracellular Ca2+ ([Ca2+]i) induced by a short tetanus (50 Hz, 1-2s), but not after treatment with ryanodine. Repetitive tetani (50 Hz for 15s every 20s) produced repetitive rises in [Ca2+]i, whose amplitude overall waxed and waned. TMB-8 blocked the waxing and waning components. Ryanodine suppressed a slow increase in end-plate potentials (EPPs) induced by stimuli (33.3 Hz, 15s) in a low Ca2+, high Mg2+ solution. KN-62, a blocker of Ca(2+)/calmoduline-activated protein kinase II (CaMKII), slightly reduced short tetanus-induced rises in [Ca2+]i, but markedly the slow waxing and waning rises produced by repetitive tetani in both normal and low Ca2+, high Mg2+ solutions. Likewise, KN-62, but not KN-04, an inactive analog, suppressed slow increases in EPP amplitude and miniature EPP frequency during long tetanus. Thus, CICR normally occurs weakly via RyR3 activation by single impulse-induced Ca2+ entry in frog motor nerve terminals and greatly after the priming of RyR via CaMKII activation by conditioning Ca2+ entry, thus, facilitating transmitter exocytosis and its plasticity.

Effects of ryanodine receptor activation on neurotransmitter release and neuronal cell death following kainic acid-induced status epilepticus.

Dynamic changes in intracellular free Ca(2+) concentration play a crucial role in various neural functions. The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and the ryanodine (Ry) receptor (RyR) are involved in Ca(2+)-induced Ca(2+)-release (CICR). Recent studies have shown that type 3 IP3R is highly expressed in rat hippocampal neurons after kainic acid (KA)-induced seizures and that dantrolene, a RyR antagonist, reduces KA-induced neuronal cell death. We investigated the RyR-associated effects of CICR agents on basal and K(+)-evoked releases of GABA and glutamate in rat hippocampus and the changes in expression of mRNA for RyRs in mouse brain after KA-induced seizures. The stimulatory effect of Ry on releases of GABA and glutamate was concentration-dependent in a biphasic manner. The inflection point in concentration-response curves for Ry on GABA release was lower than that for glutamate in both basal and K(+)-evoked conditions, suggesting that hyperactivation of RyR-associated CICR produces the imbalance between GABAergic and glutamatergic transmission. Following KA-induced seizures, transient up-regulation of brain-type RyR mRNA was observed in the hippocampal CA3 region and striatum, and signals for c-Fos mRNA increased transiently in the hippocampus, dentate gyrus and deeper layers of the neocortex. Thereafter, some dead neurons with single-stranded DNA (ssDNA) immunoreactive fragmented nuclei appeared in these areas. These findings suggest that intracellular Ca(2+) release via the RyR might be one of the mechanisms involved in KA-induced neuronal cell death.

Regulation of Ca2+ sparks by Ca2+ and Mg2+ in mammalian and amphibian muscle. An RyR isoform-specific role in excitation-contraction coupling?

Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks >100 microM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4-40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10(-7) in the frog or 10(-8) in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency.

RyR1/RyR3 chimeras reveal that multiple domains of RyR1 are involved in skeletal-type E-C coupling.

Skeletal-type E-C coupling is thought to require a direct interaction between RyR1 and the alpha(1S)-DHPR. Most available evidence suggests that the cytoplasmic II-III loop of the dihydropyridine receptor (DHPR) is the primary source of the orthograde signal. However, identification of the region(s) of RyR1 involved in bidirectional signaling with the alpha(1S)-DHPR remains elusive. To identify these regions we have designed a series of chimeric RyR cDNAs in which different segments of RyR1 were inserted into the corresponding region of RyR3 and expressed in dyspedic 1B5 myotubes. RyR3 provides a preferable background than RyR2 for defining domains essential for E-C coupling because it possesses less sequence homology to RyR1 than the RyR2 backbone used in previous studies. Our data show that two regions of RyR1 (chimera Ch-10 aa 1681-2641 and Ch-9 aa 2642-3770), were independently able to restore skeletal-type E-C coupling to RyR3. These two regions were further mapped and the critical RyR1 residues were 1924-2446 (Ch-21) and 2644-3223 (Ch-19). These results both support and refine the previous hypothesis that multiple domains of RyR1 combine to functionally interact with the DHPR during E-C coupling.

Ryanodine receptor type 3: why another ryanodine receptor isoform?

The family of ryanodine receptor (RyR) genes encodes three highly related Ca2+ release channels: RyR1, RyR2 and RyR3. Until about 10 years ago, RyRs were essentially known only for being the Ca2+ release channels of the sarcoplasmic reticulum of striated muscles, because of the high levels of expression of the RyR1 and RyR2 isoforms in skeletal and cardiac muscles, respectively. In contrast with the above picture, the RyR3 gene has been found not to be preferentially expressed in one specific tissue, but rather to be widely expressed in various cells. This wide expression pattern has been subsequently observed also for the RyR1 and RyR2 genes, which in addition to their preferential expression in striated muscles, have been found expressed also in several other cell types, although at lower levels than in striated muscles. Thus a closer look reveals that in several cells of vertebrates two or even three RyR isoforms can be co-expressed. In this chapter we will review published work on the RyR3 gene and discuss a model where co-expression of different RyR channel isoforms is interpreted as an evolutionary solution to provide, by functional interactions of distinct isoforms of Ca2+ release channels, the several types of vertebrate cells with the cell-specific Ca2+ release machinery required for generating the sophisticated intracellular Ca2+ signals needed for optimal regulation of their functions.

Regulation of mammalian ryanodine receptors.

Ryanodine receptors (RyRs) are large, high conductance Ca2+ channels that control the level of intracellular Ca2+ by releasing Ca2+ from an intracellular compartment, the sarco/endoplasmic reticulum. Mammalian tissues express 3 closely related ryanodine receptors (RyRs) known as skeletal muscle (RyR1), cardiac muscle (RyR2) and brain (RyR3). The RyRs are isolated as 30S protein complexes comprised of four 560 kDa RyR2 subunits and four 12.6 kDa FK506 binding protein (FKBP12.6) subunits. Multiple endogenous effector molecules and posttranslational modifications regulate the RyRs. This chapter reviews the regulation of the mammalian RyRs by endogenous effector molecules.

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.

Selectively suppressed Ca2+-induced Ca2+ release activity of alpha-ryanodine receptor (alpha-RyR) in frog skeletal muscle sarcoplasmic reticulum: potential distinct modes in Ca2+ release between alpha- and beta-RyR.

We reported earlier that the two ryanodine receptor (RyR) isoforms (alpha- and beta-RyR) purified from frog skeletal muscle were equipotent in the Ca(2+)-induced Ca(2+) release (CICR) activity (Murayama, T., Kurebayashi, N., and Ogawa, Y. (2000) Biophys. J. 78, 1810-1824). Whether this is also the case with the native Ca(2+) release channel in the sarcoplasmic reticulum (SR), however, remains to be determined. Taking advantage of the facts that [(3)H]ryanodine binds only to the open form of the channels and that it is practically irreversible at 4 degrees C, we devised a method to separate the total binding to contributions of alpha- and beta-RyR, using immunoprecipitation with an alpha-RyR-specific monoclonal antibody. Surprisingly, the binding of alpha-RyR was strongly suppressed to as low as approximately 4% that of beta-RyR in the SR vesicles. The two isoforms, however, showed no difference in sensitivity to Ca(2+), adenine nucleotides, or caffeine. This reduced binding of alpha-RyR was ascribed to the low affinity for [(3)H]ryanodine, with no change in the maximal binding sites. Solubilization of SR with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid partly remedied this nonequivalence, whereas 1 m NaCl was ineffective. 12-kDa FK506-binding protein (FKBP12), however, could not be responsible for it, because FK506 treatment did not eliminate the suppression, in contrast to marked removal of 12-kDa FK506-binding protein from alpha-RyR. These results suggest that alpha-RyR in the SR may serve Ca(2+) release in a mode other than CICR, being selectively suppressed in CICR.