We characterized type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RyR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe.

The effect of H2O2 was examined to elucidate the basis of muscle injury after exercise. Exposure of single fibers to 1.5-6 mM H2O2 led to twitch potentiation followed by a marked decrease. Then, fibers contracted spontaneously. BAY K 8644 augmented twitch potentiation and slowed the decay of twitches. In 5 mM dithiothreitol (DTT), twitch potentiation and spontaneous contraction were not observed on H2O2 addition. Cytoplasmic application of 1.5-3 mM H2O2 to heavy sarcoplasmic reticulum (SR) vesicles incorporated into planar lipid bilayers increased the open probability of Ca2+ release channels, an effect reversed by DTT. We investigated oxidation of sulfhydryl groups on proteins in SR membrane by H2O2 with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Pretreatment of light and heavy SR membranes with 1.5 mM H2O2 exponentially increased fluorescence intensity. The time constant of the intensity increase was increased markedly only in heavy SR in solution containing 50 microM cytoplasmic Ca2+, so Ca2+ release was associated with protein oxidation by H2O2. Thus extracellular H2O2 probably acts by oxidizing sulfhydryls of proteins at two distinct sites: the dihydropyridine receptors, oxidation of which elicits potentiation and subsequent inhibition of twitches, and Ca2+ release channels, whose oxidation elicits spontaneous contraction, resulting in muscle dysfunction.

The type 1 ryanodine receptor (RyR1) from rabbit skeletal muscle displayed two distinct degrees of response to cytoplasmic Ca2+ [high- and low-open probability ( P o) channels]. Here, we examined the effects of adenine nucleotides and caffeine on these channels and their modulations by sulfhydryl reagents. High- P o channels showed biphasic Ca2+ dependence and were activated by adenine nucleotides and caffeine. Unexpectedly, low- P o channels did not respond to either modulator. The addition of a reducing reagent, dithiothreitol, to the cis side converted the high- P o channel to a state similar to that of the low- P o channel. Treatment with p-chloromercuriphenylsulfonic acid (pCMPS) transformed low- P o channels to a high- P o channel-like state with stimulation by β,γ-methylene-ATP and caffeine. In experiments under redox control using glutathione buffers, shift of the cis potential toward the oxidative state activated the low- P ochannel, similar to that of the high- P o or the pCMPS-treated channel, whereas reductive changes inactivated the high- P o channel. Changes in transredox potential, in contrast, did not affect channel activity of either channel. In all experiments, channels with higher P o were stimulated to a great extent by modulators, but ones with lower P o were unresponsive. These results suggest that redox states of critical sulfhydryls located on the cytoplasmic side of the RyR1 may alter both gating properties of the channel and responsiveness to channel modulators.

Effects of niflumic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) on frog skeletal muscle ryanodine receptors have been studied by incorporating sarcoplasmic reticulum vesicles into planar lipid bilayers. Niflumic acid increased the mean open probability (Po) at 10 microM and decreased Po at 100 microM with no change in open time constants, unitary conductance, and reversal potential. The Po was augmented by DIDS at 5-200 microM without affecting either unitary conductance or reversal potential. DIDS induced a new third open time constant, probably contributing to a long-lived open state. Channels modified by niflumic acid or DIDS still responded to Ca2+ release channel modulators. These results provide evidence that niflumic acid and DIDS modify the gating mechanism of ryanodine receptors without affecting binding sites to the modulators and the physical pathway of the conducting pore. p-Chloromercuriphenyl sulfonic acid (pCMPS) transiently increased the Po. The channel modified by DIDS responded to pCMPS, whereas that by ryanodine did not. The long open state of the channel induced by DIDS is produced by a quite different mechanism(s) from that by ryanodine. Contrary to cardiac ryanodine receptors, Po of skeletal muscle channels was independent of voltage after DIDS modification.

Ryanodine receptor (RyR) type 1 (RyR1) exhibits a markedly lower gain of Ca(2+)-induced Ca(2+) release (CICR) activity than RyR type 3 (RyR3) in the sarcoplasmic reticulum (SR) of mammalian skeletal muscle (selective stabilization of the RyR1 channel), and this reduction in the gain is largely eliminated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). We have investigated whether the hypothesized interdomain interactions within RyR1 are involved in the selective stabilization of the channel using [(3)H]ryanodine binding, single-channel recordings, and Ca(2+) release from the SR vesicles. Like CHAPS, domain peptide 4 (DP4, a synthetic peptide corresponding to the Leu(2442)-Pro(2477) region of RyR1), which seems to destabilize the interdomain interactions, markedly stimulated RyR1 but not RyR3. Their activating effects were saturable and nonadditive. Dantrolene, a potent inhibitor of RyR1 used to treat malignant hyperthermia, reversed the effects of DP4 or CHAPS in an identical manner. These findings indicate that RyR1 is activated by DP4 and CHAPS through a common mechanism that is probably mediated by the interdomain interactions. DP4 greatly increased [(3)H]ryanodine binding to RyR1 with only minor alterations in the sensitivity to endogenous CICR modulators (Ca(2+), Mg(2+), and adenine nucleotide). However, DP4 sensitized RyR1 four- to six-fold to caffeine in the caffeine-induced Ca(2+) release. Thus the gain of CICR activity critically determines the magnitude and threshold of Ca(2+) release by drugs such as caffeine. These findings suggest that the low CICR gain of RyR1 is important in normal Ca(2+) handling in skeletal muscle and that perturbation of this state may result in muscle diseases such as malignant hyperthermia.

In a recently developed theory of light diffraction by single striated muscle fibers, we considered only the case of normal beam incidence. The present investigation represents both an experimental and theoretical extension of the previous work to arbitrary incident angle. Angle scan profiles over a 50 degrees range of incident angle (+25 degrees to -25 degrees) were obtained at different sarcomere lengths. Left and right first-order scan peak separations were found to be a function of sarcomere length (separation angle = 2 theta B), and good agreement was found between theory and experiment. Our theoretical analysis further showed that a myofibrillar population with a single common skew angle can yield an angle scan profile containing many peaks. Thus, it is not necessary to associate each peak with a different skew population. Finally, we have found that symmetry angle, theta s, also varies with sarcomere length, but not in a regular manner. Its value at a given sarcomere length is a function of a particular region of a given fiber and represents the average skew angle of all the myofibril populations illuminated. The intensity of a diffraction order line is considered to be principally the resultant of two interference phenomena. The first is a volume-grating phenomenon which results from the periodic A-I band structure of the fiber (with some contribution from Z bands and H zones). The second is Bragg reflection from skew planes, if the correct relation between incident angle and skew angle is met. This may result in intensity asymmetry between the left and right first order lines.

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.

The mechanism underlying H2O2-induced activation of frog skeletal muscle ryanodine receptors was studied using skinned fibers and by measuring single Ca2+-release channel current. Exposure of skinned fibers to 3–10 mM H2O2 elicited spontaneous contractures. H2O2 at 1 mM potentiated caffeine contracture. When the Ca2+-release channels were incorporated into lipid bilayers, open probability ( P o) and open time constants were increased on intraluminal addition of H2O2 in the presence of cis catalase, but unitary conductance and reversal potential were not affected. Exposure to cis H2O2 at 1.5 mM failed to activate the channel in the presence of trans catalase. Application of 1.5 mM H2O2 to the transside of a channel that had been oxidized by cis p-chloromercuriphenylsulfonic acid (pCMPS; 50 μM) still led to an increase in P o, comparable to that elicited by trans 1.5 mM H2O2 without pCMPS. Addition of cis pCMPS to channels that had been treated with or without trans H2O2 rapidly resulted in high P o followed by closure of the channel. These results suggest that oxidation of luminal sulfhydryls in the Ca2+-release channel may contribute to H2O2-induced channel activation and muscle contracture.