Evidence for the transport of glutathione through ryanodine receptor channel type 1

In the present study, we have investigated the role of RyR1 (ryanodine receptor calcium channel type 1) in glutathione (GSH) transport through the sarcoplasmic reticulum (SR) membrane of skeletal muscles. Lanthanum chloride, a prototypic blocker of cation channels, inhibited the influx and efflux of GSH in SR vesicles. Using a rapid-filtration-based assay and lanthanum chloride as a transport blocker, an uptake of radiolabelled GSH into SR vesicles was observed. Pretreatment of SR vesicles with the RyR1 antagonists Ruthenium Red and ryanodine as well as with lanthanum chloride blocked the GSH uptake. An SR-like GSH uptake appeared in microsomes obtained from an HEK-293 (human embryonic kidney 293) cell line after transfection of RyR1. These observations strongly suggest that RyR1 mediates GSH transport through the SR membranes of skeletal muscles.

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Role of Amino-terminal Half of the S4-S5 Linker in Type 1 Ryanodine Receptor (RyR1) Channel Gating

Journal of Biological Chemistry ◽

10.1074/jbc.m111.255240 ◽

2011 ◽

Vol 286 (41) ◽

pp. 35571-35577 ◽

Cited By ~ 14

Author(s):

Takashi Murayama ◽

Nagomi Kurebayashi ◽

Toshiharu Oba ◽

Hideto Oyamada ◽

Katsuji Oguchi ◽

...

Keyword(s):

Ryanodine Receptor ◽

Single Channel ◽

Signal Transmission ◽

Channel Gating ◽

Helical Structure ◽

Release Channel ◽

Amino Terminal ◽

Molecular Events

The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca2+ itself, i.e. Ca2+-induced Ca2+ release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr4825–Ser4829) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca2+ release, single-channel current recordings, and [3H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca2+ sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.

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The harp seal, Pagophilus groenlandicus (Erxleben, 1777). VII. A histophysiological study of certain skeletal muscles

Canadian Journal of Zoology ◽

10.1139/z71-006 ◽

1971 ◽

Vol 49 (1) ◽

pp. 25-30 ◽

Cited By ~ 17

Author(s):

J. C. George ◽

N. V. Vallyathan ◽

K. Ronald

Keyword(s):

Skeletal Muscles ◽

Biochemical Properties ◽

Histochemical Demonstration ◽

Harp Seal ◽

Activity Levels ◽

Sodium Pentobarbital ◽

Pagophilus Groenlandicus

A quantitative and histochemical study of Ms. pectoralis, gluteus, psoas, diaphragma (dorsal, lateral, and ventral parts separately) of the harp seal was carried out. Myoglobin and iron contents of all the muscles were high, highest being of M. psoas. Fat and glycogen were low in all muscles. The low glycogen value was possibly due to rapid glycolysis during struggle in capture and while the animal was under an overdose of sodium pentobarbital that was administered for killing it. Phosphorylase and succinate dehydrogenase (SDH) activity levels were also low. Lipase (tributyrinase) activity was high, that of M. diaphragma being higher than the other muscles studied. The histochemical investigation revealed some of the morphological and biochemical properties of the component fibers. Two types of fiber, the red (type 1) and white (type 2), comparable to those of other skeletal muscles were distinguished in all muscles except in the case of M. pectoralis where an intermediate type was also seen in sections treated for the histochemical demonstration of SDH activity.The low levels of fat and SDH in the muscles indicate that fat is not a favored metabolite for muscular energy. The significance of the high lipase (tributyrinase) activity is doubtful. It is suggested that the role of this enzyme is to clear the fat so as to prevent the accumulation of fat in the muscles which are, by and large, geared for a glycolytic metabolism as an adaptation for the animal's diving habit. The high myoglobin content of the muscles, however, should provide the oxygen necessary for the oxidation of glucose during the initial part of the dive.

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Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor

Biochemical Journal ◽

10.1042/bj20080489 ◽

2008 ◽

Vol 414 (3) ◽

pp. 441-452 ◽

Cited By ~ 102

Author(s):

Huihui Kong ◽

Peter P. Jones ◽

Andrea Koop ◽

Lin Zhang ◽

Henry J. Duff ◽

...

Keyword(s):

Ryanodine Receptor ◽

Cardiac Arrhythmias ◽

Single Channel ◽

Kidney Cells ◽

Human Embryonic Kidney ◽

Activation Threshold ◽

Hek 293 ◽

Human Embryonic Kidney Cells ◽

293 Cells ◽

High Concentrations

Caffeine has long been used as a pharmacological probe for studying RyR (ryanodine receptor)-mediated Ca2+ release and cardiac arrhythmias. However, the precise mechanism by which caffeine activates RyRs is elusive. In the present study, we investigated the effects of caffeine on spontaneous Ca2+ release and on the response of single RyR2 (cardiac RyR) channels to luminal or cytosolic Ca2+. We found that HEK-293 cells (human embryonic kidney cells) expressing RyR2 displayed partial or ‘quantal’ Ca2+ release in response to repetitive additions of submaximal concentrations of caffeine. This quantal Ca2+ release was abolished by ryanodine. Monitoring of endoplasmic reticulum luminal Ca2+ revealed that caffeine reduced the luminal Ca2+ threshold at which spontaneous Ca2+ release occurs. Interestingly, spontaneous Ca2+ release in the form of Ca2+ oscillations persisted in the presence of 10 mM caffeine, and was diminished by ryanodine, demonstrating that unlike ryanodine, caffeine, even at high concentrations, does not hold the channel open. At the single-channel level, caffeine markedly reduced the threshold for luminal Ca2+ activation, but had little effect on the threshold for cytosolic Ca2+ activation, indicating that the major action of caffeine is to reduce the luminal, but not the cytosolic, Ca2+ activation threshold. Furthermore, as with caffeine, the clinically relevant, pro-arrhythmic methylxanthines aminophylline and theophylline potentiated luminal Ca2+ activation of RyR2, and increased the propensity for spontaneous Ca2+ release, mimicking the effects of disease-linked RyR2 mutations. Collectively, our results demonstrate that caffeine triggers Ca2+ release by reducing the threshold for luminal Ca2+ activation of RyR2, and suggest that disease-linked RyR2 mutations and RyR2-interacting pro-arrhythmic agents may share the same arrhythmogenic mechanism.

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Human Types 1 and 3 3α-Hydroxysteroid Dehydrogenases: Differential Lability and Tissue Distribution1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.86.2.7216 ◽

2001 ◽

Vol 86 (2) ◽

pp. 841-846 ◽

Cited By ~ 1

Author(s):

Isabelle Dufort ◽

Fernand Labrie ◽

Van Luu-The

Keyword(s):

Hacat Keratinocytes ◽

Human Embryonic Kidney ◽

Intact Cells ◽

Hek 293 ◽

Human Type ◽

Hydroxysteroid Dehydrogenases ◽

293 Cells ◽

Hydroxy Compounds ◽

Type 3

3α-Hydroxysteroid dehydrogenases (3α-HSDs) catalyze the conversion of 3-ketosteroids to 3α-hydroxy compounds. The best known 3α-HSD activity is the transformation of the most potent natural androgen, dihydrotestosterone, into 5α-androstan-3α,17β-diol (3α-diol), a compound having much lower activity. Previous reports show that 3α-HSDs are involved in the metabolism of glucocorticoids, progestins, prostaglandins, bile acid precursors, and xenobiotics. 3α-HSDs could, thus, play a crucial role in the control of a series of active steroid levels in target tissues. In the human, type 1 3α-HSD was first identified as human chlordecone reductase. Recently, we have isolated and characterized type 3 3α-HSD that shares 81.7% identity with human type 1 3α-HSD. The transfection of vectors expressing types 1 and 3 3α-HSD in transformed human embryonic kidney (HEK-293) cells indicates that both enzymes efficiently catalyze the transformation of dihydrotestosterone into 3α-diol in intact cells. However, when the cells are broken, the activity of type 3 3α-HSD is rapidly lost, whereas the type 1 3α-HSD activity remains stable. We have previously found that human type 5 17β-HSD which possesses 84% and 86% identity with types 1 and 3 3α-HSD, respectively, is also labile, whereas rodent enzymes such as mouse type 5 17β-HSD and rat 3α-HSD are stable after homogenization of the cells. The variable stability of different enzymatic activities in broken cell preparations renders the comparison of different enzymes difficult. RNA expression analysis indicates that human type 1 3α-HSD is expressed exclusively in the liver, whereas type 3 is more widely expressed and is found in the liver, adrenal, testis, brain, prostate, and HaCaT keratinocytes. Based on enzymatic characteristics and sequence homology, it is suggested that type 1 3α-HSD is an ortholog of rat 3α-HSD while type 3 3α-HSD, which must have diverged recently, seems unique to human and is probably more involved in intracrine activity.

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Mg2+ activates the ryanodine receptor type 2 (RyR2) at intermediate Ca2+ concentrations

AJP Cell Physiology ◽

10.1152/ajpcell.00275.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. C535-C544 ◽

Cited By ~ 18

Author(s):

Akihito Chugun ◽

Osamu Sato ◽

Hiroshi Takeshima ◽

Yasuo Ogawa

Keyword(s):

Cardiac Muscle ◽

Ryanodine Receptor ◽

Adenine Nucleotide ◽

Receptor Type ◽

Inhibitory Effects ◽

Cardiac Muscles ◽

Partial Suppression

To clarify whether activity of the ryanodine receptor type 2 (RyR2) is reduced in the sarcoplasmic reticulum (SR) of cardiac muscle, as is the case with the ryanodine receptor type 1 (RyR1), Ca2+-dependent [3H]ryanodine binding, a biochemical measure of Ca2+-induced Ca2+ release (CICR), was determined using SR vesicle fractions isolated from rabbit and rat cardiac muscles. In the absence of an adenine nucleotide or caffeine, the rat SR showed a complicated Ca2+ dependence, instead of the well-documented biphasic dependence of the rabbit SR. In the rat SR, [3H]ryanodine binding initially increased as [Ca2+] increased, with a plateau in the range of 10–100 μM Ca2+, and thereafter further increased to an apparent peak around 1 mM Ca2+, followed by a decrease. In the presence of these modulators, this complicated dependence prevailed, irrespective of the source. Addition of 0.3–1 mM Mg2+ unexpectedly increased the binding two- to threefold and enhanced the affinity for [3H]ryanodine at 10–100 μM Ca2+, resulting in the well-known biphasic dependence. In other words, the partial suppression of RyR2 is relieved by Mg2+. Ca2+ could be a substitute for Mg2+. Mg2+ also amplifies the responses of RyR2 to inhibitory and stimulatory modulators. This stimulating effect of Mg2+ on RyR2 is entirely new, and is referred to as the third effect, in addition to the well-known dual inhibitory effects. This effect is critical to describe the role of RyR2 in excitation-contraction coupling of cardiac muscle, in view of the intracellular Mg2+ concentration.

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Role of Ryanodine Receptor Channel and Mechanisms of Vascular Diseases

Biophysical Journal ◽

10.1016/j.bpj.2017.11.1759 ◽

2018 ◽

Vol 114 (3) ◽

pp. 311a ◽

Cited By ~ 1

Author(s):

Yun-Min Zheng ◽

Yong-Xiao Wang

Keyword(s):

Ryanodine Receptor ◽

Vascular Diseases ◽

Receptor Channel

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Heat hypersensitivity of ryanodine receptor type 1 mutants implicated in malignant hyperthermia

10.1101/2020.10.29.351452 ◽

2020 ◽

Author(s):

Kotaro Oyama ◽

Vadim Zeeb ◽

Toshiko Yamazawa ◽

Takashi Murayama ◽

Hideto Oyamada ◽

...

Keyword(s):

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Positive Feedback ◽

Intracellular Signaling ◽

Feedback Loop ◽

Local Heat ◽

Receptor Type ◽

Positive Feedback Loop ◽

Hek 293

AbstractCellular heat-sensing is a universal strategy for avoiding thermal damage and adapting to environments by regulating thermogenic activities. If heat-sensing results in the acceleration of processes governing cellular thermogenesis, hyperthermia can occur. However, how this positive feedback loop contributes to hyperthermia development, especially the gap between heat-sensing and thermogenesis, remains largely unknown. Here, we show that an optically controlled local heat pulse induces an intracellular Ca2+ burst in cultured HEK 293 cells overexpressing ryanodine-receptor-type-1 (RyR1) mutants related to the life-threatening illness malignant hyperthermia (MH), and that the Ca2+ burst originates from heat-induced Ca2+-release (HICR) because of the mutant channels’ heat hypersensitivity. Furthermore, the heat hypersensitivity of the four RyR1 mutants was ranked, highlighting the complexity of MH. Our findings reveal the novel cellular heat-sensing mechanism, HICR, is essential for the functional positive feedback loop causing MH, suggesting a well-tuned HICR is fundamental for heat-mediated intracellular signaling.

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