H2O2 activates ryanodine receptor but has little effect on recovery of releasable Ca2+ content after fatigue

2002 ◽  
Vol 93 (6) ◽  
pp. 1999-2008 ◽  
Author(s):  
Toshiharu Oba ◽  
Chieko Kurono ◽  
Ritsuko Nakajima ◽  
Tetsuo Takaishi ◽  
Kazuto Ishida ◽  
...  
Keyword(s):  
Redox Potential ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Ryanodine Receptors ◽  
Control Level ◽  
Recovery Process ◽  
Channel Activity ◽  
Channel Activation ◽  
Stimulation Of

We studied whether hydrogen peroxide (H2O2) at ≤10 μM activates the ryanodine receptor and decreases releasable Ca2+ content in the sarcoplasmic reticulum after fatigue. Exposure of rabbit or frog skeletal muscle ryanodine receptors to 10 μM H2O2 enhanced channel activity in lipid bilayers when the redox potential was defined at cis = −220 mV and trans = −180 mV. Channel activation by 10 μM H2O2 was also observed when cispotential was set at −220 mV without defining transpotential, but the effect was less. Reduction of trans redox potential from −180 to −220 mV did not alter channel activity. H2O2 at 500 μM failed to activate the channel when the redox potential was not controlled. Stimulation of the frog muscle fiber for 2 min (50 Hz, a duty cycle of 200 ms/s) decreased tetanus tension by ∼50%. After 1 min, tetanus recovered rapidly to ∼70% of control and thereafter slowly approached the control level. Amplitudes of caffeine- and 4-chloro- m-cresol-induced contractures were decreased after a 60-min rest. The decrease is not enhanced by exposure to 10 μM H2O2. These results suggest that H2O2 markedly activates the ryanodine receptor under the redox control in vitro, but externally applied H2O2 may not play an important role in the postfatigue recovery process.

Na+ pump inhibition downregulates an ATP-sensitive K+ channel in rabbit proximal convoluted tubule

1993 ◽  
Vol 264 (4) ◽  
pp. F760-F764 ◽  
Author(s):  
A. M. Hurst ◽  
J. S. Beck ◽  
R. Laprade ◽  
J. Y. Lapointe
Keyword(s):  
Basolateral Membrane ◽  
Proximal Convoluted Tubule ◽  
K Channel ◽  
Bathing Solution ◽  
Channel Activity ◽  
Open Probability ◽  
Functional Link ◽  
Tightly Coupled ◽  
Stimulation Of

In several epithelial and nonepithelial tissues a functional link between the basolateral Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) and a basolateral K+ conductance has been established. However, the nature of this link is unclear. We have previously identified a K+ channel on the basolateral membrane of the proximal convoluted tubule perfused in vitro, the activity of which is increased by stimulation of Na+ transport [J. S. Beck, A. M. Hurst, J.-Y. Lapointe, and R. Laprade. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F496-F501, 1993]. In the present study we investigate whether basolateral membrane K+ channel activity is tightly coupled to Na(+)-K(+)-ATPase activity. In cell-attached patches (150 mM K+ pipette), following stimulation of channel activity by addition of Na(+)-cotransported solutes to the tubule lumen, mean channel open probability (NPo) was reduced from 0.35 +/- 0.09 to 0.14 +/- 0.06 (n = 7, P < 0.05) by blocking the Na(+)-K(+)-ATPase with 100 microM strophanthidin. In excised patches the channel was reversibly blocked by 2 mM ATP from the cytosolic face of the patch, such that NPo fell to 20.1 +/- 7.0% (n = 5, P < 0.001) of control and recovered to 52.2 +/- 11.2% (n = 5, P < 0.05) after washout of ATP. Diazoxide, a putative opener of ATP-sensitive K+ channels, when added to the bathing solution of an unstimulated tubule (microperfused in the absence of Na(+)-cotransported solutes), increased NPo from 0.046 +/- 0.035 to 0.44 +/- 0.2 (n = 6, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Different interactions of cardiac and skeletal muscle ryanodine receptors with FK-506 binding protein isoforms

1997 ◽  
Vol 272 (5) ◽  
pp. C1726-C1733 ◽  
Author(s):  
S. Barg ◽  
J. A. Copello ◽  
S. Fleischer
Keyword(s):  
Skeletal Muscle ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Ryanodine Receptors ◽  
Protein Isoforms ◽  
Channel Activity ◽  
Functional Interaction ◽  
Fk 506 ◽  
Receptor Isoforms ◽  
Functional Consequences

In the present study, we compare functional consequences of dissociation and reconstitution of binding proteins FKBP12 and FKBP12.6 with ryanodine receptors from cardiac (RyR2) and skeletal muscle (RyR1). The skeletal muscle RyR1 channel became activated on removal of endogenously bound FKBP12, consistent with previous reports. Both FKBP12 and FKBP12.6 rebind to FKBP-depleted RyR1 and restore its quiescent channel behavior by altering ligand sensitivity, as studied by single-channel recordings in planar lipid bilayers, and macroscopic behavior of the channels (ryanodine binding and net energized Ca2- uptake). By contrast, removal of FKBP12.6 from the cardiac RyR2 did not modulate the function of the channel using the same types of assays as for RyR1. FKBP12 or FKBP12.6 had no effect on channel activity of FKBP12.6-depleted cardiac RyR2, although FKBP12.6 rebinds. Our studies reveal important differences between the two ryanodine receptor isoforms with respect to their functional interaction with FKBP12 and FKBP12.6.

scholarly journals Modification of ryanodine receptor/Ca2+ release channel with dinitrofluorobenzene

1999 ◽  
Vol 342 (1) ◽  
pp. 239-248 ◽  
Author(s):  
Nurit HADAD ◽  
Wei FENG ◽  
Varda SHOSHAN-BARMATZ
Keyword(s):  
Ryanodine Receptor ◽  
Single Channel ◽  
Ruthenium Red ◽  
Amino Group ◽  
Channel Activity ◽  
Closed Channel ◽  
Release Channel ◽  
Atp Binding Site ◽  
Ph Values ◽  
Stimulation Of

Modification of the ryanodine receptor (RyR)/Ca2+ release channel with 2,4-dinitrofluorobenzene (DNFB) indicated that two classes of amino group interact with the reagent, as can be distinguished on the basis of their reactivity/accessibility and the effects on ryanodine binding and single channel activities. One group interacted very rapidly (t½ < 30 s) at 25 °C with low concentrations of DNFB [C50 (concentration of DNFB required for 50% inhibition or stimulation of ryanodine binding) = 5 μM], and at pH values of 6.2 and higher. This interaction resulted in the marked stimulation of ryanodine binding and the complete inhibition of a single Ca2+ release channel incorporated into planar lipid bilayer. The second group is accessible at higher temperatures (37 °C); at pH values higher than 7.4 it reacted slowly (t½ = 20 min) with high concentrations of DNFB (C50 = 70 μM). This interaction led to the inhibition of ryanodine binding and single channel activity. Modification of RyR with DNFB under the stimulatory conditions resulted in 3.6-fold and 6-fold increases in ryanodine-binding and Ca2+-binding affinities respectively. Modification with DNFB under the inhibitory conditions resulted in a decrease in the total ryanodine-binding sites. The exposure of the RyR single channel to DNFB under both inhibitory and stimulatory conditions led to the complete closure of the channel. However, when modified under the stimulatory conditions, but not under the inhibitory ones, the DNFB-modified closed channel could be re-activated by sub-micromolar concentrations of ryanodine, in the presence of nanomolar concentrations of Ca2+. The DNFB-modified ryanodine-activated RyR channel showed fast transitions between open, closed and several sub-conductance states, and was completely closed by Ruthenium Red. ATP re-activated the DNFB-modified closed channel or, if present during modification, prevented the inhibition of RyR channel activity by DNFB. Neither the stimulation nor the inhibition of ryanodine binding by modification with DNFB was affected by the presence of ATP. By using the photoreactive ATP analogue 3′-O-(4-benzoyl)benzoyl-[α-32P]ATP we found that DNFB modification had no effect on the ATP-binding site of RyR. The results are discussed with regard to the involvement of amino group residues in channel gating, ryanodine association/dissociation and occlusion, and the relationship between the open/closed state of the RyR and its capacity to bind ryanodine.

scholarly journals A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria

2004 ◽  
Vol 85 (2) ◽  
pp. 451-461 ◽  
Author(s):  
Stephen D. C. Griffin ◽  
Ruth Harvey ◽  
Dean S. Clarke ◽  
Wendy S. Barclay ◽  
Mark Harris ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Ion Channel ◽  
Lipid Bilayers ◽  
Mammalian Cells ◽  
Intracellular Localization ◽  
Channel Activity ◽  
Diarrhoea Virus ◽  
Basic Loop

We previously identified the function of the hepatitis C virus (HCV) p7 protein as an ion channel in artificial lipid bilayers and demonstrated that this in vitro activity is inhibited by amantadine. Here we show that the ion channel activity of HCV p7 expressed in mammalian cells can substitute for that of influenza virus M2 in a cell-based assay. This was also the case for the p7 from the related virus, bovine viral diarrhoea virus (BVDV). Moreover, amantadine was shown to abrogate HCV p7 function in this assay at a concentration that specifically inhibits M2. Mutation of a conserved basic loop located between the two predicted trans-membrane alpha helices rendered HCV p7 non-functional as an ion channel. The intracellular localization of p7 was unaffected by this mutation and was found to overlap significantly with membranes associated with mitochondria. Demonstration of p7 ion channel activity in cellular membranes and its inhibition by amantadine affirm the protein as a target for future anti-viral chemotherapy.

Spontaneous Ryanodine-Receptor-Dependent Ca2+-Activated K+ Currents and Hyperpolarizations in Rat Medial Preoptic Neurons

2010 ◽  
Vol 103 (5) ◽  
pp. 2900-2911 ◽  
Author(s):  
Göran Klement ◽  
Michael Druzin ◽  
David Haage ◽  
Evgenya Malinina ◽  
Peter Århem ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Ryanodine Receptors ◽  
Underlying Mechanism ◽  
Sk Channels ◽  
Patch Clamp Technique ◽  
Channel Activation ◽  
Impulse Generation ◽  
Outward Currents ◽  
Main Charge ◽  
Perforated Patch Clamp

The aim of the present study was to clarify the identity of slow spontaneous currents, the underlying mechanism and possible role for impulse generation in neurons of the rat medial preoptic nucleus (MPN). Acutely dissociated neurons were studied with the perforated patch-clamp technique. Spontaneous outward currents, at a frequency of ∼0.5 Hz and with a decay time constant of ∼200 ms, were frequently detected in neurons when voltage-clamped between approximately −70 and −30 mV. The dependence on extracellular K+ concentration was consistent with K+ as the main charge carrier. We concluded that the main characteristics were similar to those of spontaneous miniature outward currents (SMOCs), previously reported mainly for muscle fibers and peripheral nerve. From the dependence on voltage and from a pharmacological analysis, we concluded that the currents were carried through small-conductance Ca2+-activated (SK) channels, of the SK3 subtype. From experiments with ryanodine, xestospongin C, and caffeine, we concluded that the spontaneous currents were triggered by Ca2+ release from intracellular stores via ryanodine receptor channels. An apparent voltage dependence was explained by masking of the spontaneous currents as a consequence of steady SK-channel activation at membrane potentials > −30 mV. Under current-clamp conditions, corresponding transient hyperpolarizations occasionally exceeded 10 mV in amplitude and reduced the frequency of spontaneous impulses. In conclusion, MPN neurons display spontaneous hyperpolarizations triggered by Ca2+ release via ryanodine receptors and SK3-channel activation. Thus such events may affect impulse firing of MPN neurons.

scholarly journals PKA phosphorylation of SUR2B subunit underscores vascular KATP channel activation by beta-adrenergic receptors

2007 ◽  
Vol 293 (3) ◽  
pp. R1205-R1214 ◽  
Author(s):  
Yun Shi ◽  
Zhongying Wu ◽  
Ningren Cui ◽  
Weiwei Shi ◽  
Yang Yang ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Channel Activity ◽  
Channel Activation ◽  
Hek 293 ◽  
293 Cells ◽  
Vascular Katp Channel ◽  
Pka Pathway ◽  
Smooth Myocytes ◽  
Inside Out

ATP-sensitive K+ (KATP) channels are activated by several vasodilating hormones and neurotransmitters through the PKA pathway. Here, we show that phosphorylation at Ser1387 of the SUR2B subunit is critical for the channel activation. Experiments were performed in human embryonic kidney (HEK) 293 cells expressing the cloned Kir6.1/SUR2B channel. In whole cell patch, the Kir6.1/SUR2B channel activity was stimulated by isoproterenol via activation of β2 receptors. This effect was blocked in the presence of inhibitors for adenylyl cyclase or PKA. Similar channel activation was seen by exposing inside-out patches to the catalytic subunit of PKA. Because none of the previously suggested PKA phosphorylation sites accounted for the channel activation, we performed systematic mutational analysis on Kir6.1 and SUR2B. Two serine residues (Ser1351, Ser1387) located in the NBD2 of SUR2B were critical for the channel activation. In vitro phosphorylation experiments showed that Ser1387 but not Ser1351 was phosphorylated by PKA. The PKA-dependent activation of cell-endogenous KATP channels was observed in acutely dissociated mesenteric smooth myocytes and isolated mesenteric artery rings, where activation of these channels contributed significantly to the isoproterenol-induced vasodilation. Taken together, these results indicate that the Kir6.1/SUR2B channel is a target of β2 receptors and that the channel activation relies on PKA phosphorylation of SUR2B at Ser1387.

H2O2 and ethanol act synergistically to gate ryanodine receptor/calcium-release channel

2000 ◽  
Vol 279 (5) ◽  
pp. C1366-C1374 ◽  
Author(s):  
Toshiharu Oba ◽  
Tatsuya Ishikawa ◽  
Takashi Murayama ◽  
Yasuo Ogawa ◽  
Mamoru Yamaguchi
Keyword(s):  
Skeletal Muscle ◽  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Calcium Release ◽  
Physiological Role ◽  
Channel Activity ◽  
Calcium Release Channel ◽  
Open Probability ◽  
Skeletal Muscle Function ◽  
Release Channel

We examined the effect of low concentrations of H2O2 on the Ca2+-release channel/ryanodine receptor (RyR) to determine if H2O2 plays a physiological role in skeletal muscle function. Sarcoplasmic reticulum vesicles from frog skeletal muscle and type 1 RyRs (RyR1) purified from rabbit skeletal muscle were incorporated into lipid bilayers. Channel activity of the frog RyR was not affected by application of 4.4 mM (0.02%) ethanol. Open probability ( P o) of such ethanol-treated RyR channels was markedly increased on subsequent addition of 10 μM H2O2. Increase of H2O2to 100 μM caused a further increase in channel activity. Application of 4.4 mM ethanol to 10 μM H2O2-treated RyRs activated channel activity. Exposure to 10 or 100 μM H2O2 alone, however, failed to increase P o. Synergistic action of ethanol and H2O2 was also observed on the purified RyR1 channel, which was free from FK506 binding protein (FKBP12). H2O2 at 100–500 μM had no effect on purified channel activity. Application of FKBP12 to the purified RyR1 drastically decreased channel activity but did not alter the effects of ethanol and H2O2. These results suggest that H2O2 may play a pathophysiological, but probably not a physiological, role by directly acting on skeletal muscle RyRs in the presence of ethanol.

Involvement of the Ryanodine Receptor in Morphologic Modification of Hermissenda Type B Photoreceptors After In Vitro Conditioning

2004 ◽  
Vol 91 (2) ◽  
pp. 728-735 ◽  
Author(s):  
Ryo Kawai ◽  
Tetsuro Horikoshi ◽  
Manabu Sakakibara
Keyword(s):  
Ryanodine Receptor ◽  
Hair Cells ◽  
Ryanodine Receptors ◽  
Terminal Branch ◽  
Type B ◽  
Axon Terminals ◽  
Calcium Chelation ◽  
Intracellular Ca ◽  
Receptor Blockers

We examined whether Ca2+ induced Ca2+ release through ryanodine receptors is involved in the conditioning of specific morphologic changes at the axon terminals of type B photoreceptors in the isolated circumesophageal ganglion of Hermissenda. Calcium chelation by bis(2-aminophenoxy) ethane- N,N,N′, N′-tetraacetic acid prevented the conformational change at the terminals after five paired presentations of light and vibration, which produce terminal branch contraction of B photoreceptors. Two ryanodine receptor blockers, dantrolene and micromolar concentrations of ryanodine, depressed the increase in excitability due to in vitro conditioning and the increase in intracellular Ca2+ in response to membrane depolarization. Although the ability to increase intracellular Ca2+ was depressed, synaptic transmission was preserved in the normal state from hair cells under dantrolene and ryanodine incubation. Ryanodine receptor blockers also prevented contraction at the B photoreceptor axon terminals. These results suggest that the ryanodine receptor has a crucial role in inducing the in vitro conditioning specific changes both physiologically and morphologically, including “focusing” at the B photoreceptor axon terminal.

Actions of sulfhydryl reagents on single ryanodine receptor Ca(2+)-release channels from sheep myocardium

1997 ◽  
Vol 272 (6) ◽  
pp. C1908-C1918 ◽  
Author(s):  
K. R. Eager ◽  
L. D. Roden ◽  
A. F. Dulhunty
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptor ◽  
Channel Activity ◽  
Irreversible Loss ◽  
Sulfhydryl Reagents ◽  
Open Probability ◽  
Open Time ◽  
Wide Range ◽  
The Mean ◽  
Channel Open Time

Effects of the reactive disulfides, 2,2'- and 4,4'-dithiodipyridine, on single cardiac ryanodine receptor (RyR) ion channels incorporated into lipid bilayers are reported. RyRs are activated within minutes of addition of the reactive disulfides (10(-7) to 10(-3) M) with an irreversible loss of channel activity after the activation at concentrations > or = 10(-4) M. This activation, followed by loss of activity, is seen over a wide range of cytoplasmic (cis) Ca2+ concentration between 10(-9) and 2 x 10(-2) M and occurs more rapidly with higher reactive disulfide concentrations or when RyRs are initially active at 10(-5) or 10(-3) M Ca2+. The reactive disulfides increase the channel open probability by introducing long components into the open time distributions, increasing the mean channel open time by up to 50-fold. Closed time distributions are not altered by the sulfhydryl reagents. The effects of the reactive disulfides are prevented by the reducing agents dithiothreitol and glutathione (1–10 mM). The results suggest that cysteine residues on the RyR complex can regulate the ion channel gating mechanisms.

scholarly journals Niflumic acid differentially modulates two types of skeletal ryanodine-sensitive Ca2+-release channels

1997 ◽  
Vol 273 (5) ◽  
pp. C1588-C1595 ◽  
Author(s):  
Toshiharu Oba
Keyword(s):  
Lipid Bilayers ◽  
Ryanodine Receptors ◽  
Reversal Potential ◽  
Current Treatment ◽  
Ruthenium Red ◽  
Niflumic Acid ◽  
Open Probability ◽  
Activation Curve ◽  
Release Channel ◽  
Channel Activation

The effects of niflumic acid on ryanodine receptors (RyRs) of frog skeletal muscle were studied by incorporating sarcoplasmic reticulum (SR) vesicles into planar lipid bilayers. Frog muscle had two distinct types of RyRs in the SR: one showed a bell-shaped channel activation curve against cytoplasmic Ca2+ or niflumic acid, and its mean open probability ( P o) was increased by perchlorate at 20–30 mM (termed “α-like” RyR); the other showed a sigmoidal activation curve against Ca2+ or niflumic acid, with no effect on perchlorate (termed “β-like” RyR). The unitary conductance and reversal potential of both channel types were unaffected after exposure to niflumic acid when clamped at 0 mV. When clamped at more positive potentials, the β-like RyR channel rectified this, increasing the unitary current. Treatment with niflumic acid did not inhibit the response of both channels to Ca2+ release channel modulators such as caffeine, ryanodine, and ruthenium red. The different effects of niflumic acid on P o and the unitary current amplitude in both types of channels may be attributable to the lack or the presence of inactivation sites and/or distinct responses to agonists.

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