Single-channel properties of skeletal muscle ryanodine receptor pore Δ4923FF4924 in two brothers with a lethal form of fetal akinesia

Ca2+ release from intracellular stores is controlled by complex interactions between multiple proteins. Triadin is a transmembrane glycoprotein of the junctional sarcoplasmic reticulum of striated muscle that interacts with both calsequestrin and the type 1 ryanodine receptor (RyR1) to communicate changes in luminal Ca2+ to the release machinery. However, the potential impact of the triadin association with RyR1 in skeletal muscle excitation–contraction coupling remains elusive. Here we show that triadin binding to RyR1 is critically important for rapid Ca2+ release during excitation–contraction coupling. To assess the functional impact of the triadin-RyR1 interaction, we expressed RyR1 mutants in which one or more of three negatively charged residues (D4878, D4907, and E4908) in the terminal RyR1 intraluminal loop were mutated to alanines in RyR1-null (dyspedic) myotubes. Coimmunoprecipitation revealed that triadin, but not junctin, binding to RyR1 was abolished in the triple (D4878A/D4907A/E4908A) mutant and one of the double (D4907A/E4908A) mutants, partially reduced in the D4878A/D4907A double mutant, but not affected by either individual (D4878A, D4907A, E4908A) mutations or the D4878A/E4908A double mutation. Functional studies revealed that the rate of voltage- and ligand-gated SR Ca2+ release were reduced in proportion to the degree of interruption in triadin binding. Ryanodine binding, single channel recording, and calcium release experiments conducted on WT and triple mutant channels in the absence of triadin demonstrated that the luminal loop mutations do not directly alter RyR1 function. These findings demonstrate that junctin and triadin bind to different sites on RyR1 and that triadin plays an important role in ensuring rapid Ca2+ release during excitation–contraction coupling in skeletal muscle.

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A central core disease mutation in the Ca2+-binding site of skeletal muscle ryanodine receptor impairs single-channel regulation

AJP Cell Physiology ◽

10.1152/ajpcell.00052.2019 ◽

2019 ◽

Vol 317 (2) ◽

pp. C358-C365 ◽

Cited By ~ 3

Author(s):

Venkat R. Chirasani ◽

Le Xu ◽

Hannah G. Addis ◽

Daniel A. Pasek ◽

Nikolay V. Dokholyan ◽

...

Keyword(s):

Skeletal Muscle ◽

Amino Acid ◽

Missense Mutation ◽

Ryanodine Receptor ◽

Single Channel ◽

Central Core ◽

Central Core Disease ◽

Hek293 Cells ◽

Amino Acid Residues ◽

Single Channel Recordings

Cryoelectron microscopy and mutational analyses have shown that type 1 ryanodine receptor (RyR1) amino acid residues RyR1-E3893, -E3967, and -T5001 are critical for Ca2+-mediated activation of skeletal muscle Ca2+ release channel. De novo missense mutation RyR1-Q3970K in the secondary binding sphere of Ca2+ was reported in association with central core disease (CCD) in a 2-yr-old boy. Here, we characterized recombinant RyR1-Q3970K mutant by cellular Ca2+ release measurements, single-channel recordings, and computational methods. Caffeine-induced Ca2+ release studies indicated that RyR1-Q3970K formed caffeine-sensitive, Ca2+-conducting channel in HEK293 cells. However, in single-channel recordings, RyR1-Q3970K displayed low Ca2+-dependent channel activity and greatly reduced activation by caffeine or ATP. A RyR1-Q3970E mutant corresponds to missense mutation RyR2-Q3925E associated with arrhythmogenic syndrome in cardiac muscle. RyR1-Q3970E also formed caffeine-induced Ca2+ release in HEK293 cells and exhibited low activity in the presence of the activating ligand Ca2+ but, in contrast to RyR1-Q3970K, was activated by ATP and caffeine in single-channel recordings. Computational analyses suggested distinct structural rearrangements in the secondary binding sphere of Ca2+ of the two mutants, whereas the interaction of Ca2+ with directly interacting RyR1 amino acid residues Glu3893, Glu3967, and Thr5001 was only minimally affected. We conclude that RyR1-Q3970 has a critical role in Ca2+-dependent activation of RyR1 and that a missense RyR1-Q3970K mutant may give rise to myopathy in skeletal muscle.

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Nicotinic acid–adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor

Biochemical Journal ◽

10.1042/bj20020584 ◽

2002 ◽

Vol 367 (2) ◽

pp. 423-431 ◽

Cited By ~ 79

Author(s):

Martin HOHENEGGER ◽

Josef SUKO ◽

Regina GSCHEIDLINGER ◽

Helmut DROBNY ◽

Andreas ZIDAR

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Nicotinic Acid ◽

Ryanodine Receptor ◽

Spatial Information ◽

Single Channel ◽

Reversal Potential ◽

Open Probability ◽

Release Channel

Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca2+-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca2+-release from intracellular Ca2+ stores can be triggered by diffusible second messengers like InsP3, cyclic ADP-ribose or nicotinic acid—adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca2+-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca2+-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca2+-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC5030nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel.

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Chloroform extract of hog barn dust modulates skeletal muscle ryanodine receptor calcium-release channel (RyR1)

Journal of Applied Physiology ◽

10.1152/japplphysiol.00123.2010 ◽

2010 ◽

Vol 109 (3) ◽

pp. 830-839 ◽

Cited By ~ 4

Author(s):

Chengju Tian ◽

Chun Hong Shao ◽

Danielle S. Fenster ◽

Mark Mixan ◽

Debra J. Romberger ◽

...

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Muscle Weakness ◽

Calcium Release ◽

Single Channel ◽

Calcium Release Channel ◽

Open Probability ◽

Skeletal Muscle Function ◽

Release Channel ◽

Skeletal Muscle Weakness

Skeletal muscle weakness is a reported ailment in individuals working in commercial hog confinement facilities. To date, specific mechanisms responsible for this symptom remain undefined. The purpose of this study was to assess whether hog barn dust (HBD) contains components that are capable of binding to and modulating the activity of type 1 ryanodine receptor Ca2+-release channel (RyR1), a key regulator of skeletal muscle function. HBD collected from confinement facilities in Nebraska were extracted with chloroform, filtered, and rotary evaporated to dryness. Residues were resuspended in hexane-chloroform (20:1) and precipitates, referred to as HBDorg, were air-dried and studied further. In competition assays, HBDorg dose-dependently displaced [3H]ryanodine from binding sites on RyR1 with an IC50 of 1.5 ± 0.1 μg/ml ( Ki = 0.4 ± 0.0 μg/ml). In single-channel assays using RyR1 reconstituted into a lipid bilayer, HBDorg exhibited three distinct dose-dependent effects: first it increased the open probability of RyR1 by increasing its gating frequency and dwell time in the open state, then it induced a state of reduced conductance (55% of maximum) that was more likely to occur and persist at positive holding potentials, and finally it irreversibly closed RyR1. In differentiated C2C12 myotubes, addition of HBD triggered a rise in intracellular Ca2+ that was blocked by pretreatment with ryanodine. Since persistent activation and/or closure of RyR1 results in skeletal muscle weakness, these new data suggest that HBD is responsible, at least in part, for the muscle ailment reported by hog confinement workers.

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Reconstitution of the ATP-sensitive potassium channel of skeletal muscle. Activation by a G protein-dependent process.

The Journal of General Physiology ◽

10.1085/jgp.94.3.445 ◽

1989 ◽

Vol 94 (3) ◽

pp. 445-463 ◽

Cited By ~ 36

Author(s):

L Parent ◽

R Coronado

Keyword(s):

Skeletal Muscle ◽

G Protein ◽

Muscle Activation ◽

Open Channel ◽

Single Channel ◽

Rabbit Skeletal Muscle ◽

S 100 ◽

Internal Side ◽

Gamma S ◽

Channel Properties

Potassium channels inhibited by adenosine-5'-trisphosphate, K(ATP), found in the transverse tubular membrane of rabbit skeletal muscle were studied using the planar bilayer recording technique. In addition to the single-channel properties of K(ATP) we report its regulation of Mg2+ and by the guanosine-5'-trisphosphate analogue, GTP-y(gamma)-S. The K(ATP) channel (a) has a conductance of 67 pS in 250 mM internal, 50 mM external KCl, and rectifies weakly at holding potentials more positive than 50 mV, (b) is not activated by internal Ca2+ or membrane depolarization, (c) has a permeability ratio PK/PNa greater than 50, and (d) is inhibited by millimolar internal ATP. Activity of K(ATP), measured as open channel probability as a function of time, was unstable at all holding potentials and decreases continuously within a few minutes after a recording is initiated. After a decrease in activity, GTP-y-S (100 microM) added to the internal side reactivated K(ATP) channels but only transiently. In the presence of internal 1 mM Mg2+, GTP-y-S produced a sustained reactivation lasting 20-45 min. Incubation of purified t-tubule vesicles with AlF4 increased the activity of K(ATP) channels, mimicking the effect of GTP-y-S. The effect of AlF4 and the requirement of GTP-y-S plus Mg2+ for sustained channel activation suggests that a nucleotide-binding G protein regulates ATP-sensitive K channels in the t-tuble membrane of rabbit skeletal muscle.

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Activation and labelling of the purified skeletal muscle ryanodine receptor by an oxidized ATP analogue

Biochemical Journal ◽

10.1042/bj3080119 ◽

1995 ◽

Vol 308 (1) ◽

pp. 119-125 ◽

Cited By ~ 3

Author(s):

M Hohenegger ◽

A Herrmann-Frank ◽

M Richter ◽

F Lehmann-Horn

Keyword(s):

Skeletal Muscle ◽

Ryanodine Receptor ◽

Single Channel ◽

Adenine Nucleotide ◽

Nucleotide Binding ◽

Binding Pocket ◽

Hill Coefficient ◽

Dependent Manner ◽

Open Probability ◽

Release Channel

We have tested the periodate-oxidized ATP analogue 2′,3′-dialdehyde adenosine triphosphate (oATP) as a ligand for the skeletal muscle ryanodine receptor/Ca(2+)-release channel. Ca2+ efflux from passively loaded heavy sarcoplasmic reticulum vesicles of skeletal muscle is biphasic. oATP stimulates the initial phase of Ca2+ release in a concentration-dependent manner (EC50 160 microM), and the efflux proceeds with a half-time in the range 100-200 ms. This oATP-modulated initial rapid Ca2+ release was specifically inhibited by millimolar concentrations of Mg2+ and micromolar concentrations of Ruthenium Red, indicating that the effect of oATP was mediated via the ryanodine receptor. The purified Ca(2+)-release channel was incorporated into planar lipid bilayers, and single-channel recordings were carried out to verify a direct interaction of oATP with the ryanodine receptor. Addition of oATP to the cytoplasmic side activated the channel with an EC50 of 76 microM, which is roughly 30-fold higher than the apparent affinity of ATP. The oATP-induced increase in the open probability of the ryanodine receptor displays a steep concentration-response curve with a Hill coefficient of approximately 2, which suggests a co-operativity of the ATP binding sites in the tetrameric protein. oATP binds to the ryanodine receptor in a quasi-irreversible manner via Schiff base formation between the aldehyde groups of oATP and amino groups in the nucleotide binding pocket. This allows for the covalent specific incorporation of [alpha-32P]oATP by borhydride reduction. A typical adenine nucleotide binding site cannot be identified in the primary sequence of the ryanodine receptor. Our results demonstrate that oATP can be used to probe the structure and function of the nucleotide binding pocket of the ryanodine receptor and presumably of other ATP-regulated ion channels.

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Chicken skeletal muscle ryanodine receptor isoforms: ion channel properties

Biophysical Journal ◽

10.1016/s0006-3495(94)80665-4 ◽

1994 ◽

Vol 67 (5) ◽

pp. 1834-1850 ◽

Cited By ~ 64

Author(s):

A.L. Percival ◽

A.J. Williams ◽

J.L. Kenyon ◽

M.M. Grinsell ◽

J.A. Airey ◽

...

Keyword(s):

Skeletal Muscle ◽

Ion Channel ◽

Ryanodine Receptor ◽

Receptor Isoforms ◽

Ion Channel Properties ◽

Channel Properties ◽

Chicken Skeletal Muscle

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Simulation of Calcium Sparks in Cut Skeletal Muscle Fibers of the Frog

The Journal of General Physiology ◽

10.1085/jgp.200308787 ◽

2003 ◽

Vol 121 (4) ◽

pp. 311-324 ◽

Cited By ~ 29

Author(s):

W.K. Chandler ◽

S. Hollingworth ◽

S.M. Baylor

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Ryanodine Receptor ◽

Single Channel ◽

Total Concentration ◽

Extreme Case ◽

Volume Integral ◽

Calcium Sparks ◽

Proportionality Constant ◽

Buffering Power

Spark mass, the volume integral of ΔF/F, was investigated theoretically and with simulations. These studies show that the amount of Ca2+ bound to fluo-3 is proportional to mass times the total concentration of fluo-3 ([fluo-3T]); the proportionality constant depends on resting Ca2+ concentration ([Ca2+]R). In the simulation of a Ca2+ spark in an intact frog fiber with [fluo-3T] = 100 μM, fluo-3 captures approximately one-fourth of the Ca2+ released from the sarcoplasmic reticulum (SR). Since mass in cut fibers is several times that in intact fibers, both with similar values of [fluo-3T] and [Ca2+]R, it seems likely that SR Ca2+ release is larger in cut fiber sparks or that fluo-3 is able to capture a larger fraction of the released Ca2+ in cut fibers, perhaps because of reduced intrinsic Ca2+ buffering. Computer simulations were used to identify these and other factors that may underlie the differences in mass and other properties of sparks in intact and cut fibers. Our spark model, which successfully simulates calcium sparks in intact fibers, was modified to reflect the conditions of cut fiber measurements. The results show that, if the protein Ca2+-buffering power of myoplasm is the same as that in intact fibers, the Ca2+ source flux underlying a spark in cut fibers is 5–10 times that in intact fibers. Smaller source fluxes are required for less buffer. In the extreme case in which Ca2+ binding to troponin is zero, the source flux needs to be 3–5 times that in intact fibers. An increased Ca2+ source flux could arise from an increase in Ca2+ flux through one ryanodine receptor (RYR) or an increase in the number of active RYRs per spark, or both. These results indicate that the gating of RYRs, or their apparent single channel Ca2+ flux, is different in frog cut fibers—and, perhaps, in other disrupted preparations—than in intact fibers.

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Postulated role of interdomain interactions within the type 1 ryanodine receptor in the low gain of Ca2+-induced Ca2+ release activity of mammalian skeletal muscle sarcoplasmic reticulum

AJP Cell Physiology ◽

10.1152/ajpcell.00415.2004 ◽

2005 ◽

Vol 288 (6) ◽

pp. C1222-C1230 ◽

Cited By ~ 17

Author(s):

Takashi Murayama ◽

Toshiharu Oba ◽

Shigeki Kobayashi ◽

Noriaki Ikemoto ◽

Yasuo Ogawa

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Malignant Hyperthermia ◽

Ryanodine Receptor ◽

Single Channel ◽

Common Mechanism ◽

Mammalian Skeletal Muscle ◽

Selective Stabilization ◽

Interdomain Interactions

Ryanodine receptor (RyR) type 1 (RyR1) exhibits a markedly lower gain of Ca2+-induced Ca2+ 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 [3H]ryanodine binding, single-channel recordings, and Ca2+ release from the SR vesicles. Like CHAPS, domain peptide 4 (DP4, a synthetic peptide corresponding to the Leu2442-Pro2477 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 [3H]ryanodine binding to RyR1 with only minor alterations in the sensitivity to endogenous CICR modulators (Ca2+, Mg2+, and adenine nucleotide). However, DP4 sensitized RyR1 four- to six-fold to caffeine in the caffeine-induced Ca2+ release. Thus the gain of CICR activity critically determines the magnitude and threshold of Ca2+ release by drugs such as caffeine. These findings suggest that the low CICR gain of RyR1 is important in normal Ca2+ handling in skeletal muscle and that perturbation of this state may result in muscle diseases such as malignant hyperthermia.

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