Activation and inhibition of purified skeletal muscle calcium release channel by NO donors in single channel current recordings

AbstractThe actions of the nitric oxide (NO) donors 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3 methyl-1-triazine (NOC-7), S-nitrosoacetylcysteine (CySNO) and S-nitrosoglutathione (GSNO) on the purified calcium release channel (ryanodine receptor) of rabbit skeletal muscle were determined by single channel current recordings. In addition, the activation of the NO donor modulated calcium release channel by the sulfhydryl oxidizing organic mercurial compound 4-(chloromercuri)phenylsulfonic acid (4-CMPS) was investigated. NOC-7 (0.1 and 0.3 mM) and CySNO (0.4 and 0.8 mM) increased the open probability (Po) of the calcium release channel at activating calcium concentrations (20–100 μM Ca2+) by 60–100%, with no effect on the current amplitude; this activation was abolished by the specific sulfhydryl reducing agent DTT. High concentrations of CySNO (1.6–2 mM) decreased Po. Activation by GSNO (1 mM) was observed in two thirds of the experiments, but 2 mM and 4 mM GSNO markedly reduced Po at activating Ca2+ (20–100 μM). In contrast to 4-CMPS, NOC-7 or GSNO had no effect at subactivating free Ca2+ (0.6 μM). 4-CMPS further increased the open probability of NOC-7- or CySNO-stimulated channels and reversed transiently the reduced open probability of CySNO or GSNO inhibited channels at activating free Ca2+. High concentrations of GSNO did not prevent channel activation of 4-CMPS at subactivating free Ca2+. The NOC-7-, CySNO- or GSNO-modified channels were completely blocked by ruthenium red. It is suggested that nitrosylation/oxidation of sulfhydryls by NO donors and oxidation of sulfhydryls by 4-CMPS affect different cysteine residues essential in the gating of the calcium release channel

Short-and Long-Term Functional Alterations of the Skeletal Muscle Calcium Release Channel (Ryanodine Receptor) by Suramin: Apparent Dissociation of Single Channel Current Recording and [ 3 H]Ryanodine Binding

ABSTRACT The present study demonstrates the following characteristic suramin actions on the purified skeletal muscle calcium release channel in single-channel current recordings and

Electron Spin Resonance Studies on Conformational Changes of the Sarcoplasmic Reticulum Ca2+Ca^{2+} – ATPase Induced by Synergistic Action of Calcium and ATP

Abstract Changes in motional properties of the -SH group environment in sarcoplasmic reticulum ATPase [1] induced by addition of specific ligands to sarcoplasmic reticulum membrane vesicles were investigated systematically by electron spin resonance (e.p.r.) spectroscopy. Alternatively, two kinds of iodoacetamide analog spin labels, 4-(2-iodoacetamido)- 2,2,5,5-tetramethyl-1-pyrrolidinyl-N-oxyl (label I) and 4-(2-iodoacetamido)-2,2,6,6-tetramethyl- 1-piperidinyl-N-oxyl (label II) were used. The labeling conditions were chosen such that less than three moles of -SH groups per mole of ATPase reacted with the spin labels. A marked increase in isotropic motion of either spin label was observed on addition of calcium in millimolar concentrations plus ATP or β-γ-methylene ATP. Qualitatively similar but smaller changes were also observed with inosine 5'-triphosphate (ITP), acetylphosphate, or ADP in the presence of calcium. These effects were independent of added magnesium. The spectral changes induced by β-γ-methylene ATP, which binds to the ATPase but is not hydrolyzed, and those of calcium plus ADP suggest a conformational change due to simultaneous binding of these ligands in the absence of enzyme phosphorylation. These changes in e.p.r. spectra were different in quality and magnitude from those observed upon separate binding of calcium or adenine nucleotides. The two spin labels, differing only in their numbers of heterocyclic ring atoms, were found to reflect environmental changes in different ways. This demonstrates the usefulness of employing different spin labels to detect and interpret structural transitions in macromolecular assemblies.</jats:p

Nicotinic acid-adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor.

Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca(2+)-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca(2+)-release from intracellular Ca(2+) stores can be triggered by diffusible second messengers like Ins P (3), 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 Ca(2+)-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca(2+)-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 Ca(2+)-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC(50) approximately 30 nM) 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 0.25 nmol/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