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

Frontiers in Pharmacology / Electrophysiological Investigation of the Subcellular Fine Tuning of Sympathetic Neurons by Hydrogen Sulfide

HS is well-known as hypotensive agent, whether it is synthetized endogenously or administered systemically. Moreover, the HS donor NaHS has been shown to inhibit vasopressor responses triggered by stimulation of preganglionic sympathetic fibers. In contradiction with this latter result, NaHS has been reported to facilitate transmission within sympathetic ganglia. To resolve this inconsistency, HS and NaHS were applied to primary cultures of dissociated sympathetic ganglia to reveal how this gasotransmitter might act at different subcellular compartments of such neurons. At the somatodendritic region of ganglionic neurons, NaHS raised the frequency, but not the amplitudes, of cholinergic miniature postsynaptic currents via a presynaptic site of action. In addition, the HS donor as well as HS itself caused membrane hyperpolarization and decreased action potential firing in response to current injection. Submillimolar NaHS concentrations did not affect currents through K7 channels, but did evoke currents through KATP channels. Similarly to NaHS, the KATP channel activator diazoxide led to hyperpolarization and decreased membrane excitability; the effects of both, NaHS and diazoxide, were prevented by the KATP channel blocker tolbutamide. At postganglionic sympathetic nerve terminals, HS and NaHS enhanced noradrenaline release due to a direct action at the level of vesicle exocytosis. Taken together, HS may facilitate transmitter release within sympathetic ganglia and at sympatho-effector junctions, but causes hyperpolarization and reduced membrane excitability in ganglionic neurons. As this latter action was due to KATP channel gating, this channel family is hereby established as another previously unrecognized determinant in the function of sympathetic ganglia.(VLID)486035

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

Diatom Mimics: Directing the Formation of Biosilica Nanoparticles by Controlled Folding of Lysine-Leucine Peptides

Silaffins, long chain polyamines, and other biomolecules found in diatoms are involved in the assembly of a large number of silica nanostructures under mild, ambient conditions. Nanofabrication researchers have sought to mimic the diatom’s biosilica production capabilities by engineering proteins to resemble aspects of naturally occurring biomolecules. Such mimics can produce monodisperse biosilica nanospheres, but in vitro production of the variety of intricate biosilica nanostructures that compose the diatom frustule is not yet possible. In this study we demonstrate how LK peptides, composed solely of lysine (K) and leucine (L) amino acids arranged with varying hydrophobic periodicities, initiate the formation of different biosilica nanostructures in vitro. When L and K residues are arranged with a periodicity of 3.5 the α-helical form of the LK peptide produces monodisperse biosilica nanospheres. However, when the LK periodicity is changed to 3.0, corresponding to a 3₁₀ helix, the morphology of the nanoparticles changes to elongated rod-like structures. β-strand LK peptides with a periodicity of 2.0 induce wire-like silica morphologies. This study illustrates how the morphology of biosilica can be changed simply by varying the periodicity of polar and nonpolar amino acids

Induction by low Na(+) or Cl(−) of cocaine sensitive carrier-mediated efflux of amines from cells transfected with the cloned human catecholamine transporters

1. COS-7 cells transfected with the cDNA of the human dopamine transporter (DAT cells) or the human noradrenaline transporter (NAT cells) were loaded with [(3)H]-dopamine or [(3)H]-noradrenaline and superfused with buffers of different ionic composition. 2. In DAT cells lowering the Na(+) concentration to 0, 5 or 10 mM caused an increase in (3)H-efflux. Cocaine (10 μM) or mazindol (0.3 μM) blocked the efflux at low Na(+), but not at 0 Na(+). Lowering the Cl(−) concentration to 0, 5 or 10 mM resulted in an increased efflux, which was blocked by cocaine or mazindol. Desipramine (0.1 μM) was without effect in all the conditions tested. 3. In NAT cells, lowering the Na(+) concentration to 0, 5 or 10 mM caused an increase in (3)H-efflux, which was blocked by cocaine or mazindol. Desipramine produced a partial block, its action being stronger at 5 or 10 mM Na(+) than at 0 mM Na(+). Efflux induced by 0, 5 or 10 mM Cl(−) was completely blocked by all three uptake inhibitors. 4. In cross-loading experiments, 5 mM Na(+)- or 0 Cl(−)-induced efflux was much lower from [(3)H]-noradrenaline-loaded DAT, than NAT cells and was sensitive to mazindol, but not to desipramine. Efflux from [(3)H]-dopamine-loaded NAT cells elicited by 5 mM Na(+) or 0 Cl(−) was blocked by mazindol, as well as by desipramine. 5. Thus, cloned catecholamine transporters display carrier-mediated efflux of amines if challenged by lowering the extracellular Na(+) or Cl(−), whilst retaining their pharmacological profile. The transporters differ with regard to the ion dependence of the blockade of reverse transport by uptake inhibitors