S -nitrosylation and S -glutathionylation of Cys134 on troponin I have opposing competitive actions on Ca 2+ sensitivity in rat fast-twitch muscle fibers

S -nitrosylation and S -glutathionylation of Cys134 on troponin I have opposing competitive actions on Ca 2+ sensitivity in rat fast-twitch muscle fibers

Nitric oxide is generated in skeletal muscle with activity and decreases Ca sensitivity of the contractile apparatus, putatively by nitrosylation of an unidentified protein. We investigated the mechanistic basis of this effect and its relationship to the oxidation-induced increase in Ca sensitivity...

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Bibliographic Details
Published in:American Journal of Physiology: Cell Physiology Vol. 312; no. 3; p. C316
Main Authors: Dutka, T L, Mollica, J P, Lamboley, C R, Weerakkody, V C, Greening, D W, Posterino, G S, Murphy, R M, Lamb, G D
Format: Journal Article
Language:English
Published: United States 01-03-2017
Subjects:
Animals
Binding Sites
Calcium - metabolism
Calcium Signaling - physiology
Cells, Cultured
Chickens
Cysteine - chemistry
Cysteine - metabolism
Glutathione - metabolism
Humans
Isometric Contraction - physiology
Male
Muscle Fibers, Fast-Twitch - physiology
Nitric Oxide - metabolism
Protein Binding
Rats
Rats, Long-Evans
Rats, Sprague-Dawley
Species Specificity
Troponin I - chemistry
Troponin I - metabolism
Young Adult
oxidation
muscle fatigue
contractile apparatus
skinned muscle fiber
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Summary:Nitric oxide is generated in skeletal muscle with activity and decreases Ca sensitivity of the contractile apparatus, putatively by nitrosylation of an unidentified protein. We investigated the mechanistic basis of this effect and its relationship to the oxidation-induced increase in Ca sensitivity in mammalian fast-twitch (FT) fibers mediated by glutathionylation of Cys134 on fast troponin I (TnI ). Force-[Ca ] characteristics of the contractile apparatus in mechanically skinned fibers were assessed by direct activation with heavily Ca -buffered solutions. Treatment with nitrosylating agents, nitrosoglutathione (GSNO) or nitroso- -acetyl-penicillamine (SNAP), decreased pCa ( = -log [Ca ] at half-maximal activation) by ~-0.07 pCa units in rat and human FT fibers without affecting maximum force, but had no effect on rat and human slow-twitch fibers or toad or chicken FT fibers, which all lack Cys134. The Ca sensitivity decrease was ) fully reversed with dithiothreitol or reduced glutathione, ) at least partially reversed with ascorbate, indicative of involvement of S-nitrosylation, and ) irreversibly blocked by low concentration of the alkylating agent, -ethylmaleimide (NEM). The biotin-switch assay showed that both GSNO and SNAP treatments caused nitrosylation of TnI glutathionylation pretreatment blocked the effects of nitrosylation on Ca sensitivity, and vice-versa. nitrosylation pretreatment prevented NEM from irreversibly blocking glutathionylation of TnI and its effects on Ca sensitivity, and likewise glutathionylation pretreatment prevented NEM block of nitrosylation. Following substitution of TnI into rat slow-twitch fibers, nitrosylation treatment caused decreased Ca sensitivity. These findings demonstrate that nitrosylation and glutathionylation exert opposing effects on Ca sensitivity in mammalian FT muscle fibers, mediated by competitive actions on Cys134 of TnI .
ISSN:1522-1563
DOI:10.1152/ajpcell.00334.2016