Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+

Jose M. Eltit, Wei Feng, Jose R. Lopez, Isela T. Padilla, Isaac N Pessah, Tadeusz F. Molinski, Bradley R. Fruen, Paul D. Allen, Claudio F. Perez

Research output: Contribution to journalArticle

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Abstract

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca 2+([Ca2+ rest), suggesting a role for triadins in modulating global intracellular Ca2+ homeostasis. To understand this mechanism, we study here how triadin alters [Ca2+ rest, Ca2+ release, and Ca2+ entry pathways using a combination of Ca2+ microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca2+, and Mn2+ imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca2+] rest that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn2+ quench rates under resting conditions indicate that triadin-null cells also have higher Ca2+ entry rates and lower sarcoplasmic reticulum Ca2+ load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca2+ entry, recovering sarcoplasmic reticulum Ca2+ content levels, and restoring near normal [Ca2+]rest. Exogenous FKBP12.6 also reduced the RyR1 channel Po but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced Po nor recovered multiple subconductance gating. These data suggest that elevated [Ca2+] rest in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca2+ entry at rest.

Original languageEnglish (US)
Pages (from-to)38453-38462
Number of pages10
JournalJournal of Biological Chemistry
Volume285
Issue number49
DOIs
StatePublished - Dec 3 2010

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Tacrolimus Binding Protein 1A
Ryanodine Receptor Calcium Release Channel
Ablation
Muscle
Skeletal Muscle
Null Lymphocytes
Skeletal Muscle Fibers
Lipid bilayers
Lipid Bilayers
Sarcoplasmic Reticulum
Membrane Lipids
Ryanodine
triadin
Microelectrodes
Muscle Cells
Homeostasis
Cells
Phenotype
Imaging techniques
Muscles

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+ . / Eltit, Jose M.; Feng, Wei; Lopez, Jose R.; Padilla, Isela T.; Pessah, Isaac N; Molinski, Tadeusz F.; Fruen, Bradley R.; Allen, Paul D.; Perez, Claudio F.

In: Journal of Biological Chemistry, Vol. 285, No. 49, 03.12.2010, p. 38453-38462.

Research output: Contribution to journalArticle

Eltit, Jose M. ; Feng, Wei ; Lopez, Jose R. ; Padilla, Isela T. ; Pessah, Isaac N ; Molinski, Tadeusz F. ; Fruen, Bradley R. ; Allen, Paul D. ; Perez, Claudio F. / Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+ In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 49. pp. 38453-38462.
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abstract = "Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca 2+([Ca2+ rest), suggesting a role for triadins in modulating global intracellular Ca2+ homeostasis. To understand this mechanism, we study here how triadin alters [Ca2+ rest, Ca2+ release, and Ca2+ entry pathways using a combination of Ca2+ microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca2+, and Mn2+ imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca2+] rest that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn2+ quench rates under resting conditions indicate that triadin-null cells also have higher Ca2+ entry rates and lower sarcoplasmic reticulum Ca2+ load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca2+ entry, recovering sarcoplasmic reticulum Ca2+ content levels, and restoring near normal [Ca2+]rest. Exogenous FKBP12.6 also reduced the RyR1 channel Po but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced Po nor recovered multiple subconductance gating. These data suggest that elevated [Ca2+] rest in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca2+ entry at rest.",
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AU - Padilla, Isela T.

AU - Pessah, Isaac N

AU - Molinski, Tadeusz F.

AU - Fruen, Bradley R.

AU - Allen, Paul D.

AU - Perez, Claudio F.

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