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 language | English (US) |
|---|---|
| Pages (from-to) | 38453-38462 |
| Number of pages | 10 |
| Journal | Journal of Biological Chemistry |
| Volume | 285 |
| Issue number | 49 |
| DOIs | |
| State | Published - Dec 3 2010 |
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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 journal › Article
}
TY - JOUR
T1 - Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+
AU - Eltit, Jose M.
AU - Feng, Wei
AU - Lopez, Jose R.
AU - Padilla, Isela T.
AU - Pessah, Isaac N
AU - Molinski, Tadeusz F.
AU - Fruen, Bradley R.
AU - Allen, Paul D.
AU - Perez, Claudio F.
PY - 2010/12/3
Y1 - 2010/12/3
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=78649638443&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=78649638443&partnerID=8YFLogxK
U2 - 10.1074/jbc.M110.164525
DO - 10.1074/jbc.M110.164525
M3 - Article
C2 - 20926377
AN - SCOPUS:78649638443
VL - 285
SP - 38453
EP - 38462
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 49
ER -