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

doi:10.1074/jbc.m110.164525

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+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](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. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](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 Ca(2+) entry at rest.

Highlights

In skeletal muscle, where control of cytosolic Ca2ϩ concentration is key to muscle contraction, the overall Ca2ϩ homeostasis is preserved by a concerted action of several ionic channels and transporters within the plasma membrane and the sarcoplasmic reticulum (SR).3 These proteins, including plasma membrane Ca2ϩ ATPase, Naϩ/Ca2ϩ exchanger, dihydropyridine receptor, ryanodine receptor (RyRs), and the sarcoendoplasmic reticulum Ca2ϩ ATPase, among others, interact functionally and physically with each other and with a plethora of regulatory proteins that modulate their activity, the resting intracellular Ca2ϩ levels
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
In a recent study aimed at unmasking the role of triadin in EC coupling, we found that the lack of triadin expression in skeletal myotubes and adult fibers caused a significantly elevated [Ca2ϩ]rest with no obvious changes in RyR1 expression level or activity [14], suggesting a role for triadin in regulating resting Ca2ϩ homeostasis

Summary

Introduction

In skeletal muscle, where control of cytosolic Ca2ϩ concentration is key to muscle contraction, the overall Ca2ϩ homeostasis is preserved by a concerted action of several ionic channels and transporters within the plasma membrane and the sarcoplasmic reticulum (SR).3 These proteins, including plasma membrane Ca2ϩ ATPase, Naϩ/Ca2ϩ exchanger, dihydropyridine receptor, ryanodine receptor (RyRs), and the sarcoendoplasmic reticulum Ca2ϩ ATPase, among others, interact functionally and physically with each other and with a plethora of regulatory proteins that modulate their activity, the resting intracellular Ca2ϩ levels. Whereas overnight incubation with 15 ␮M ryanodine, a condition that totally inhibited caffeine-induced Ca2ϩ release (data not shown), did not seem to affect [Ca2ϩ]rest in WT cells (116 Ϯ 7 nM versus 123 Ϯ 8 nM, p Ͼ 0.05), the triadin-null myotubes showed a significant reduction in [Ca2ϩ]rest from 181 Ϯ 11 nM to 167 Ϯ 10 nM (p Ͻ 0.05, Fig. 1), revealing an increased basal activity of the RyR1 channel population.

Results

Conclusion