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

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.

FIGURE 1.

Average resting free Ca²⁺ concentration measured from individual myotubes of WT and triadin-null myotubes in the presence (+) and in the absence (−) of the indicated concentrations of Ca²⁺, ryanodine (Rya), and bastadin-5 (B-5). Ca²⁺-free solutions were also supplemented with 0.5 mm Cd²⁺ and 0.1 mm La³⁺ to block Ca²⁺ entry. Calcium measurements were regularly acquired 5–10 min post-modification of the extracellular medium. Numbers on the bars indicate the number of cells analyzed per each condition. Data are presented as mean ± S.D. **, p < 0.01; ***, p < 0.001 with respect to untreated cells. a, data from Ref. .

FIGURE 2.

Effect of triadin on FKBP12 expression and its binding to RyR1. A, equivalent amounts of crude membrane homogenates from WT and triadin-null myotubes were loaded and immunoblotted for expression of RyR1, DHPR α_1S, CSQ-1, junctin, and FKBP12. Band intensity is expressed as fraction of the WT signal (dotted line). Numbers on the bars indicate the number of blots analyzed per protein. Data are presented as mean ± S.E. *, p < 0.05; ***, p < 0.01. B, Western blot analysis of the protein complex pulled down by 34C antibody from 300 μg of crude homogenates of WT and triadin-null (KO) muscles. SR, 2 μg of SR vesicle. Average band intensity of FKBP12 signal that co-precipitate with RyR1 in WT and triadin-null (KO) homogenates is expressed as fraction of the RyR1 signal (p > 0.05). C, binding of a fluorescent FKBP12 to crude membrane homogenates isolated from WT and triadin-null muscle (○, total binding; ●, specific binding; □, nonspecific binding). Data are expressed as a function of the relative amount of RyR1 measure by [³H]ryanodine binding. Values of K_d and B_max (mean ± S.E.) are based on fits of averaged data (n = three experiments) to a model assuming a single, saturable binding site (SigmaPlot software, San Jose, CA).

FIGURE 3.

RyR1 triadin-null channel exhibits more predominantly uncoordinated conduction. RyR1 single channels from either WT or triadin-null skeletal muscle membrane preps were induced to incorporate into BLM as described under “Experimental Procedures.” Free Ca²⁺ in cis and trans were 1 and 100 μm, respectively. Channel opening and closing fluctuations were indicated by an arrow with “o” and “c”, respectively. The tetrameric RyR1 channel subconductance levels (G_n = i, ii, iii, and iv) were indicated with dashed lines. P_o, mean open and closed dwell times, and current amplitude histograms were obtained with the software pClamp 9.

FIGURE 4.

FKPB12.6 but not FKBP12 reduces P_o of RyR1 triadin-null channels, although neither eliminates pronounced subconductance gating. RyR1 triadin-null channels were incorporated from fused heavy SR fraction vesicles in bilayers and were recorded for at least 2 min before being exposed to 200 nm FKBP12.6 or 400 nm FKBP12 cis. A, shown is a representative channel (from n = five independent single channel measurements) in the presence of 1/100 μm Ca²⁺ (cis/trans) and 2 mm Na₂ATP (cis). B, shown is a representative single channel recorded (from n = four independent measurements) in the presence of 0.05/1 mm Ca²⁺ (cis/trans) and 2 mm Na₂ATP (cis). C, the mean P_o ± S.D. before and after addition of FKBP12.6 (n = 5) or FKBP12 (n = 4) is shown.

FIGURE 5.

Triadin ablation reduces SR Ca²⁺ content in null myotubes. A, representative traces of caffeine-induced Ca²⁺ transients of Fura-4F loaded cells. B, effect of FKBP12.6 overexpression on the average SR Ca²⁺ content of WT and triadin-null myotubes, estimated from the peak Ca²⁺ transient amplitude. ***, p < 0.001. Data are presented as mean ± S.D. Numbers on the bars indicate the number of cells analyzed per condition.

FIGURE 6.

Effect of FKBP12.6 expression on resting Ca²⁺ levels in cultured myotubes. Overexpression of FKBP12 significantly lowered resting Ca²⁺ concentration in triadin-null myotubes but not in WT cells. Prevention of global Ca²⁺ entry with 0.5 mm Cd²⁺ and 0.1 mm La³⁺ lowered Ca²⁺ concentrations even further to levels similar to that of WT cells. Unlike Cd²⁺/La³⁺, the inhibition of SOCE with BTP-2 failed to restore normal resting Ca²⁺. ***, p < 0.001. Data are presented as mean ± S.D. Numbers on the bars indicate the number of cells analyzed per condition.

FIGURE 7.

Effect of BTP-2 and FKBP12.6 expression on Ca²⁺ entry in cultured myotubes. A, representative traces of Fura-2 fluorescence quench by 500 μm Mn²⁺ measured under resting condition. Triadin-null myotubes (KO) displayed faster rates of Mn²⁺ entry (slope indicated by dashed lines) than WT cells. B, comparison of the average rate of Mn²⁺ entry between WT and triadin-null myotubes before and after a 5-min incubation with 2 μm BTP-2. C, effect of overexpression of FKBP12.6 on average rate of Mn²⁺ entry in the absence and in the presence of BTP-2. *, p < 0.05; **, p < 0.01; ***, p < 0.001 with respect to untreated myotubes. Data are presented as mean ± S.D. Numbers on the bars indicate the number of cells analyzed per condition.