A Skeletal Muscle Ryanodine Receptor Interaction Domain in Triadin

Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200-232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200-232 or 200-231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49 ± 0.11-fold and 1.8 ± 0.15-fold respectively, when added to its luminal side. A similar increase in [(3)H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95's 200-231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction.This work was supported by the Australian Research Council (DP1094219 to A.F.D. and N.A.B.)

Dichloroacetate prevents cisplatin-induced nephrotoxicity without compromising cisplatin anticancer properties

Cisplatin is an effective anticancer drug; however, cisplatin use often leads to nephrotoxicity, which limits its clinical effectiveness. In this study, we determined the effect of dichloroacetate, a novel anticancer agent, in a mouse model of cisplatin-induced AKI. Pretreatment with dichloroacetate significantly attenuated the cisplatin-induced increase in BUN and serum creatinine levels, renal tubular apoptosis, and oxidative stress. Additionally, pretreatment with dichloroacetate accelerated tubular regeneration after cisplatin-induced renal damage. Whole transcriptome sequencing revealed that dichloroacetate prevented mitochondrial dysfunction and preserved the energy-generating capacity of the kidneys by preventing the cisplatin-induced downregulation of fatty acid and glucose oxidation, and of genes involved in the Krebs cycle and oxidative phosphorylation. Notably, dichloroacetate did not interfere with the anticancer activity of cisplatin in vivo. These data provide strong evidence that dichloroacetate preserves renal function when used in conjunction with cisplatin

Identifying the in vitro interactions between the skeletal muscle ryanodine receptor, triadin and calsequestrin

Excitation-contraction coupling (EC coupling) is a process linking depolarization of a muscle fibre membrane with eventual mechanical contraction of that fibre. Key in this process is calcium release into the muscle fibre cytoplasm from an intracellular store (sarcoplasmic reticulum, SR) via a ligand gated calcium channel, the ryanodine receptor (RyR). Numerous proteins engage in RyR regulation to ensure healthy calcium release during EC coupling. One such protein is triadin, which binds both the cytoplasmic and SR luminal domains of the RyR. Triadin has been shown to engage in direct RyR regulation in vitro, however, in vivo it has been associated with many roles not directly related to RyR regulation or EC coupling. As such, it has proven difficult to correlate triadin's protein-protein interactions to functional effects in vivo. In an attempt to better understand the nature of the luminal triadin-RyR association, regulatory interactions between the major rabbit skeletal muscle triadin isoform, Trisk 95, and the skeletal RyR isoform (RyR1) were investigated. Using a triadin peptide corresponding to residues 200 to 232 of rabbit Trisk 95, it was found that this region was sufficient to replicate specific RyR1 activation as mediated by full length Trisk 95 in vitro. Furthermore, using a set of mutant peptides, it was discovered that K218, K220 and K224 were minimally required to recapitulate full length Trisk 95 mediated RyR1 regulation. Peptides incorporating only one or two of these mutations retained RyR1 regulation, but highlighted a tendency for K220 to be more critical in maintaining RyR1 regulation than K218 and K224. This matched well with previous work showing three RyR1 residues contribute unequally to Trisk 95 binding. Consequently, it was hypothesised that the luminal association between Trisk 95 and RyR1 requires three charged pair interactions, with each contributing differently to the overall interaction. It is hoped that the identification of Trisk 95's RyR1 binding residues will enable specific mutational targeting of the luminal association between Trisk 95 and RyR1 in future studies, thereby identifying the function of this interaction in vivo. Interestingly, one of Trisk 95's other protein binding partners, calsequestrin (CSQ1), has been predicted to bind Trisk 95 at residues overlapping those here identified as critical for RyR1 regulation. Investigating whether the same Trisk 95 residues critical for RyR1 regulation are also critical for CSQ1 binding, it was found that a triadin peptide incorporating mutation of K218, K220 and K224 was still able to bind substantial amounts of CSQ1. This suggested that Trisk 95 binds RyR1 and CSQ1 via different residues. However, the RyR1 regulating and CSQ1 binding residues were in close proximity, leaving the possibility that CSQ1 or RyR1 may occlude the other's binding site on Trisk 95 when both are present. Consequently, a model is proposed to explain how an inability of Trisk 95 to simultaneously bind RyR1 and CSQ1 might result in dynamic RyR1 regulation. The model presents a new set of hypotheses which, upon testing, are expected to expand current knowledge regarding Trisk 95's role in EC coupling

A skeletal muscle ryanodine receptor interaction domain in triadin.

Excitation-contraction coupling in skeletal muscle depends, in part, on a functional interaction between the ligand-gated ryanodine receptor (RyR1) and integral membrane protein Trisk 95, localized to the sarcoplasmic reticulum membrane. Various domains on Trisk 95 can associate with RyR1, yet the domain responsible for regulating RyR1 activity has remained elusive. We explored the hypothesis that a luminal Trisk 95 KEKE motif (residues 200-232), known to promote RyR1 binding, may also form the RyR1 activation domain. Peptides corresponding to Trisk 95 residues 200-232 or 200-231 bound to RyR1 and increased the single channel activity of RyR1 by 1.49 ± 0.11-fold and 1.8 ± 0.15-fold respectively, when added to its luminal side. A similar increase in [(3)H]ryanodine binding, which reflects open probability of the channels, was also observed. This RyR1 activation is similar to activation induced by full length Trisk 95. Circular dichroism showed that both peptides were intrinsically disordered, suggesting a defined secondary structure is not necessary to mediate RyR1 activation. These data for the first time demonstrate that Trisk 95's 200-231 region is responsible for RyR1 activation. Furthermore, it shows that no secondary structure is required to achieve this activation, the Trisk 95 residues themselves are critical for the Trisk 95-RyR1 interaction

Channel open probability parameter values for native and purified RyR1 measured in the presence and absence of 63 nM Triadin peptide and 63 nM full length Trisk 95.

<p>The average open probability (<i>P_o</i>) values calculated from lipid bilayer experiments are shown. <i>P_o</i> for each channel type and presence or absence of peptide/Trisk 95 is measured from 90 s of activity at both +40 mV and −40 mV (pooled). The fold change is the <i>P_o</i> value after <i>trans</i> addition of triadin peptide or full length Trisk 95, relative to <i>P_o</i> in the absence of triadin peptide/Trisk 95. Trisk 95 was isolated from rabbit skeletal muscle and these data (<i>in italics</i>) have been previously published in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043817#pone.0043817-Wei2" target="_blank">[13]</a> and are included here for comparison. Significant (p≤0.05) differences in <i>P_o</i> upon addition of triadin peptide or Trisk 95 are indicated for each condition compared to its control (*).</p

Triadin peptide modulates purified RyR1 single channel open time, closed time and closed frequency.

<p>(A) Average data for open probability (<i>P_o</i>) in presence of triadin^200–232 and triadin^200–231 (n = 10), data from both peptides combined (see results text, collectively termed triadin peptide, n = 11–20) for each of the following parameters; open time (<i>T_o</i>), close time (<i>T_c</i>) and open frequency (<i>F_o</i>), collected at −40 mV and +40 mV. All data is expressed as relative mean data (Log rel (parameter)). Relative mean <i>P_o</i> (log rel <i>P_o</i>) is the average of differences between the log₁₀ of the <i>P_o</i> in the presence of either 63 nM or 252 nM triadin peptide (log₁₀<i>P_o</i>_Pep) and log₁₀ of the control <i>P_o</i> (log₁₀<i>P_o</i>_Con) for each channel. Log rel <i>T_o</i> is log₁₀<i>T_o</i>_Pep-log₁₀<i>T_o</i>_Con, log rel <i>T_c</i> is log₁₀<i>T_c</i>_Pep-log₁₀<i>T_c</i>_Con and log rel <i>F_o</i> is log₁₀<i>F_o</i>_Pep-log₁₀<i>F_o</i>_Con. (B) [³H]ryanodine binding to purified RyR1 in the absence and presence of 63 nM of triadin peptide. [³H]ryanodine binding is measured as pmol ryanodine/mg RyR1. Data is expressed relative to binding recorded in the absence of peptides (rel ryanodine binding). Significance (p≤0.05) is indicated for each concentration compared to activity recorded prior to addition of peptide (*).</p

Triadin peptides modulate purified RyR1.

<p>(A) Running histogram of a typical bilayer experiment. Open probability (<i>P_o</i>) was measured every 30 s throughout the lifetime of the experiment before and after the addition of 63 nM and then 252 nM triadin^200–231 peptide (arrows) at +40 mV (light grey bins) and −40 mV (dark grey bins). Data averages for each condition are shown as horizontal broken lines for +40 mV and −40 mV, and median is presented as a horizontal solid line. (B, C) 3 s traces of purified RyR1 channel activity. Activity was recorded at +40 mV (left) where channels are opening upward from zero current (c, continuous line) to maximum open conductance (o, broken line) and at −40 mV (right), where channel openings are downwards from zero current (c, continuous line) to maximum open conductance (o, broken line). <i>Top panel –</i> control recording of purified RyR1 prior to the addition of triadin peptide; <i>middle and bottom panel</i> – after the addition of 63 nM and 252 nM triadin peptide to the <i>trans</i> chamber. (B) Shows addition of triadin^200–232 peptide, and (C) shows addition of triadin^200–231. Control <i>P_o</i> in the absence of peptide was 0.46±0.12 at +40 mV and 0.38±0.17 at −40 mV (for triadin^200–232), and 0.14±0.07 at +40 mV and 0.29±0.17 at −40 mV (for triadin^200–231).</p

Triadin^200–232 and triadin^200–231 peptides bind to RyR1 and exhibit a disordered secondary structure.

<p>(A) Circular dichroism spectra averaged from four scans and corrected for the 10 mM sodium phosphate buffer are shown. Both triadin peptides (triadin^200–231 (black trace) and triadin^200–232 (grey trace); 0.03 mg/ml) show a negative peak at ∼197 nm, consistent with an intrinsically disordered structure. A positive peak at ∼190 nm and negative peaks at ∼208 and ∼223 nm (indicative of α-helical secondary structure) or a positive peak at ∼195 nm and a negative peak at ∼217 nm (indicative of β-sheet secondary structure), are absent. (B) Western blot, following streptavidin-agarose affinity chromatography, showing the association of RyR1 with biotin tagged triadin peptide. The upper half of the membrane was probed with anti-RyR1 antibody and the lower half was probed with Streptactin-HRP conjugate to identify the biotin tagged peptides. <i>Lane 1</i> protein sample eluted from streptavidin-agarose incubated with RyR1 alone; <i>Lane 2</i> purified RyR1 alone (control); <i>Lane 3</i> biotin tagged triadin^200–231 peptide alone (control); <i>Lanes 4 and 5</i> protein sample eluted from streptavidin-agarose affinity chromatography, where streptavidin-agarose was incubated with biotin tagged triadin^200–232 or triadin^200–231 peptide respectively, prior to incubation with RyR1.</p

Triadin peptide does not activate mutant or native RyR1, or when its RyR1 binding site is blocked.

<p>(A–B) 3 s traces of RyR1 channel activity at −40 mV. Channels are opening downwards from zero current (c, continuous line) to maximum open conductance (o, broken line). (A) RyR1 ΔM_1,2,3 mutants (with Trisk 95 binding residues mutated) in absence of triadin peptide and in the presence of <i>trans</i> 63 nM and 252 nM peptide. (B) Native RyR1 in the absence and presence of <i>trans</i> 63 nM triadin peptide. Average open probability (<i>P_o</i>) of data, collected at −40 mV and +40 mV (n = 6–10) is displayed below each trace. (C) [³H]ryanodine binding to purified RyR with triadin binding blocking peptide (RyR1^4871–4910 ) in the absence and presence of 63 nM triadin peptide. [³H]ryanodine binding is measured as pmol ryanodine/mg RyR1 and data is expressed relative to binding recorded in the absence of either peptide (rel ryanodine binding). All values are presented as relative to control. Asterisks (*) indicate a significant difference (p≤0.05) in binding from RyR1 control (no peptide); crosshatch (#) indicates a significant difference (p≤0.05) in binding from RyR1 in the presence of RyR1^4871–4910.</p