Structural and functional properties of ryanodine receptor type 3 in zebrafish tail muscle
Stefano PerniKurt C. MarsdenMatías EscobarStephen HollingworthStephen M. BaylorClara Franzini‐Armstrong
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The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca2+ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as “junctional feet” (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation–contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca2+ sparks, which are elementary SR Ca2+ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca2+ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere−1 s−1. The few Ca2+ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca2+ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca2+ flux underlying Ca2+ sparks detected in fully differentiated frog and fish fibers.Keywords:
Sarcoplasm
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Malignant hyperthermia
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Regions of ryanodine receptors that influence activation by the dihydropyridine receptor β1a subunit
Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca(2+) release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR β1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of β1a activation of RyRs and the β1a binding site on RyR1.We used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca(2+) currents and Ca(2+) transients in myotubes.We demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10-100 nM of the β1a subunit. Activation of RyR2 by 10 nM β1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for β1a. A reduction in activation was also observed when 10 nM β1a was added to the alternatively spliced (ASI(-)) isoform of RyR1, which lacks ASI residues (A3481-Q3485). It is notable that the equivalent region of RyR2 also lacks four of five ASI residues, suggesting that the absence of these residues may contribute to the reduced 10 nM β1a activation observed for both RyR2 and ASI(-)RyR1 compared to ASI(+)RyR1. We also investigated the influence of a polybasic motif (PBM) of RyR1 (K3495KKRRDGR3502) that is located immediately downstream from the ASI residues and has been implicated in EC coupling. We confirmed that neutralizing the basic residues in the PBM (RyR1 K-Q) results in an ~50 % reduction in Ca(2+) transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for β1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of β1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca(2+) release generated during skeletal EC coupling.We conclude that the β1a subunit likely binds to a region that is largely conserved in RyR1 and RyR2 and that this region is influenced by the presence of the ASI residues and the PBM in RyR1.
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INTRODUCTION Titin is a protein that spans the length of a half sarcomere in skeletal muscle myofibrils. It behaves like a molecular spring within the myofibril, playing a role in stabilizing sarcomeres and regulating passive force [1, 2]. Isolated titin has been shown to be essentially elastic if immunoglobulin (Ig) domain unfolding/refolding is prevented [3]. In its native, sarcomeric environment, it has been suggested that stretching and holding a myofibril at very long lengths produces a time-dependent unfolding of all Ig domains, thus, allowing titin’s elastic behavior to be exhibited [4]. Experiments on active myofibrils showed a decrease in force and a persistent hysteresis throughout a stretch-shortening (SS) protocol, suggesting a time-dependent unfolding of Ig domains [5]. Holding active myofibrils at long lengths prior to stretch-shortening cycles should allow most (all) of the Ig domains to unfold thus reducing (eliminating) force loss and hysteresis. The goal of this study was to test the hypothesis that holding myofibrils at long lengths prior to small stretch-shortening cycles would result in essentially elastic properties of myofibrils, compared to the highly visco-elastic properties for conditions without holding. METHODS Rabbit psoas muscle myofibrils (n = 5) with clear striation patterns were tested. Single myofibrils were attached at one end to a glass needle (to control length) and at the other end to a nanolever (to quantify force). Myofibrils were activated at an average sarcomere length of 2.7 µm, and then stretched to a length of 5.2 µm/sarcomere, where they were held for 2 minutes to allow for Ig domain unfolding to occur. The myofibril then underwent a SS protocol with amplitude of ± 0.25 µm (10 cycles) before being shortened to its original length. Myofibril length, diameter, and force were quantified. Diameter was used to calculate cross-sectional area, which accommodated the calculation of myofibril stress from force. Hystereses were calculated as the difference in area under the loading and unloading curves for each SS cycle of the force-length plots. RESULTS Peak stress throughout the 10 cycles remained approximately constant, averaging 102 % relative to the first cycle (Fig 1a). Hysteresis did not follow a specific trend throughout the 10 SS cycles (Fig 1b). DISCUSSION AND CONCLUSIONS The “constant” peak forces are indicative of elastic recoil of myofibrils during the SS cycles. However, the persistent and random hystereses are indicative of viscous properties. If Ig domains were still unfolding during the SS cycles, peak stresses should also decrease. Since this is not observed, we suggest that all Ig domains are unfolded in this experiment, and that the viscous behaviour producing the hystereses must come from a source other than titin. At this point, any proposition as to the origin of the remnant hystereses is highly speculative but might be associated with titin binding-unbinding to another structural (titin) or contractile (actin) protein that is forming and breaking continuously during the SS cycles.
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The cardiac and skeletal muscle sarcoplasmic reticulum ryanodine receptor Ca2+ release channels contain thiols that are potential targets of endogenously produced reactive oxygen and nitrogen intermediates. Previously, we showed that the skeletal muscle ryanodine receptor (RyR1) has O2-sensitive thiols; only when these thiols are in the reduced state (pO2 ∼ 10 mmHg) can physiological concentrations of NO (nanomolar) activate RyR1. Here, we report that cardiac muscle ryanodine receptor (RyR2) activity also depends on pO2, but unlike RyR1, RyR2 was not activated or S-nitrosylated directly by NO. Rather, activation and S-nitrosylation of RyR2 required S-nitrosoglutathione. The effects of peroxynitrite were indiscriminate on RyR1 and RyR2. Our results indicate that both RyR1 and RyR2 are pO2-responsive yet point to different mechanisms by which NO and S-nitrosoglutathione influence cardiac and skeletal muscle sarcoplasmic reticulum Ca2+ release.
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Six chimeras of the skeletal muscle (RyR1) and cardiac muscle (RyR2) Ca 2+ release channels (ryanodine receptors) previously used to identify RyR1 dihydropyridine receptor interactions [Nakai et al. (1998) J. Biol. Chem. 273, 13403] were expressed in HEK293 cells to assess their Ca 2+ dependence in [ 3 H]ryanodine binding and single channel measurements. The results indicate that the C‐terminal one‐fourth has a major role in Ca 2+ activation and inactivation of RyR1. Further, our results show that replacement of RyR1 regions with corresponding RyR2 regions can result in loss and/or reduction of [ 3 H]ryanodine binding affinity while maintaining channel activity.
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