Effects of anti-triadin antibody on Ca2+ release from sarcoplasmic reticulum

AbstractThe monoclonal antibody, mAb GE 4.90, raised against triadin, a 95 kDa protein of sarcoplasmic reticulum (SR), inhibits the slow phase of Ca2+ release from SR following depolarization of the T-tubule moiety of the triad. The antibody has virtually no effect on the fast phase of depolarization-induced Ca2+ release nor on caffeine-induced Ca2+ release. Since the slow phase of depolarization-induced Ca2+ release is also inhibited by dihydropyridines (DHP), these results suggest that triadin may be involved in the functional coupling between the DHP receptor and the SR Ca2+ channel

Antibody probe study of Ca2+ channel regulation by interdomain interaction within the ryanodine receptor.

N-terminal and central domains of ryanodine receptor 1 (RyR1), where many reported malignant hyperthermia (MH) mutations are localized, represent putative channel regulatory domains. Recent domain peptide (DP) probe studies led us to the hypothesis that these domains interact to stabilize the closed state of channel (zipping), while weakening of domain-domain interactions (unzipping) by mutation de-stabilizes the channel, making it leaky to Ca2+ or sensitive to the agonists of RyR1. As shown previously, DP1 (N-terminal domain peptide) and DP4 (central domain peptide) produced MH-like channel activation/sensitization effects, presumably by peptide binding to sites critical to stabilizing domain-domain interactions and resultant loss of conformational constraints. Here we report that polyclonal anti-DP1 and anti-DP4 antibodies also produce MH-like channel activation and sensitization effects as evidenced by about 4-fold enhancement of high affinity [3H]ryanodine binding to RyR1 and by a significant left-shift of the concentration-dependence of activation of sarcoplasmic reticulum Ca2+ release by polylysine. Fluorescence quenching experiments demonstrate that the accessibility of a DP4-directed, conformationally sensitive fluorescence probe linked to the RyR1 N-terminal domain is increased in the presence of domain-specific antibodies, consistent with the view that these antibodies produce unzipping of interacting domains that are of hindered accessibility to the surrounding aqueous environment. Our results suggest that domain-specific antibody binding induces a conformational change resulting in channel activation, and are consistent with the hypothesis that interacting N-terminal and central domains are intimately involved in the regulation of RyR1 channel function

Effects of a domain peptide of the ryanodine receptor on Ca2+ release in skinned skeletal muscle fibers

Published abstract reprinted by permissionMutations in the central domain of the skeletal muscle ryanodine receptor (RyR) cause malignant hyperthermia (MH). A synthetic peptide (DP4) in this domain (Leu-2442-Pro-2477) produces enhanced ryanodine binding and sensitized Ca2+ release in isolated sarcoplasmic reticulum, similar to the properties in MH, possibly because the peptide disrupts the normal interdomain interactions that stabilize the closed state of the RyR (Yamamoto T, El-Hayek R, and Ikemoto N. J Biol Chem 275: 11618-11625, 2000). Here, DP4 was applied to mechanically skinned fibers of rat muscle that had the normal excitation-contraction coupling mechanism still functional to determine whether muscle fiber responsiveness was enhanced. DP4 (100 µM) substantially potentiated the Ca2+ release and force response to caffeine (8 mM) and to low [Mg2+] (0.2 mM) in every fiber examined, with no significant effect on the properties of the contractile apparatus. DP4 also potentiated the response to submaximal depolarization of the transverse tubular system by ionic substitution. Importantly, DP4 did not significantly alter the size of the twitch response elicited by action potential stimulation. These results support the proposal that DP4 causes an MH-like aberration in RyR function and are consistent with the voltage sensor triggering Ca2+ release by destabilizing the closed state of the RyRs.Graham D. Lamb, Giuseppe S. Posterino, Takeshi Yamamoto, and Noriaki Ikemot

A domain peptide of the cardiac ryanodine receptor regulates channel sensitivity to luminal Ca²⁺ via cytoplasmic Ca²⁺ sites

The clustering of cardiac RyR mutations, linked to sudden cardiac death (SCD), into several regions in the amino acid sequence underlies the hypothesis that these mutations interfere with stabilising interactions between different domains of the RyR2. SCD mutations cause increased channel sensitivity to cytoplasmic and luminal Ca²⁺. A synthetic peptide corresponding to part of the central domain (DPc10:²⁴⁶⁰ G-P²⁴⁹⁵) was designed to destabilise the interaction of the N-terminal and central domains of wild-type RyR2 and mimic the effects of SCD mutations. With Ca²⁺ as the sole regulating ion, DPc10 caused increased channel activity which could be reversed by removal of the peptide whereas in the presence of ATP DPc10 caused no activation. In support of the domain destablising hypothesis, the corresponding peptide (DPc10-mut) containing the CPVT mutation R2474S did not affect channel activity under any circumstances. DPc10-induced activation was due to a small increase in RyR2 sensitivity to cytoplasmic Ca²⁺ and a large increase in the magnitude of luminal Ca²⁺ activation. The increase in the luminal Ca²⁺ response appeared reliant on the luminal-to-cytoplasmic Ca²⁺ flux in the channel, indicating that luminal Ca²⁺ was activating the RyR2 via its cytoplasmic Ca²⁺ sites. DPc10 had no significant effect on the RyR2 gating associated with luminal Ca2+ sensing sites. The results were fitted by the luminal-triggered Ca²⁺ feed-through model and the effects of DPc10 were explained entirely by perturbations in cytoplasmic Ca²⁺ -activation mechanism