Arg615Cys Substitution in Pig Skeletal Ryanodine Receptors Increases Activation of Single Channels by a Segment of the Skeletal DHPR II-III Loop

AbstractThe effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca2+ release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr671-Leu690; 36μM) enhanced the rate of Ca2+ release from normal SR (SRN) and from SR of MH-susceptible muscle (SRMH) by 10±3.2 nmole/mg/min and 76±9.7 nmole/mg/min, respectively. Ca 2+ release from SRN or SRMH was not increased by control peptide NB (Gly689-Lys708). AS (scrambled A sequence; 36μM) did not alter Ca 2+ release from SRN, but increased release from SRMH by 29±4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyRMH) were up to about fourfold more strongly activated by peptide A (≥1 nM) than normal RyR channels (RyRN) at −40mV. Neither NB or AS activated RyRN. RyRMH showed an ∼1.8-fold increase in mean current with 30μM AS. Inhibition at +40mV was stronger in RyRMH and seen with peptide A (≥0.6μM) and AS (≥0.6μM), but not NB. These results show that the Arg615Cys substitution in RyRMH has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca2+ release from SR in MH-susceptible muscle

Dissection of the inhibition of cardiac ryanodine receptors by human glutathione transferase GSTM2-2

The muscle specific glutathione transferase GSTM2-2 inhibits the activity of cardiac ryanodine receptor (RyR2) calcium release channels with high affinity and activates skeletal RyR (RyR1) channels with lower affinity. To determine which overall region of the GSTM2-2 molecule supports binding to RyR2, we examined the effects of truncating GSTM2-2 on its ability to alter Ca2+ release from sarcoplasmic reticulum (SR) vesicles and RyR channel activity. The C-terminal half of GSTM2-2 which lacks the critical GSH binding site supported the inhibition of RyR2, but did not support activation of RyR1. Smaller fragments of GSTM2-2 indicated that the C-terminal helix 6 was crucial for the action of GSTM2-2 on RyR2. Only fragments containing the helix 6 sequence inhibited Ca2+ release from cardiac SR. Single RyR2 channels were strongly inhibited by constructs containing the helix 6 sequence in combination with adjacent helices (helices 5-8 or 4-6). Fragments containing helices 5-6 or helix 6 sequences alone had less well-defined effects. Chemical cross-linking indicated that C-terminal helices 5-8 bound to RyR2, but not RyR1. Structural analysis with circular dichroism showed that the helical content was greater in the longer helix 6 containing constructs, while the helix 6 sequence alone had minimal helical structure. Therefore the active centre of GSTM2-2 for inhibition of cardiac RyR2 involves the helix 6 sequence and the helical nature of this region is essential for its efficacy. GSTM2-2 helices 5-8 may provide the basis for RyR2-specific compounds for experimental and therapeutic use

In vitro modulation of the cardiac ryanodine receptor activity by Homer1

The Homer protein family allows clustering and/or functional modulation of many proteins from different calcium signalling complexes including those formed by the ryanodine receptor (RyR) Ca2+ release channel in skeletal muscle and the heart. Homer1b/c and the cardiac RyR (RyR2) are strongly expressed in the heart and neurons where their interaction with each other may modulate Ca2+ signalling. However, functional interactions between Homer1b and RyR2 have been poorly defined. Our preliminary data and similar consensus binding sites for Homer in RyR2 and skeletal RyR (RyR1) proteins, led to the hypothesis that Homer may similarly regulate both RyR isoforms. Single-channel and [3H]ryanodine binding data showed that RyR2 and RyR1 activity increased to a maximum with ~50-100 nM Homer1b and fell with Homer1b > 200 nM. Homer1b (50 nM) activated RyR2 and RyR1 at all cytosolic [Ca2+]; estimated EC50 value of RyR2 diminished from ~2.8 μM Ca2+ (control) to ~1.9 μM Ca2+ in the presence of 50 nM Homer1b. Short Homer1 (lacking the coiled-coil multimerisation domain) and Homer1b similarly modulated RyR2, indicating an action through ligand binding, not mutimerisation. These actions of Homer were generally similar in RyR2 and RyR1. The strong functional interactions suggest that Homer1 is likely to be an endogenous modulator of RyR channels in the heart and neurons as well as in skeletal muscle

Structural Determinants for Activation or Inhibition of Ryanodine Receptors by Basis Residues in the Dihydropyridine Receptor II-III Loop

The structures of peptide A, and six other 7-20 amino acid peptides corresponding to sequences in the A region (Thr671-Leu690) of the skeletal muscle dihydropyridine receptor II-III loop have been examined, and are correlated with the ability of the peptides to activate or inhibit skeletal ryanodine receptor calcium release channels. The peptides adopted either random coil or nascent helix-like structures, which depended upon the polarity of the terminal residues as well as the presence and ionisation state of two glutamate residues. Enhanced activation of Ca2+ release from sarcoplasmic reticulum, and activation of current flow through single ryanodine receptor channels (at -40 mV), was seen with peptides containing the basic residues681Arg Lys Arg Arg Lys685, and was strongest when the residues were a part of an α-helix. Inhibition of channels (at +40 mV) Was also seen with peptides containing the five positively charged residues, but was not enhanced in helical peptides. These results confirm the hypothesis that activation of ryanodine receptor channels by the II-III loop peptides requires both the basic residues and their participation in helical structure, and show for the first time that inhibition requires the basic residues, but is not structure-dependent. These findings imply that activation and inhibition result from peptide binding to separate sites on the ryanodine receptor

Characteristics of Irreversible ATP Activation Suggest that Native Skeletal Ryanodine Receptors Can Be Phosphorylated via an Endogenous CaMKII

Phosphorylation of skeletal muscle ryanodine receptor (RyR) calcium release channels by endogenous kinases incorporated into lipid bilayers with native sarcoplasmic reticulum vesicles was investigated during exposure to 2 mM cytoplasmic ATP. Activation of RyRs after 1-min exposure to ATP was reversible upon ATP washout. In contrast, activation after 5 to 8 min was largely irreversible: the small fall in activity with washout was significantly less than that after brief ATP exposure. The irreversible activation was reduced by acid phosphatase and was not seen after exposure to nonhydrolyzable ATP analogs. The data suggested that the channel complex was phosphorylated after addition of ATP and that phosphorylation reduced the RyR's sensitivity to ATP, adenosine, and Ca2+. The endogenous kinase was likely to be a calcium calmodulin kinase II (CaMKII) because the CaMKII inhibitor KN-93 and an inhibitory peptide for CaMKII prevented the phosphorylation-induced irreversible activation. In contrast, phosphorylation effects remained unchanged with inhibitory peptides for protein kinase C and A. The presence of CaMKIIβ in the SR vesicles was confirmed by immunoblotting. The results suggest that CaMKII is anchored to skeletal muscle RyRs and that phosphorylation by this kinase alters the enhancement of channel activity by ATP and Ca2+

Effects of an α-helical ryanodine receptor C-terminal tail peptide on ryanodine receptor activity: Modulation by Homer

We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the ryanodine receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly a-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1-10 mu M) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca2+ concentration was 1 mu M; but with 10 mu M cytoplasmic Ca2+, skeletal ryanodine receptors were activated b

Glutathione transferase M2 variants inhibit ryanodine receptor function in adult mouse cardiomyocytes

Release of Ca2+ from the sarcoplasmic reticulum (SR) through the cardiac ryanodine receptor (RyR2) is an essential step in cardiac excitation-contraction coupling. Excess Ca2+ release due to overactive RyR2 can cause arrhythmia that can lead to cardiac arrest. Fragments derived from the carboxy-terminal domain of human glutathione transferase M2 (GSTM2C) specifically inhibit RyR2 activity. Our aim was to further improve this inhibition by mutagenesis and to assess the therapeutic potential of GSTM2C based peptides to treat Ca2+ release-based arrhythmia. We generated several mutant variants of the C-terminal fragment GSTM2C H5-8 and from those mutant proteins we identified two (RM13 and SM2) that exhibited significantly greater inhibition of cardiac SR Ca2+ release and single RyR2 channel activity. Flow cytometry analysis showed that these two mutant proteins as well as GSTM2C H5-8 are taken up by isolated adult mouse cardiomyocytes without the aid of any additional compounds, Ca2+ imaging and isolated cell contraction measurements revealed that GSTM2C H5-8, SM2 and RM13 reduce the SR Ca2+ release rate and the fractional shortening of adult mouse cardiomyocytes, while importantly increasing the rate of Ca2+ removal from the sarcoplasm. These observations indicate that peptides derived from GSTM2C inhibit RyR2 at a cellular level and thus they may provide the basis for a novel therapeutic agent to treat arrhythmia and heart attack