Flecainide Paradoxically Activates Cardiac Ryanodine Receptor Channels under Low Activity Conditions: A Potential Pro-Arrhythmic Action.

Cardiac ryanodine receptor (RyR2) mutations are implicated in the potentially fatal catecholaminergic polymorphic ventricular tachycardia (CPVT) and in atrial fibrillation. CPVT has been successfully treated with flecainide monotherapy, with occasional notable exceptions. Reported actions of flecainide on cardiac sodium currents from mice carrying the pro-arrhythmic homozygotic RyR2-P2328S mutation prompted our explorations of the effects of flecainide on their RyR2 channels.

Neutralizing the pathological effects of extracellular histones with small polyanions.

Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9-1.

Ion channel gating in cardiac ryanodine receptors from the arrhythmic RyR2-P2328S mouse.

Mutations in the cardiac ryanodine receptor Ca release channel (RyR2) can cause deadly ventricular arrhythmias and atrial fibrillation (AF). The RyR2-P2328S mutation produces catecholaminergic polymorphic ventricular tachycardia (CPVT) and AF in hearts from homozygous RyR2 (denoted RyR2) mice. We have now examined P2328S RyR2 channels from RyR2 hearts.

Association of FK506 binding proteins with RyR channels - effect of CLIC2 binding on sub-conductance opening and FKBP binding.

Ryanodine receptor (RyR) Ca channels are central to striated muscle function and influence signalling in neurons and other cell types. Beneficially low RyR activity and maximum conductance opening may be stabilised when RyRs bind to FK506 binding proteins (FKBPs) and destabilised by FKBP dissociation, with submaximal opening during RyR hyperactivity associated with myopathies and neurological disorders. However, the correlation with submaximal opening is debated and quantitative evidence is lacking.

A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels.

Junctin, a non-catalytic splice variant encoded by the aspartate-β-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca(2+) store where it modifies Ca(2+) signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca(2+) release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca(2+) signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen.

Cardiac ryanodine receptor activation by a high Ca²⁺ store load is reversed in a reducing cytoplasmic redox environment.

Here, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca(2+)]. Basal leak from the sarcoplasmic reticulum is required for normal Ca(2+) handling, but excess diastolic Ca(2+) leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca(2+) release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca(2+)] under reducing or oxidising cytoplasmic redox conditions.

Multiple actions of phi-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins.

We recently reported the isolation of a scorpion toxin named U1-liotoxin-Lw1a (U1-LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U1-LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U1-LITX-Lw1a, now described as ϕ-liotoxin-Lw1a (ϕ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state.

An X-linked channelopathy with cardiomegaly due to a CLIC2 mutation enhancing ryanodine receptor channel activity.

Chloride intracellular channel 2 (CLIC2) protein is a member of the glutathione transferase class of proteins. Its' only known function is the regulation of ryanodine receptor (RyR) intracellular Ca(2+) release channels. These RyR proteins play a major role in the regulation of Ca(2+) signaling in many cells.

The β(1a) subunit of the skeletal DHPR binds to skeletal RyR1 and activates the channel via its 35-residue C-terminal tail.

Although it has been suggested that the C-terminal tail of the β(1a) subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca(2+) release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β(1a) bound to RyR1 in affinity chromatography.

The elusive role of the SPRY2 domain in RyR1.

The second of three SPRY domains (SPRY2, S1085 -V1208) located in the skeletal muscle ryanodine receptor (RyR1) is contained within regions of RyR1 that influence EC coupling and bind to imperatoxin A, a toxin probe of RyR1 channel gating. We examined the binding of the F loop (P1107-A1121) in SPRY2 to the ASI/basic region in RyR1 (T3471-G3500, containing both alternatively spliced (ASI) residues and neighboring basic amino acids). We then investigated the possible influence of this interaction on excitation contraction (EC) coupling.

Junctin and triadin each activate skeletal ryanodine receptors but junctin alone mediates functional interactions with calsequestrin.

Normal Ca(2+) signalling in skeletal muscle depends on the membrane associated proteins triadin and junctin and their ability to mediate functional interactions between the Ca(2+) binding protein calsequestrin and the type 1 ryanodine receptor in the lumen of the sarcoplasmic reticulum. This important mechanism conserves intracellular Ca(2+) stores, but is poorly understood. Triadin and junctin share similar structures and are lumped together in models of interactions between skeletal muscle calsequestrin and ryanodine receptors, however their individual roles have not been examined at a molecular level.

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 Ca(2+) 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.

Redox potential and the response of cardiac ryanodine receptors to CLIC-2, a member of the glutathione S-transferase structural family.

The type 2 chloride intracellular channel, CLIC-2, is a member of the glutathione S-transferase structural family and a suppressor of cardiac ryanodine receptor (RyR2) Ca2+ channels located in the membrane of the sarcoplasmic reticulum (SR). Modulators of RyR2 activity can alter cardiac contraction. Since both CLIC-2 and RyR2 are modified by redox reactions, we speculated that the action of CLIC-2 on RyR2 may depend on redox potential.

Muscle-specific GSTM2-2 on the luminal side of the sarcoplasmic reticulum modifies RyR ion channel activity.

We show that a glutathione transferase (GST) protein, which is recognised by an antibody against the muscle-specific human GSTM2-2 (hGSTM2-2), is associated with the lumen of the sarcoplasmic reticulum (SR) of cardiac muscle, but not skeletal muscle. We further show that hGSTM2-2 modifies both cardiac and skeletal ryanodine receptor (RyR) activity when it binds to the luminal domain of the RyR channel complex. The properties of hGSTM2-2 were compared with those of the calsequestrin (CSQ), a Ca(2+) binding protein also present in the lumen of the SR which, like GSTM2-2, contains a thioredoxin-fold structure and modifies RyR activity (Wei, L.

The Mu class glutathione transferase is abundant in striated muscle and is an isoform-specific regulator of ryanodine receptor calcium channels.

Members of the glutathione transferase (GST) structural family are novel regulators of cardiac ryanodine receptor (RyR) calcium channels. We present the first detailed report of the effect of endogenous muscle GST on skeletal and cardiac RyRs. An Mu class glutathione transferase is specifically expressed in human muscle.

Exclusion of linkage of the RYR1, CACNA1S, and ATP2A1 genes to recurrent exertional rhabdomyolysis in Thoroughbreds.

Objective: To determine whether there was genetic linkage between the recurrent exertional rhabdomyolysis (RER) trait in Thoroughbred horse pedigrees and DNA markers in genes (the sarcoplasmic reticulum calcium release channel [RYR1] gene, the sarcoplasmic reticulum calcium ATPase [ATP2A1] gene, and the transverse tubule dihydropyridine receptor-voltage sensor [CACNA1S] gene) that are important in myoplasmic calcium regulation.

Animals: 34 horses in the University of Minnesota RER resource herd and 62 Thoroughbreds from 3 families of Thoroughbreds outside of the university in which RER-affected status was assigned after 2 or more episodes of ER had been observed.

Procedures: Microsatellite DNA markers from the RYR1, ATP2A1, and CACNA1S gene loci on equine chromosomes 10, 13, and 30 were identified.

Effects of an alpha-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 alpha-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels.

The cysteine-rich secretory protein domain of Tpx-1 is related to ion channel toxins and regulates ryanodine receptor Ca2+ signaling.

The cysteine-rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract as well as in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionary diverse N-terminal domain and a characteristic cysteine-rich C-terminal domain, which we refer to as the Crisp domain. We presented the NMR solution structure of the Crisp domain of mouse Tpx-1, and we showed that it contains two subdomains, one of which has a similar fold to the ion channel regulators BgK and ShK.

Inheritance of recurrent exertional rhabdomyolysis in thoroughbreds.

Objective: To develop a diagnostic test for recurrent exertional rhabdomyolysis (RER) in Thoroughbreds that relied on in vitro contracture of muscle biopsy specimens and determine whether the inheritance pattern of RER diagnosed on the basis of this contracture test was consistent with an autosomal dominant trait.

Design: Clinical trial.

Animals: 8 adult horses with RER and 16 control adult horses for development of the contracture test; 23 foals for inheritance of RER.

Caffeine sensitivity of native RyR channels from normal and malignant hyperthermic pigs: effects of a DHPR II-III loop peptide.

Enhanced sensitivity to caffeine is part of the standard tests for susceptibility to malignant hyperthermia (MH) in humans and pigs. The caffeine sensitivity of skeletal muscle contraction and Ca(2+) release from the sarcoplasmic reticulum is enhanced, but surprisingly, the caffeine sensitivity of purified porcine ryanodine receptor Ca(2+)-release channels (RyRs) is not affected by the MH mutation (Arg(615)Cys). In contrast, we show here that native malignant hyperthermic pig RyRs (incorporated into lipid bilayers with RyR-associated lipids and proteins) were activated by caffeine at 100- to 1000-fold lower concentrations than native normal pig RyRs.

Myoplasmic calcium regulation in myotubes from horses with recurrent exertional rhabdomyolysis.

Objective: To determine whether alterations in myoplasmic calcium regulation can be identified in muscle cell cultures (myotubes) and intact muscle fiber bundles derived from Thoroughbreds affected with recurrent exertional rhabdomyolysis (RER).

Animals: 6 related Thoroughbreds with RER and 8 clinically normal (control) Thoroughbred or crossbred horses.

Procedures: Myotube cell cultures were grown from satellite cells obtained from muscle biopsy specimens of RER-affected and control horses.