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

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. Lipid bilayer electrophysiology techniques demonstrated a novel, paradoxical increase in RyR2 activity. Preceding flecainide exposure, channels were mildly activated by 1 mM luminal Ca2+ and 1 µM cytoplasmic Ca2+, with open probabilities (Po) of 0.03 ± 0.01 (wild type, WT) or 0.096 ± 0.024 (P2328S). Open probability (Po) increased within 0.5 to 3 min of exposure to 0.5 to 5.0 µM cytoplasmic flecainide, then declined with higher concentrations of flecainide. There were no such increases in a subset of high Po channels with Po ≥ 0.08, although Po then declined with ≥5 µM (WT) or ≥50 µM flecainide (P2328S). On average, channels with Po < 0.08 were significantly activated by 0.5 to 10 µM of flecainide (WT) or 0.5 to 50 µM of flecainide (P2328S). These results suggest that flecainide can bind to separate activation and inhibition sites on RyR2, with activation dominating in lower activity channels and inhibition dominating in more active channels

Open cholecystectomy: exposure and confidence of surgical trainees and new fellows

The laparoscopic approach to cholecystectomy has overtaken open procedures in terms of frequency, despite open procedures playing an important role in certain clinical situations. This study explored exposure and confidence of Australasian surgical trainees and new fellows in performing an open versus laparoscopic cholecystectomy.An online survey was disseminated via the Royal Australasian College of Surgeons to senior general surgery trainees (years 3-5 of surgical training) and new fellows (fellowship within the previous 5 years). The survey included questions regarding level of experience and confidence in performing an open cholecystectomy and converting from a laparoscopic to an open approach.A total of 135 participants responded; 58 (43%) were surgical trainees, 58 (43%) were fellows and 19 (14%) did not specify their level of training. Respondents who were involved in more than 20 open cholecystectomy procedures as an assistant or independent operator compared with those less exposed were more likely to feel confident to independently perform an elective open cholecystectomy (87.8% vs. 57.3%, P = 0.001), independently convert from a laparoscopic to open cholecystectomy (87.8% vs. 58.7%, P = 0.001) and independently perform an open cholecystectomy as a surgical consultant based on their level of exposure as a trainee (73.2% vs. 45.3%, P = 0.004).This study suggests the need to ensure surgical trainees are exposed to sufficient open cholecystectomies to enable confidence and skill with performing these procedures when indicated. Greater recognition of the need for exposure during training, including meaningful simulation, may assist

Structure of the Janus Protein Human CLIC2

Chloride intracellular channel (CLIC) proteins possess the remarkable property of being able to convert from a water-soluble state to a membrane channel state. We determined the three-dimensional structure of human CLIC2 in its water-soluble form by X-ray crystallography at 1.8-Å resolution from two crystal forms. In contrast to the previously characterized CLIC1 protein, which forms a possibly functionally important disulfide-induced dimer under oxidizing conditions, we show that CLIC2 possesses an intramolecular disulfide and that the protein remains monomeric irrespective of redox conditions. Site-directed mutagenesis studies show that removal of the intramolecular disulfide or introduction of cysteine residues in CLIC2, equivalent to those that form the intramolecular disulfide in CLIC1, does not cause dimer formation under oxidizing conditions.We also show that CLIC2 forms pH-dependent chloride channels in vitro with higher channel activity at low pH levels and that the channels are subject to redox regulation. In both crystal forms, we observed an extended loop region from the C-terminal domain, called the foot loop, inserting itself into an interdomain crevice of a neighboring molecule. The equivalent region in the structurally related glutathione transferase superfamily corresponds to the active site. This so-called foot-in-mouth interaction suggests that CLIC2 might recognize other proteins such as the ryanodine receptor through a similar interaction

DHPR activation underlies SR Ca2+ release induced by osmotic stress in isolated rat skeletal muscle fibers

Changes in skeletal muscle volume induce localized sarcoplasmic reticulum (SR) Ca2+ release (LCR) events, which are sustained for many minutes, suggesting a possible signaling role in plasticity or pathology. However, the mechanism by which cell volume influences SR Ca2+ release is uncertain. In the present study, rat flexor digitorum brevis fibers were superfused with isoosmotic Tyrode's solution before exposure to either hyperosmotic (404 mOsm) or hypoosmotic (254 mOsm) solutions, and the effects on cell volume, membrane potential (Em), and intracellular Ca2+ ([Ca2+]i) were determined. To allow comparison with previous studies, solutions were made hyperosmotic by the addition of sugars or divalent cations, or they were made hypoosmotic by reducing [NaCl]o. All hyperosmotic solutions induced a sustained decrease in cell volume, which was accompanied by membrane depolarization (by 14–18 mV; n = 40) and SR Ca2+ release. However, sugar solutions caused a global increase in [Ca2+]i, whereas solutions made hyperosmotic by the addition of divalent cations only induced LCR. Decreasing osmolarity induced an increase in cell volume and a negative shift in Em (by 15.04 ± 1.85 mV; n = 8), whereas [Ca2+]i was unaffected. However, on return to the isoosmotic solution, restoration of cell volume and Em was associated with LCR. Both global and localized SR Ca2+ release were abolished by the dihydropyridine receptor inhibitor nifedipine by sustained depolarization of the sarcolemmal or by the addition of the ryanodine receptor 1 inhibitor tetracaine. Inhibitors of the Na-K-2Cl (NKCC) cotransporter markedly inhibited the depolarization associated with hyperosmotic shrinkage and the associated SR Ca2+ release. These findings suggest (1) that the depolarization that accompanies a decrease in cell volume is the primary event leading to SR Ca2+ release, and (2) that volume-dependent regulation of the NKCC cotransporter contributes to the observed changes in Em. The differing effects of the osmotic agents can be explained by the screening of fixed charges by divalent ions