The V2475F CPVT1 mutation yields distinct RyR2 channel populations that differ in their responses to cytosolic Ca2+ and Mg2.

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is a lethal genetic disease causing arrhythmias and sudden cardiac death in children and young adults and is linked to mutations in the cardiac ryanodine receptor (RyR2). The effects of CPVT1 mutations on RyR2 ion-channel function are often investigated using purified recombinant RyR2 channels homozygous for the mutation. However, CPVT1 patients are heterozygous for the disease, so this approach does not reveal the true changes to RyR2 function across the entire RyR2 population of channels in the heart. We therefore investigated the native cardiac RyR2 single-channel abnormalities in mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2, and applied molecular modelling techniques to investigate the possible structural changes that could initiate any altered function. We observed that increased sensitivity of cardiac V2475F(+/-)-RyR2 channels to both activating and inactivating levels of cytosolic Ca2+ , plus attenuation of Mg2+ inhibition, were the most marked changes. Severity of abnormality was not uniform across all channels, giving rise to multiple sub-populations with differing functional characteristics. For example, 46% of V2475F(+/-)-RyR2 channels exhibited reduced Mg2+ inhibition and 23% were actually activated by Mg2+ . Using homology modelling, we discovered that V2475 is situated at a hinge between two regions of the RyR2 helical domain 1 (HD1). Our model proposes that detrimental functional changes to RyR2 arise because mutation at this critical site reduces the angle between these regions. Our results demonstrate the necessity of characterising the total heterozygous population of CPVT1-mutated channels in order to understand CPVT1 phenotypes in patients. KEY POINTS: RyR2 mutations can cause type-1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a lethal, autosomal-dominant arrhythmic disease. However, the changes in RyR2 ion-channel function that result from the many different patient mutations are rarely investigated in detail and often only recombinant RyR2, homozygous for the mutation, is studied. As CPVT1 is a heterozygous disease and the tetrameric RyR2 channels expressed in the heart will contain varying numbers of mutated monomers, we have investigated the range of RyR2 single-channel abnormalities found in the hearts of mice heterozygous for the CPVT1 mutation, V2475F(+/-)-RyR2. Specific alterations to ligand regulation of V2475F(+/-)-RyR2 were observed. Multiple sub-populations of channels exhibited varying degrees of abnormality. In particular, an increased sensitivity to activating and inactivating cytosolic [Ca2+ ], and reduced sensitivity to Mg2+ inhibition were evident. Our results provide mechanistic insight into the changes to RyR2 gating that destabilise sarcoplasmic reticulum Ca2+ -release causing life-threatening arrhythmias in V2475F(+/-)-CPVT1 patients.

Quantitative RyR1 reduction and loss of calcium sensitivity of RyR1Q1970fsX16+A4329D cause cores and loss of muscle strength.

Recessive ryanodine receptor 1 (RYR1) mutations cause congenital myopathies including multiminicore disease (MmD), congenital fiber-type disproportion and centronuclear myopathy. We created a mouse model knocked-in for the Q1970fsX16+A4329D RYR1 mutations, which are isogenic with those identified in a severely affected child with MmD. During the first 20 weeks after birth the body weight and the spontaneous running distance of the mutant mice were 20% and 50% lower compared to wild-type littermates. Skeletal muscles from mutant mice contained 'cores' characterized by severe myofibrillar disorganization associated with misplacement of mitochondria. Furthermore, their muscles developed less force and had smaller electrically evoked calcium transients. Mutant RyR1 channels incorporated into lipid bilayers were less sensitive to calcium and caffeine, but no change in single-channel conductance was observed. Our results demonstrate that the phenotype of the RyR1Q1970fsX16+A4329D compound heterozygous mice recapitulates the clinical picture of multiminicore patients and provide evidence of the molecular mechanisms responsible for skeletal muscle defects.

Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC-A.

KEY POINTS: The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric-a gene knockout (KO) alters the single-channel function of skeletal RyR (RyR1). We find that RyR1 from Tric-a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+ , ATP, caffeine and luminal Ca2+ . In the presence of Mg2+ , ATP cannot effectively activate RyR1 from Tric-a KO mice. Additionally, RyR1 from Tric-a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of ß-adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+ -release. The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+ -release observed in skeletal muscle from Tric-a KO mice, further highlighting the importance of TRIC-A for normal physiological regulation of SR Ca2+ -release in skeletal muscle. ABSTRACT: The type A trimeric intracellular cation channel (TRIC-A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric-a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric-a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage-clamp conditions. We find that RyR1 channels from Tric-a KO mice respond normally to cytosolic Ca2+ , ATP, adenine, caffeine and to luminal Ca2+ . However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+ , ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC-A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC-A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC-A.

Modulation of a critical period for motor development in Drosophila by BK potassium channels.

Critical periods are windows of heightened plasticity occurring during neurodevelopment. Alterations in neural activity during these periods can cause long-lasting changes in the structure, connectivity, and intrinsic excitability of neurons, which may contribute to the pathology of neurodevelopmental disorders. However, endogenous regulators of critical periods remain poorly defined. Here, we study this issue using a fruit fly (Drosophila) model of an early-onset movement disorder caused by BK potassium channel gain of function (BK GOF). Deploying a genetic method to place robust expression of GOF BK channels under spatiotemporal control, we show that adult-stage neuronal expression of GOF BK channels minimally disrupts fly movement. In contrast, limiting neuronal expression of GOF BK channels to a short window during late neurodevelopment profoundly impairs locomotion and limb kinematics in resulting adult flies. During this critical period, BK GOF perturbs synaptic localization of the active zone protein Bruchpilot and reduces excitatory neurotransmission. Conversely, enhancing neural activity specifically during development rescues motor defects in BK GOF flies. Collectively, our results reveal a critical developmental period for limb control in Drosophila that is influenced by BK channels and suggest that BK GOF causes movement disorders by disrupting activity-dependent aspects of synaptic development.

Acidic environments trigger intracellular H+-sensing FAK proteins to re-balance sarcolemmal acid-base transporters and auto-regulate cardiomyocyte pH.

AIMS: In cardiomyocytes, acute disturbances to intracellular pH (pHi) are promptly corrected by a system of finely tuned sarcolemmal acid-base transporters. However, these fluxes become thermodynamically re-balanced in acidic environments, which inadvertently causes their set-point pHi to fall outside the physiological range. It is unclear whether an adaptive mechanism exists to correct this thermodynamic challenge, and return pHi to normal. METHODS AND RESULTS: Following left ventricle cryo-damage, a diffuse pattern of low extracellular pH (pHe) was detected by acid-sensing pHLIP. Despite this, pHi measured in the beating heart (13C NMR) was normal. Myocytes had adapted to their acidic environment by reducing Cl-/HCO3- exchange (CBE)-dependent acid-loading and increasing Na+/H+ exchange (NHE1)-dependent acid-extrusion, as measured by fluorescence (cSNARF1). The outcome of this adaptation on pHi is revealed as a cytoplasmic alkalinization when cells are superfused at physiological pHe. Conversely, mice given oral bicarbonate (to improve systemic buffering) had reduced myocardial NHE1 expression, consistent with a needs-dependent expression of pHi-regulatory transporters. The response to sustained acidity could be replicated in vitro using neonatal ventricular myocytes incubated at low pHe for 48 h. The adaptive increase in NHE1 and decrease in CBE activities was linked to Slc9a1 (NHE1) up-regulation and Slc4a2 (AE2) down-regulation. This response was triggered by intracellular H+ ions because it persisted in the absence of CO2/HCO3- and became ablated when acidic incubation media had lower chloride, a solution manoeuvre that reduces the extent of pHi-decrease. Pharmacological inhibition of FAK-family non-receptor kinases, previously characterized as pH-sensors, ablated this pHi autoregulation. In support of a pHi-sensing role, FAK protein Pyk2 (auto)phosphorylation was reduced within minutes of exposure to acidity, ahead of adaptive changes to pHi control. CONCLUSIONS: Cardiomyocytes fine-tune the expression of pHi-regulators so that pHi is at least 7.0. This autoregulatory feedback mechanism defines physiological pHi and protects it during pHe vulnerabilities.

Synthesis and use of isotope-labelled substrates for a mechanistic study on human alpha-methylacyl-CoA racemase 1A (AMACR; P504S).

Alpha-Methylacyl-CoA racemase (AMACR) is an important enzyme for the metabolism of branched-chain lipids and drugs. The enzyme is over-expressed in prostate and other cancers. AMACR 1A, the major splice variant, was purified from recombinant E. coli cells as a His-tag protein. Purified enzyme catalysed chiral inversion of both S- and R-2-methyldecanoyl-CoA, with an equilibrium constant of 1.09 +/- 0.14 (2S/2R). Reactions with (2)H-labelled substrate showed that loss of the alpha-proton was a prerequisite for chiral inversion. Reactions conducted in (2)H(2)O indicated that reprotonation was not stereospecific. These results are the first mechanistic study on any recombinant mammalian alpha-methylacyl-CoA racemase.

Simvastatin activates single skeletal RyR1 channels but exerts more complex regulation of the cardiac RyR2 isoform.

BACKGROUND AND PURPOSE: Statins are amongst the most widely prescribed drugs for those at risk of cardiovascular disease, lowering cholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase. Although effective at preventing cardiovascular disease, statin use is associated with muscle weakness, myopathies and, occasionally, fatal rhabdomyolysis. As simvastatin, a commonly prescribed statin, promotes Ca2+ release from sarcoplasmic reticulum (SR) vesicles, we investigated if simvastatin directly activates skeletal (RyR1) and cardiac (RyR2) ryanodine receptors. EXPERIMENTAL APPROACH: RyR1 and RyR2 single-channel behaviour was investigated after incorporation of sheep cardiac or mouse skeletal SR into planar phospholipid bilayers under voltage-clamp conditions. LC-MS was used to monitor the kinetics of interconversion of simvastatin between hydroxy-acid and lactone forms during these experiments. Cardiac and skeletal myocytes were permeabilised to examine simvastatin modulation of SR Ca2+ release. KEY RESULTS: Hydroxy acid simvastatin (active at HMG-CoA reductase) significantly and reversibly increased RyR1 open probability (Po) and shifted the distribution of Ca2+ spark frequency towards higher values in skeletal fibres. In contrast, simvastatin reduced RyR2 Po and shifted the distribution of spark frequency towards lower values in ventricular cardiomyocytes. The lactone pro-drug form of simvastatin (inactive at HMG-CoA reductase) also activated RyR1, suggesting that the HMG-CoA inhibitor pharmacophore was not responsible for RyR1 activation. CONCLUSION AND IMPLICATIONS: Simvastatin interacts with RyR1 to increase SR Ca2+ release and thus may contribute to its reported adverse effects on skeletal muscle. The ability of low concentrations of simvastatin to reduce RyR2 Po may also protect against Ca2+ -dependent arrhythmias and sudden cardiac death.