Sarcoplasmic Reticulum Vesicles Research Articles

Establishment and evaluation of targeted molecular screening model for the ryanodine receptor or sarco/endoplasmic reticulum calcium ATPase.

Endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) is crucial for maintaining intracellular calcium homeostasis due to the calcium-signaling-related proteins on its membrane. While ryanodine receptors (RyR) on insect ER/SR membranes are well-known as targets for diamide insecticides, little is known about other calcium channels. Given the resistance of diamide insecticides, the establishment of molecular screening models targeting RyR or sarco/endoplasmic reticulum calcium ATPase (SERCA) is conducive to the discovery of new insecticidal molecules. The morphological features of Mythimna separata SR have closed vesicles with integrity and high density. The 282 proteins in the SR component contained RyR and SERCA. A measurement model for the release and uptake of calcium was successfully established by detecting calcium ions outside the SR membrane using a fluorescence spectrophotometer. In vitro testing systems using SR vesicles found that diamide insecticides could activate dose-dependently RyR, with EC50 values of 0.14 μM (Chlorantraniliprole), 0.21 μM (Flubendiamide), and 0.57 μM (Cyantraniliprole), respectively. However, dantrolene inhibited RyR-mediated calcium release with an IC50 value of 353.9 μM, suggesting that dantrolene can weakly antagonize RyR. Moreover, cyclopiazonic acid significantly reduced the enzyme activity and calcium uptake capacity of SERCA. On the contrary, CDN1163 markedly activated the enzyme activity and improved the calcium transport capacity of SERCA. SR vesicles can be used to study the function of unknown proteins on the SR membranes, as well as for high-throughput screening of highly active compounds targeting RyR or SERCA. © 2024 Society of Chemical Industry.

SERCA-1 conformational change exerted by the Ca2+-channel blocker diltiazem affects mammalian skeletal muscle function

In skeletal muscle (SM), inward Ca2+-currents have no apparent role in excitation-contraction coupling (e-c coupling), however the Ca2+-channel blocker can affect twitch and tetanic muscle in mammalian SM. Experiments were conducted to study how diltiazem (DLZ) facilitates e-c coupling and inhibits contraction. 1) In complete Extensor Digitorum Longus (EDL) muscle and single intact fibres, 0.03 mM DLZ causes twitch potentiation and decreases force during tetanic activity, with increased fatigue. 2) In split open fibres isolated from EDL fibres, DLZ inhibits sarcoplasmic reticulum (SR) Ca2+-loading in a dose-dependent manner and has a potentiating effect on caffeine-induced SR Ca2+-release. 3) In isolated light SR (LSR) vesicles, SERCA1 hydrolytic activity is not affected by DLZ up to 0.2 mM. However, ATP-dependent Ca2+-uptake was inhibited in a dose-dependent manner at a concentration where e-c coupling is changed. 4) The passive Ca2+-efflux from LSR was reduced by half with 0.03 mM diltiazem, indicating that SR leaking does not account for the decreased Ca2+-uptake. 5) The denaturation profile of the SERCA Ca2+-binding domain has lower thermal stability in the presence of DLZ in a concentration-dependent manner, having no effect on the nucleotide-binding domain. We conclude that the effect of DLZ on SM is exerted by crossing the sarcolemma and interacting directly with the SERCA Ca2+-binding domain, affecting SR Ca2+-loading during relaxation, which has a consequence on SM contractility. Diltiazem effect on SM could be utilized as a tool to understand SM e-c coupling and muscle fatigue.

Impact of nicotine and cotinine on macrophage inflammatory plasticity via vesicular modifications in gastrointestinal bacteria

ObjectivesThis study aimed to elucidate mechanistic explanation(s) for compositional changes to enteric microbiota by determining the impacts of continuous nicotine/cotinine exposure on representative gastrointestinal bacteria and how these alterations impact innate immune cell plasticity. MethodsIn vitro cultures of the gastrointestinal bacteria (Bacteroides fragilis 25285, Prevotella bryantii B14, and Acetoanaerobium sticklandii SR) were continuously exposed to nicotine or cotinine. Supernatant samples were collected for fermentation acid analysis. Vesicles were collected and analyzed for physiological changes in number, size, and total protein cargo. Cultured macrophages were stimulated to a tolerogenic phenotype, exposed to control or altered (nicotine or cotinine – exposed) vesicles, and inflammatory plasticity assessed via inflammatory cytokine production. ResultsNicotine/cotinine exposure differentially affected metabolism of all bacteria tested in a Gram (nicotine) and concentration-dependent (cotinine) manner. Physiological studies demonstrated changes in vesiculation number and protein cargo following nicotine/cotinine exposures. Continuous exposure to 1 μM nicotine and 10 μM cotinine concentrations reduced total protein cargo of Gram (-) – 25285 and B14 vesicles, while cotinine generally increased total protein in Gram (+) - SR vesicles. We found that theses physiological changes to the vesicles of 25285 and SR formed under nicotine and cotinine, respectively, challenged the plasticity of tolerogenic macrophages. Tolerogenic macrophages exposed to vesicles from 1 μM nicotine, and 5 or 10 μΜ cotinine cultures produced significantly less IL-12p70, TNFα, or KC/GRO, regardless of macrophage exposure to nicotine/cotinine. ConclusionsNicotine/cotinine exposure differentially alters bacterial metabolism and vesicle physiology, ultimately impacting the inflammatory response of tolerogenic macrophages.

Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State.

Ca2+ leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide (DPc10) binding promotes leaky RyR2 in cardiomyocytes and reports on that endogenous state. Conversely, calmodulin (CaM) binding inhibits RyR2 leak and low CaM affinity is diagnostic of leaky RyR2. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. We used FRET to clarify the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca2+, DPc10 decreased both FRETmax (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca2+, DPc10 decreased FRETmax without affecting CaM/RyR2 binding affinity. This correlates with the analysis of fluorescence-lifetime-detected FRET, indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca2+ and lengthens D-FKBP/A-CaM distances independent of [Ca2+]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, with this effect being larger in micromolar versus nanomolar Ca2+. Moreover, A-DPc10/RyR2 binding is cooperative in a CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by the analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.

A bioelectrochemical approach based on a solid supported membrane to evaluate the effect of natural products on Ca2+-ATPase: The case of 6-gingerol

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) plays an essential role in maintaining the low cytosolic Ca2+ level that enables a variety of cellular processes. SERCA couples ATP hydrolysis to the transport of two Ca2+ ions against their electrochemical potential gradient from the cytoplasm into the lumen of the sarco/endoplasmic reticulum (SR/ER). Because of its central role in regulating cytoplasmic Ca2+ concentration, SERCA dysfunction has been associated with several pathological conditions. Stimulation of SERCA activity may represent a potential therapeutic strategy in various disease states connected with dysfunctional SERCA. The natural phenolic compound 6-gingerol, the most abundant and the major biologically active compound of ginger, was reported to activate the SERCA enzyme. The present study aimed at investigating the effect of 6-gingerol on SERCA transport activity using a bioelectrochemical approach based on a solid supported membrane (SSM). We first performed a voltammetric characterization of 6-gingerol to better understand its electrochemical behavior. We then studied the interaction of 6-gingerol with SR vesicles containing SERCA adsorbed on the SSM electrode. The measured current signals indicated that ATP-dependent Ca2+ translocation by SERCA was remarkably increased in the presence of 6-gingerol at low micromolar concentration. We also found that 6-gingerol has a rather high affinity for SERCA (EC50 of 1.8 ± 0.3 µM), and SERCA activation by 6-gingerol is reversible. The observed stimulatory effect of 6-gingerol on SERCA Ca2+-translocating activity may be beneficial in the prevention and/or treatment of pathological conditions related to SERCA dysfunction.

Ca2+ Dependent Formation/Collapse of Cylindrical Ca2+-ATPase Crystals in Scallop Sarcoplasmic Reticulum (SR) Vesicles: A Possible Dynamic Role of SR in Regulation of Muscle Contraction.

[Ca2+]-dependent crystallization of the Ca2+-ATPase molecules in sarcoplasmic reticulum (SR) vesicles isolated from scallop striated muscle elongated the vesicles in the absence of ATP, and ATP stabilized the crystals. Here, to determine the [Ca2+]-dependence of vesicle elongation in the presence of ATP, SR vesicles in various [Ca2+] environments were imaged using negative stain electron microscopy. The images obtained revealed the following phenomena. (i) Crystal-containing elongated vesicles appeared at ≤1.4 µM Ca2+ and almost disappeared at ≥18 µM Ca2+, where ATPase activity reaches its maximum. (ii) At ≥18 µM Ca2+, almost all SR vesicles were in the round form and covered by tightly clustered ATPase crystal patches. (iii) Round vesicles dried on electron microscopy grids occasionally had cracks, probably because surface tension crushed the solid three-dimensional spheres. (iv) [Ca2+]-dependent ATPase crystallization was rapid (<1 min) and reversible. These data prompt the hypothesis that SR vesicles autonomously elongate or contract with the help of a calcium-sensitive ATPase network/endoskeleton and that ATPase crystallization may modulate physical properties of the SR architecture, including the ryanodine receptors that control muscle contraction.

CNS-focused high throughput screening for RyR-targeted therapeutics.

Using high-throughput screening (HTS) assays based on fluorescence lifetime (FLT)-detected FRET, we have identified compounds that allosterically modulate ryanodine receptor (RyR) calcium release channels, to prevent or reduce the elevated Ca2+ leak that fuels age-related neurodegeneration. RyRs are responsible for Ca release from intracellular storage organelles (endoplasmic reticulum – ER; sarcoplasmic reticulum – SR), and the resulting Ca signaling is essential for many cellular processes. RyRs have been intensively studied for decades in SR vesicles purified from skeletal muscle (RyR1) or heart (RyR2), where dysregulated RyR function – particularly a leaky state of the resting RyR – has been identified as a targetable culprit to interrupt the vicious cycle that compromises cell physiology in age-related pathologies. Recently RyR2 dysfunction in neurons has been implicated as a major contributor to Alzheimer's disease. Our FRET-based HTS detects the RyR leaky state by monitoring binding of the accessory proteins calmodulin, FKBP12.6, and DPc10. Under conditions that mimic a pathological state, we have carried out HTS of the 50k-compound CNS library (ChemBridge), to identify modulators of RyR2 in native SR membranes. This screen yielded 603 compounds (Hits) that reproducibly altered FRET between screens. With therapeutic feasibility a priority, freshly prepared stock solutions of 85 of the most promising compounds, validated for purity and chemical structure, were then evaluated for their dose-response in FRET and functional assays measuring 3[H]ryanodine binding. The resulting Hit compounds comprised seven unique chemical clusters that were either RyR isoform-specific or altered both RyR1 and RyR2 activity. Structure-activity relationship studies are ongoing. Good candidate compounds for treatment of neurodegeneration will progress to testing on RyR function in neurons from AD mouse models. This work is supported by NIH grant R43AG069582 (JJT/RLC).

Junctophilin-2 allosterically interacts with ryanodine receptor type 2 to regulate calcium release units in mouse cardiomyocytes.

The interaction between junctophilin-2 (JPH2) and ryanodine receptor type 2 (RyR2) regulated Ca2+ signaling in mouse cardiomyocytes. However, their exact interaction remains unclear. This study elucidates the interaction between JPH2 with RyR2 using co-immunoprecipitation of cardiac sarcoplasmic reticulum vesicles. Additionally, a glutathione S-transferase (GST) pull-down analysis was performed to investigate the physical interaction between RyR2 and JPH2 fragments. JPH2 interacted with RyR2 and the C terminus of the JPH2 protein can pull-down RyR2 receptors. Confocal immunofluorescence imaging indicated that the majority of JPH2 and RyR2 proteins were colocalized near Z-lines in isolated mouse cardiomyocytes. Knockdown of JPH2 reduced the amplitude of Ca2+ transients and disrupted its interaction with RyR2. Therefore, the C-terminus domain of JPH2 is required for interactions with RyR2 in mouse cardiomyocytes, which provides a molecular mechanism for seeking Ca2+-related disease prevention strategies.

New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca2+ dynamics by increasing SERCA2a Ca2+ pumping

The type 2a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a central role in the intracellular Ca2+ homeostasis of cardiac myocytes, pumping Ca2+ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca2+ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca2+-ATPase and Ca2+ transport activities in cardiac SR vesicles, and on Ca2+ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca2+-ATPase and Ca2+ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca2+ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca2+ load by enhancing SERCA2a-mediated Ca2+ transport. Analysis of cytosolic Ca2+ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca2+ transients and SR Ca2+ load, with negligible effects on L-type Ca2+ channels and Na+/Ca2+ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca2+ transients and SR Ca2+ load, but it decreased the propensity toward Ca2+ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [3H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca2+ and a small increase in μM Ca2+. Accordingly, compound 1 slightly decreased Ca2+ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca2+ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca2+ uptake, as found in failing hearts.

Decavanadate and metformin-decavanadate effects in human melanoma cells

Decavanadate is a polyoxometalate (POMs) that has shown extensive biological activities, including antidiabetic and anticancer activity. Importantly, vanadium-based compounds as well as antidiabetic biguanide drugs, such as metformin, have shown to exert therapeutic effects in melanoma. A combination of these agents, the metformin-decavanadate complex, was also recognized for its antidiabetic effects and recently described as a better treatment than the monotherapy with metformin enabling lower dosage in rodent models of diabetes. Herein, we compare the effects of decavanadate and metformin-decavanadate on Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from rabbit skeletal muscles and on cell signaling events and viability in human melanoma cells. We show that unlike the decavanadate-mediated non-competitive mechanism, metformin-decavanadate inhibits Ca2+-ATPase by a mixed-type competitive–non-competitive inhibition with an IC50 value about 6 times higher (87 μM) than the previously described for decavanadate (15 μM). We also found that both decavanadate and metformin-decavanadate exert antiproliferative effects on melanoma cells at 10 times lower concentrations than monomeric vanadate. Western blot analysis revealed that both, decavanadate and metformin-decavanadate increased phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine protein kinase AKT signaling proteins upon 24 h drug exposure, suggesting that the anti-proliferative activities of these compounds act independent of growth-factor signaling pathways.

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

Sarcoplasmic Reticulum from Horse Gluteal Muscle Is Poised for Enhanced Calcium Transport

We have analyzed the enzymatic activity of the sarcoplasmic reticulum (SR) Ca2+-transporting ATPase (SERCA) from the horse gluteal muscle. Horses are bred for peak athletic performance yet exhibit a high incidence of exertional rhabdomyolysis, with elevated levels of cytosolic Ca2+ proposed as a correlative linkage. We recently reported an improved protocol for isolating SR vesicles from horse muscle; these horse SR vesicles contain an abundant level of SERCA and only trace-levels of sarcolipin (SLN), the inhibitory peptide subunit of SERCA in mammalian fast-twitch skeletal muscle. Here, we report that the in vitro Ca2+ transport rate of horse SR vesicles is 2.3 ± 0.7-fold greater than rabbit SR vesicles, which express close to equimolar levels of SERCA and SLN. This suggests that horse myofibers exhibit an enhanced SR Ca2+ transport rate and increased luminal Ca2+ stores in vivo. Using the densitometry of Coomassie-stained SDS-PAGE gels, we determined that horse SR vesicles express an abundant level of the luminal SR Ca2+ storage protein calsequestrin (CASQ), with a CASQ-to-SERCA ratio about double that in rabbit SR vesicles. Thus, we propose that SR Ca2+ cycling in horse myofibers is enhanced by a reduced SLN inhibition of SERCA and by an abundant expression of CASQ. Together, these results suggest that horse muscle contractility and susceptibility to exertional rhabdomyolysis are promoted by enhanced SR Ca2+ uptake and luminal Ca2+ storage.

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca2+ release during excitation–contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose–response profiles and previously unreported effects on RyR were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential enhancers of excitation–contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation–contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.

Stimulation of Ca2+ -ATPase Transport Activity by a Small-Molecule Drug.

The sarco(endo)plasmic reticulum Ca2+−ATPase (SERCA) hydrolyzes ATP to transport Ca2+ from the cytoplasm to the sarcoplasmic reticulum (SR) lumen, thereby inducing muscle relaxation. Dysfunctional SERCA has been related to various diseases. The identification of small‐molecule drugs that can activate SERCA may offer a therapeutic approach to treat pathologies connected with SERCA malfunction. Herein, we propose a method to study the mechanism of interaction between SERCA and novel SERCA activators, i. e. CDN1163, using a solid supported membrane (SSM) biosensing approach. Native SR vesicles or reconstituted proteoliposomes containing SERCA were adsorbed on the SSM and activated by ATP concentration jumps. We observed that CDN1163 reversibly interacts with SERCA and enhances ATP‐dependent Ca2+ translocation. The concentration dependence of the CDN1163 effect provided an EC50=6.0±0.3 μM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca2+ concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca2+ release into the SR lumen.

Ca2+-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum.

The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca2+ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca2+ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.

Purification of sarcoplasmic reticulum vesicles from horse gluteal muscle

We have analyzed protein expression and enzyme activity of the sarcoplasmic reticulum Ca2+-transporting ATPase (SERCA) in horse gluteal muscle. Horses exhibit a high incidence of recurrent exertional rhabdomyolysis, with myosolic Ca2+ proposed, but yet to be established, as the underlying cause. To better assess Ca2+ regulatory mechanisms, we developed an improved protocol for isolating sarcoplasmic reticulum (SR) vesicles from horse skeletal muscle, based on mechanical homogenization and optimized parameters for differential centrifugation. Immunoblotting identified the peak subcellular fraction containing the SERCA1 protein (fast-twitch isoform). Gel analysis using the Stains-all dye demonstrated that calsequestrin (CASQ) and phospholipids are highly enriched in the SERCA-containing subcellular fraction isolated from horse gluteus. Immunoblotting also demonstrated that these horse SR vesicles show low content of glycogen phosphorylase (GP), which is likely an abundant contaminating protein of traditional horse SR preps. The maximal Ca2+-activated ATPase activity (Vmax) of SERCA in horse SR vesicles isolated using this protocol is 5‒25-fold greater than previously-reported SERCA activity in SR preps from horse skeletal muscle. We propose that this new protocol for isolating SR vesicles will be useful for determining enzymatic parameters of horse SERCA with high fidelity, plus assessing regulatory effect of SERCA peptide subunit(s) expressed in horse muscle.

Peptide mimetic compounds can activate or inhibit cardiac and skeletal ryanodine receptors

AimsOur aim was to characterise the actions of novel BIT compounds with structures based on peptides and toxins that bind to significant regulatory sites on ryanodine receptor (RyR) Ca2+ release channels. RyRs, located in sarcoplasmic reticulum (SR) Ca2+ store membranes of striated muscle, are essential for muscle contraction. Although severe sometimes-deadly myopathies occur when the channels become hyperactive following genetic or acquired changes, specific inhibitors of RyRs are rare. Main methodsThe effect of BIT compounds was determined by spectrophotometric analysis of Ca2+ release from isolated SR vesicles, analysis of single RyR channel activity in artificial lipid bilayers and contraction of intact and skinned skeletal muscle fibres. Key findingsThe inhibitory compounds reduced: (a) Ca2+ release from SR vesicles with IC50s of 1.1–2.5 μM, competing with activation by parent peptides and toxins; (b) single RyR ion channel activity with IC50s of 0.5–1.5 μM; (c) skinned fibre contraction. In contrast, activating BIT compounds increased Ca2+ release with an IC50 of 5.0 μM and channel activity with AC50s of 2 to 12 nM and enhanced skinned fibre contraction. Sub-conductance activity dominated channel activity with both inhibitors and activators. Effects of all compounds on skeletal and cardiac RyRs were similar and reversible. Competition experiments suggest that the BIT compounds bind to the regulatory helical domains of the RyRs that impact on channel gating mechanisms through long-range allosteric interactions. SignificanceThe BIT compounds are strong modulators of RyR activity and provide structural templates for novel research tools and drugs to combat muscle disease.

Effects of Acute Exercise and Training on the Sarcoplasmic Reticulum Ca2+ Release and Uptake Rates in Highly Trained Endurance Athletes.

Little is presently known about the effects of acute high-intensity exercise or training on release and uptake of Ca2+ by the sarcoplasmic reticulum (SR). The aims here were to characterize this regulation in highly trained athletes following (1) repeated bouts of high-intensity exercise and (2) a period of endurance training including high-intensity sessions. Eleven cross-country skiers (25 ± 4 years, 65 ± 4 mL O2⋅kg−1⋅min–1) performed four self-paced sprint time-trials (STT 1-4) lasting ≈ 4 min each (STT 1–4) and separated by 45 min of recovery; while 19 triathletes and road cyclists (25 ± 4 years, 65 ± 5 mL O2⋅kg−1⋅min–1) completed 4 weeks of endurance training in combination with three sessions of high-intensity interval cycling per week. Release (μmol⋅g–1 prot⋅min–1) and uptake [tau (s)] of Ca2+ by SR vesicles isolated from m. triceps brachii and m. vastus lateralis were determined before and after STT 1 and 4 in the skiers and in m. vastus lateralis before and after the 4 weeks of training in the endurance athletes. The Ca2+ release rate was reduced by 17–18% in both limbs already after STT 1 (arms: 2.52 ± 0.74 to 2.08 ± 0.60; legs: 2.41 ± 0.45 to 1.98 ± 0.51, P < 0.0001) and attenuated further following STT 4 (arms: 2.24 ± 0.67 to 1.95 ± 0.45; legs: 2.13 ± 0.51 to 1.83 ± 0.36, P < 0.0001). Also, there was a tendency toward an impairment in the SR Ca2+ uptake from pre STT1 to post STT4 in both arms and legs (arms: from 22.0 ± 3.7 s to 25.3 ± 6.0 s; legs: from 22.5 ± 4.7 s to 25.5 ± 7.7 s, P = 0.05). Endurance training combined with high-intensity exercise increased the Ca2+ release rate by 9% (1.76 ± 0.38 to 1.91 ± 0.44, P = 0.009), without altering the Ca2+ uptake (29.6 ± 7.0 to 29.1 ± 8.7 s; P = 0.98). In conclusion, the Ca2+ release and uptake rates by SR in exercising limbs of highly trained athletes declines gradually by repetitive bouts of high-intensity exercise. We also demonstrate, for the first time, that the SR Ca2+ release rate can be enhanced by a specific program of training in highly trained athletes, which may have important implications for performance parameters.

ANS Interacts with the Ca2+-ATPase Nucleotide Binding Site.

The binding of 8-anilino-1-naphthalene sulfonate (ANS) to the nucleotide binding domain (N-domain) of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) was studied. Molecular docking predicted two ANS binding modes (BMI and BMII) in the nucleotide binding site. The molecular interaction was confirmed as the fluorescence intensity of ANS was dramatically increased when in the presence of an engineered recombinant N-domain. Molecular dynamics simulation showed BMI (which occupies the ATP binding site) as the mode that is stable in solution. The above was confirmed by the absence of ANS fluorescence in the presence of a fluorescein isothiocyanate (FITC)-labeled N-domain. Further, the labeling of the N-domain with FITC was hindered by the presence of ANS, i.e., ANS was bound to the ATP binding site. Importantly, ANS displayed a higher affinity than ATP. In addition, ANS binding led to quenching the N-domain intrinsic fluorescence displaying a FRET pattern, which suggested the existence of a Trp-ANS FRET couple. Nonetheless, the chemical modification of the sole Trp residue with N-bromosuccinimide (NBS) discarded the existence of FRET and instead indicated structural rearrangements in the nucleotide binding site during ANS binding. Finally, Ca2+-ATPase kinetics in the presence of ANS showed a partial mixed-type inhibition. The Dixon plot showed the ANS-Ca2+-ATPase complex as catalytically active, hence supporting the existence of a functional dimeric Ca2+-ATPase in sarcoplasmic reticulum vesicles. ANS may be used as a molecular platform for the development of more effective inhibitors of Ca2+-ATPase and appears to be a new fluorescent probe for the nucleotide binding site. Graphical Abstract Molecular docking of ANS to the nucleotide binding site of Ca2+-ATPase. ANS fluorescence increase reveals molecular interaction.

Development of a High-Throughput Assay using R-CEPIA1er Fluorescence to Assess Small-Molecule Modulators of SERCA Activity in Living Cells

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is an essential component of Ca2+ signaling in most eukaryotic cells. Dysregulation of intracellular Ca2+ is associated with specific health problems, thus attracting attention for SERCA as a promising therapeutic target for skeletal and cardiac myopathies (e.g., Duchenne muscular dystrophy, sarcopenia, heart failure and atrial fibrillation). Here, we report on the development of a high-throughput assay to characterize novel drug-like small molecules that modulate SERCA-Ca2+ transport. This assay is based on microplate fluorescence measurement of ER luminal Ca2+ concentration ([Ca]ER) in HEK293 cells that co-express human SERCA2a and the ER-targeted Ca2+ biosensor R-CEPIA1er (genetically encoded, organelle-entrapped Ca2+ biosensor). We validated the cellular localization, biosensor response, and sensitivity limit of the R-CEPIA assays using fluorescence microscopy and plate-reader spectroscopy, including effects of the well-known Ca2+ pump inhibitors thapsigargin (TG) and cyclopiazonic acid (CPA). Microplate measurement and fluorescence intensity analysis revealed highly reproducible results for R-CEPIA1er detection of [Ca]ER inhibition by TG and CPA. Co-expression of the SERCA inhibitory peptide phospholamban (PLB) in HEK cells decreased [Ca]ER, demonstrating PLB-specific cardiac-type inhibition that was relieved by the known PLB antagonist, ellagic acid. We also analyzed the effect of additional small-molecule modulators of SERCA function and obtained results that are consistent with Ca2+-ATPase and Ca2+-transport assays using isolated sarcoplasmic reticulum vesicles. Thus, our new microplate-compatible, live-cell fluorescence assay offers a powerful combination of sensitivity and speed for high-throughput analysis of pre-clinical hit compounds that control SERCA function.