Receptor Ca2 Research Articles

In vivo Ca2+ dynamics during cooling after eccentric contractions in rat skeletal muscle.

The effect of cooling on in vivo intracellular calcium ion concentration [Ca2+]i after eccentric contractions (ECs) remains to be determined. We tested the hypothesis that cryotherapy following ECs promotes an increased [Ca2+]i and induces greater muscle damage in two muscles with substantial IIb and IIx fiber populations. The thin spinotrapezius (SPINO) muscles of Wistar rats were used for in vivo [Ca2+]i imaging, and tibialis anterior (TA) muscles provided greater fidelity and repeatability of contractile function measurements. SPINO [Ca2+]i was estimated using fura 2-AM and the magnitude, location, and temporal profile of [Ca2+]i determined as the temperature near the muscle surface post-ECs was decreased from 30°C (control) to 20°C or 10°C. Subsequently, in the TA, the effect of post-ECs cooling to 10°C on muscle contractile performance was determined at 1 and 2 days after ECs. TA muscle samples were examined by hematoxylin and eosin staining to assess damage. In SPINO, reducing the muscle temperature from 30°C to 10°C post-ECs resulted in a 3.7-fold increase in the spread of high [Ca2+]i sites generated by ECs (P < 0.05). These high [Ca2+]i sites demonstrated partial reversibility when rewarmed to 30°C. Dantrolene, a ryanodine receptor Ca2+ release inhibitor, reduced the presence of high [Ca2+] sites at 10°C. In the TA, cooling exacerbated ECs-induced muscle strength deficits via enhanced muscle fiber damage (P < 0.05). By demonstrating that cooling post-ECs potentiates [Ca2+]i derangements, this in vivo approach supports a putative mechanistic basis for how postexercise cryotherapy might augment muscle fiber damage and decrease subsequent exercise performance.

Abstract 17220: Functional Roles and Molecular Mechanisms of Calcium Release Signaling in Hypoxic Cellular Responses in Pulmonary Artery Smooth Muscle Cells

Introduction: Hypoxia causes contractile and proliferative responses in pulmonary arterial smooth muscle cells (PASMCs), but not systemic artery SMCs, thereby leading to pulmonary hypertension (PH), but not systemic hypertension. However, the underlying mechanisms remain unclear. Hypothesis: Ca 2+ release signaling in PASMCs may mediate hypoxic pulmonary artery vasoconstriction, remodeling and hypertension. Methods: Single cell RT-PCR and Western blot analysis, FRET and immunoprecipitation, tissue contraction assay, immunohistochemistry staining and pressure-volume loop analysis, and genetic and pharmacological approaches, were, respectively, used to determine targeted molecular expression, interaction and function. Results: All ryanodine receptor (RyR1, RyR2 and RyR3) Ca 2+ release channels are expressed in mouse PASMCs. RyR1 or RyR3 knockout (KO) partially inhibits, while RyR2 KO completely blocks, hypoxic cellular responses. SMC-specific RyR2 KO restores abolishes hypoxic pulmonary artery vasoconstriction, remodeling and hypertension in mice. RyR2 KO also inhibits hypoxic nuclear factor (NF)-κB and downstream cyclin D1 activation. FK506 binding protein 12.6 (FKBP12.6) is an endogenous RyR2 inhibitor; its KO or FK506 treatment enhances, and treatment of S107 (a specific RyR2/FKBP12.6 stabilizer) inhibits hypoxic PH. Lentiviral shRNA-mediated SMC-specific Rieske iron-sulfur protein (RISP) knockdown diminishes mitochondrial reactive oxygen species (ROS) generation, RyR2/FKBP12.6 complex dissociation, and RyR2 activation, thereby blocking hypoxic PH. Conclusions: RyR1, RyR2 and RyR3 are all involved in hypoxic contractile and proliferative responses in PASMCs, but RyR2 is a most important player. RISP-dependent mitochondrial ROS - FKBP12.6/RyR2 complex dissociation - RyR2 hyperfunction signaling mediate PA vasoconstriction. RyR2 hyperfunction also activates Ca2+-dependent NF-κB/cyclinD1 pathway to provoke PA remodeling; PA vasoconstriction and remodeling together cause PH. Furthermore, RISP-mediated ROS inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blockade may be novel and effective options for the treatment of PH.

A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types.

Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.

G protein-coupled receptor signal transduction and Ca2+ signaling pathways of the allatotropin/orexin system in Hydra

Allatotropin is a pleiotropic peptide originally characterized in insects. The existence of AT neuropeptide signaling was proposed in other invertebrates. In fact, we previously proposed the presence of an AT-like system regulating feeding behavior in Hydra sp. Even in insects, the information about the AT signaling pathway is incomplete. The aim of this study is to analyze the signaling cascade activated by AT in Hydra plagiodesmica using a pharmacological approach. The results show the involvement of Ca2+ and IP3 signaling in the transduction pathway of the peptide. Furthermore, we confirm the existence of a GPCR system involved in this pathway, that would be coupled to a Gq subfamily of Gα protein, which activates a PLC, inducing an increase in IP3 and cytosolic Ca2+. To the best of our knowledge, this work represents the first in vivo approach to study the overall signaling pathway and intracellular events involved in the myoregulatory effect of AT in Hydra sp.

Metabolic syndrome inhibits store-operated Ca2+ entry and calcium-induced calcium-release mechanism in coronary artery smooth muscle

Background and purposeMetabolic syndrome causes adverse effects on the coronary circulation including altered vascular responsiveness and the progression of coronary artery disease (CAD). However the underlying mechanisms linking obesity with CAD are intricated. Augmented vasoconstriction, mainly due to impaired Ca2+ homeostasis in coronary vascular smooth muscle (VSM), is a critical factor for CAD. Increased calcium–induced calcium release (CICR) mechanism has been associated to pathophysiological conditions presenting persistent vasoconstriction while increased store operated calcium (SOC) entry appears to activate proliferation and migration in coronary vascular smooth muscle (VSM). We analyze here whether metabolic syndrome might alter SOC entry as well as CICR mechanism in coronary arteries, contributing thus to a defective Ca2+ handling and therefore accelerating the progression of CAD. Experimental approachMeasurements of intracellular Ca2+ ([Ca2+]i) and tension and of Ca2+ channels protein expression were performed in coronary arteries (CA) from lean Zucker rats (LZR) and obese Zucker rats (OZR). Key resultsSOC entry stimulated by emptying sarcoplasmic reticulum (SR) Ca2+ store with cyclopiazonic acid (CPA) was decreased and associated to decreased STIM-1 and Orai1 protein expression in OZR CA. Further, CICR mechanism was blunted in these arteries but Ca2+ entry through voltage-dependent L-type channels was preserved contributing to maintain depolarization-induced increases in [Ca2+]i and vasoconstriction in OZR CA. These results were associated to increased expression of voltage-operated L-type Ca2+ channel alpha 1C subunit (CaV1.2) but unaltered ryanodine receptor (RyR) and sarcoendoplasmic reticulum Ca2+‐ATPase (SERCA) pump protein content in OZR CA. Conclusion and implicationsThe present manuscript provides evidence of impaired Ca2+ handling mechanisms in coronary arteries in metabolic syndrome where a decrease in both SOC entry and CICR mechanism but preserved vasoconstriction are reported in coronary arteries from obese Zucker rats. Remarkably, OZR CA VSM at this state of metabolic syndrome seemed to have developed a compensation mechanism for impaired CICR by overexpressing CaV1.2 channels.

Preserved Ca2+ handling and excitation-contraction coupling in muscle fibres from diet-induced obese mice.

Disrupted intracellular Ca2+ handling is known to play a role in diabetic cardiomyopathy but it has also been postulated to contribute to obesity- and type 2 diabetes-associated skeletal muscle dysfunction. Still, there is so far very limited functional insight into whether, and if so to what extent, muscular Ca2+ homeostasis is affected in this situation, so as to potentially determine or contribute to muscle weakness. In differentiated muscle, force production is under the control of the excitation-contraction coupling process: upon plasma membrane electrical activity, the CaV1.1 voltage sensor/Ca2+ channel in the plasma membrane triggers opening of the ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. Opening of the ryanodine receptor triggers the rise in cytosolic Ca2+, which activates contraction while Ca2+ uptake by the SR ATPase Ca2+-pump promotes relaxation. These are the core mechanisms underlying the tight control of muscle force by neuronal electrical activity. This study aimed at characterising their inherent physiological function in a diet-induced mouse model of obesity and type 2 diabetes. Intact muscle fibres were isolated from mice fed either with a standard chow diet or with a high-fat, high-sucrose diet generating obesity, insulin resistance and glucose intolerance. Properties of muscle fibres were investigated with a combination of whole-cell voltage-clamp electrophysiology and confocal fluorescence imaging. The integrity and density of the plasma membrane network (transverse tubules) that carries the membrane excitation throughout the muscle fibres was assessed with the dye Di-8-ANEPPS. CaV1.1 Ca2+ channel activity was studied by measuring the changes in current across the plasma membrane elicited by voltage-clamp depolarising pulses of increasing amplitude. SR Ca2+ release through ryanodine receptors was simultaneously detected with the Ca2+-sensitive dye Rhod-2 in the cytosol. CaV1.1 voltage-sensing activity was separately characterised from the properties of intra-plasma-membrane charge movement produced by short voltage-clamp depolarising pulses. Spontaneous Ca2+ release at rest was assessed with the Ca2+-sensitive dye Fluo-4. The rate of SR Ca2+ uptake was assessed from the time course of cytosolic Ca2+ recovery after the end of voltage excitation using the Ca2+-sensitive dye Fluo-4FF. The response to a fatigue-stimulation protocol was determined from the time course of decline of the peak Fluo-4FF Ca2+ transients elicited by 30 trains of 5-ms-long depolarising pulses delivered at 100Hz. The transverse tubule network architecture and density were well preserved in the fibres from the obese mice. The CaV1.1 Ca2+ current and voltage-sensing properties were also largely unaffected with mean values for maximum conductance and maximum amount of charge of 234 ± 12 S/F and 30.7 ± 1.6 nC/μF compared with 196 ± 13 S/F and 32.9 ± 2.0 nC/μF in fibres from mice fed with the standard diet, respectively. Voltage-activated SR Ca2+ release through ryanodine receptors also exhibited very similar properties in the two groups with mean values for maximum rate of Ca2+ release of 76.0 ± 6.5 and 78.1 ± 4.4μmoll-1ms-1, in fibres from control and obese mice, respectively. The response to a fatigue protocol was also largely unaffected in fibres from the obese mice, and so were the rate of cytosolic Ca2+ removal and the spontaneous Ca2+ release activity at rest. The functional properties of the main mechanisms involved in the control of muscle Ca2+ homeostasis are well preserved in muscle fibres from obese mice, at the level of both the plasma membrane and of the SR. We conclude that intracellular Ca2+ handling and excitation-contraction coupling in skeletal muscle fibres are not primary targets of obesity and type 2 diabetes. Graphical abstract.

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.

Mitochondrial calcium drives clock gene-dependent activation of pyruvate dehydrogenase and of oxidative phosphorylation

Regulation of metabolism is emerging as a major output of circadian clock circuitry in mammals. Accordingly, mitochondrial oxidative metabolism undergoes both in vivo and in vitro daily oscillatory activities. In a previous study we showed that both glycolysis and mitochondrial oxygen consumption display a similar time-resolved rhythmic activity in synchronized HepG2 cell cultures, which translates in overall bioenergetic changes as here documented by measurement of the ATP level. Treatment of synchronized cells with specific metabolic inhibitors unveiled pyruvate as a major source of reducing equivalents to the respiratory chain with its oxidation driven by the rhythmic (de)phosphorylation of pyruvate dehydrogenase. Further investigation enabled to causally link the autonomous cadenced mitochondrial respiration to a synchronous increase of the mitochondrial Ca2+. The rhythmic change of the mitochondrial respiration was dampened by inhibitors of the mitochondrial Ca2+ uniporter as well as of the ryanodine receptor Ca2+ channel or the ADPR cyclase, indicating that the mitochondrial Ca2+ influx originated from the ER store, likely at contact sites with the mitochondrial compartment. Notably, blockage of the mitochondrial Ca2+ influx resulted in deregulation of the expression of canonical clock genes such as BMALl1, CLOCK, NR1D1. All together our findings unveil a hitherto unexplored function of Ca2+-mediated signaling in time keeping the mitochondrial metabolism and in its feed-back modulation of the circadian clockwork.

Rieske iron-sulfur protein induces FKBP12.6/RyR2 complex remodeling and subsequent pulmonary hypertension through NF-\u03baB/cyclin D1 pathway

Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) plays an important role in pulmonary hypertension (PH). However, the underlying specific ion channel mechanisms remain largely unknown. Here, we report ryanodine receptor (RyR) channel activity and Ca2+ release both are increased, and association of RyR2 by FK506 binding protein 12.6 (FKBP12.6) is decreased in PASMCs from mice with chronic hypoxia (CH)-induced PH. Smooth muscle cell (SMC)-specific RyR2 knockout (KO) or Rieske iron-sulfur protein (RISP) knockdown inhibits the altered Ca2+ signaling, increased nuclear factor (NF)-κB/cyclin D1 activation and cell proliferation, and CH-induced PH in mice. FKBP12.6 KO or FK506 treatment enhances CH-induced PH, while S107 (a specific stabilizer of RyR2/FKBP12.6 complex) produces an opposite effect. In conclusion, CH causes RISP-dependent ROS generation and FKBP12.6/RyR2 dissociation, leading to PH. RISP inhibition, RyR2/FKBP12.6 complex stabilization and Ca2+ release blockade may be potentially beneficial for the treatment of PH.

NMDA Receptor Modulates Spinal Iron Accumulation Via Activating DMT1(-)IRE in Remifentanil-Induced Hyperalgesia

N-methyl-D-aspartate (NMDA) receptor activation is known to be critical in remifentanil-induced hyperalgesia. Evidence indicates that iron accumulation participates in NMDA neurotoxicity. This study aims to investigate the role of iron accumulation in remifentanil-induced hyperalgesia. Remifentanil was delivered intravenously in rats to induce hyperalgesia. The NMDA receptor antagonist MK-801 was intrathecally administrated. The levels of divalent metal transporter 1 without iron-responsive element [DMT1(-)IRE] and iron were detected. Behavior testing was performed in DMT1(-)IRE knockdown rats and rats treated with iron chelator DFO. Meanwhile, the spinal dorsal horn neurons were cultured and transfected with DMT1(-)IRE siRNA, and then respectively incubated with remifentanil and MK-801. The levels of intracellular Ca2+ and iron were assessed by fluorescence imaging. Our data revealed that spinal DMT1(-)IRE and iron content significantly increased in remifentanil-treated rats, and MK-801 inhibited the enhancements. DMT1(-)IRE knockdown and DFO prevented against remifentanil-induced hyperalgesia. Notably, the levels of Ca2+ and iron increased in remifentanil-incubated neurons, and these growths can be blocked by MK-801. DMT1(-)IRE knockdown attenuated iron accumulation but did not influence Ca2+ influx. This study suggests that DMT1(-)IRE-mediated iron accumulation is likely to be the downstream event following NMDA receptor activation and Ca2+ influx, contributing to remifentanil-induced hyperalgesia. PerspectiveRemifentanil-induced hyperalgesia is common even when used within clinical accepted doses. This study presents that aberrant iron accumulation is involved in the development of remifentanil-induced hyperalgesia in vivo and in vitro. Iron chelation may be a potential therapeutic strategy for the prevention of hyperalgesia in populations at high risk.

The TNFSF Members APRIL and BAFF and Their Receptors TACI, BCMA, and BAFFR in Oncology, With a Special Focus in Breast Cancer.

Tumor necrosis factor (TNF) superfamily consists of 19 ligands and 29 receptors and is related to multiple cellular events from proliferation and differentiation to apoptosis and tumor reduction. In this review, we overview the whole system, and we focus on A proliferation-inducing ligand (APRIL, TNFSF13) and B cell-activating factor (BAFF, TNFSF13B) and their receptors transmembrane activator and Ca2+ modulator (CAML) interactor (TACI, TNFRSF13B), B cell maturation antigen (BCMA, TNFRSF17), and BAFF receptor (BAFFR, TNFRSF13C). We explore their role in cancer and novel biological therapies introduced for multiple myeloma and further focus on breast cancer, in which the modulation of this system seems to be of potential interest, as a novel therapeutic target. Finally, we discuss some precautions which should be taken into consideration, while targeting the APRIL–BAFF system.

ER-luminal [Ca2+] regulation of InsP3 receptor gating mediated by an ER-luminal peripheral Ca2+-binding protein.

Modulating cytoplasmic Ca2+ concentration ([Ca2+]i) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP3R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca2+ concentration ([Ca2+]ER) is poorly understood and controversial. We discovered that the InsP3R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca2+]ER. The widely-expressed Ca2+-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca2+]ER inhibition of InsP3R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP3-mediated Ca2+ signaling events. Thus, [Ca2+]ER is a major regulator of InsP3R channel activity and InsP3R-mediated [Ca2+]i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca2+-binding protein, likely ANXA1.

Effect of carvedilol on atrial excitation-contraction coupling, Ca2+ release, and arrhythmogenicity.

Carvedilol is an FDA-approved β-blocker commonly used for treatment of high blood pressure, congestive heart failure, and cardiac tachyarrhythmias, including atrial fibrillation. We investigated at the cellular level the mechanisms through which carvedilol interferes with sarcoplasmic reticulum (SR) Ca2+ release during excitation-contraction coupling (ECC) in single rabbit atrial myocytes. Carvedilol caused concentration-dependent (1-10 µM) failure of SR Ca2+ release. Failure of ECC and Ca2+ release was the result of dose-dependent inhibition of voltage-gated Na+ (INa) and L-type Ca2+ (ICa) currents that are responsible for the rapid depolarization phase of the cardiac action potential (AP) and the initiation of Ca2+-induced Ca2+ release from the SR, respectively. Carvedilol (1 µM) led to AP duration shortening, AP failures, and peak INa inhibition by ~80%, whereas ICa was not markedly affected. Carvedilol (10 µM) blocked INa almost completely and reduced ICa by ~40%. No effect on Ca2+-transient amplitude, ICa, and INa was observed in control experiments with the β-blocker metoprolol, suggesting that the carvedilol effect on ECC is unlikely the result of its β-blocking property. The effects of carvedilol (1 µM) on subcellular SR Ca2+ release was spatially inhomogeneous, where a selective inhibition of peripheral subsarcolemmal Ca2+ release from the junctional SR accounted for the cell-averaged reduction in Ca2+-transient amplitude. Furthermore, carvedilol significantly reduced the probability of spontaneous arrhythmogenic Ca2+ waves without changes of SR Ca2+ load. The data suggest a profound antiarrhythmic action of carvedilol in atrial myocytes resulting from an inhibitory effect on the SR Ca2+ release channel.NEW & NOTEWORTHY Here we show that the clinically widely used β-blocker carvedilol has profound effects on Ca2+ signaling and ion currents, but also antiarrhythmic effects in adult atrial myocytes. Carvedilol inhibits sodium and calcium currents and leads to failure of ECC but also prevents spontaneous Ca2+ release from cellular sarcoplasmic reticulum (SR) Ca2+ stores in form of arrhythmogenic Ca2+ waves. The antiarrhythmic effect occurs by carvedilol acting directly on the SR ryanodine receptor Ca2+ release channel.

Relaxant and antiadrenergic effects of ranolazine in human saphenous vein.

Ranolazine improves vascular function in animal models. We evaluate the effects of ranolazine on vascular function and adrenergic response in human saphenous vein. Rings from 53 patients undergoing coronary artery bypass grafting were mounted in organ baths. Concentration-response curves to ranolazine were constructed in rings precontracted with phenylephrine, endothelin-1, vasopressin, KCl and the thromboxane A2 analogue U-46619. In rings precontracted with phenylephrine, relaxation to ranolazine was tested in the absence and presence of endothelial factors inhibitors, K+ channel blockers and verapamil. The effects of ranolazine on frequency-response and concentration-response curves to phenylephrine were performed in the absence and presence of endothelial factors inhibitors and K+ channel blockers. Endothelial nitric oxide synthase, α1 adrenergic receptor and large conductance Ca2+-activated K+ channel protein expressions were measured by Western blotting. Ranolazine (10-9-10-4 M) produced a concentration-dependent relaxation only in rings precontracted with phenylephrine that was reduced by endothelial denudation, NG-nitro-l-arginine methyl ester (10-4 M), charybdotoxin (10-7 M) and verapamil (10-6 M). Ranolazine diminished adrenergic contractions induced by electrical field stimulation (2-4 Hz) and phenylephrine (10-9-10-5 M) that were prevented by tetraethylammonium (10-3 M) and charybdotoxin (10-7 M). Ranolazine significantly decreased α1 adrenergic receptor and increased large conductance Ca2+-activated K+ channel protein expression in the saphenous vein. Ranolazine diminishes the adrenergic vasoconstriction, acting as α1 antagonist, and by increasing large conductance Ca2+-activated K+ channel involvement. The relaxant effects of ranolazine are partially mediated by endothelial nitric oxide, large conductance Ca2+-activated K+ channels and the blockade of voltage-dependent Ca2+ channels.

Regulation of Human RyR2 by Calmodulin

In cardiomyocytes, cellular contraction requires the process of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum via ryanodine receptor (RyR2) Ca2+ channels. Conversely, cellular rest requires tight control over CICR to avoid spontaneous sarcoplasmic reticulum Ca2+ release. One such control mechanism is calmodulin (CaM)-mediated inhibition of RyR2. Here, we studied how this regulatory mechanism is affected by phosphorylation and oxidation of human (h)RyR2 recombinantly expressed in HEK293 cells. We monitored hRyR2-CaM binding and endoplasmic reticulum (ER) Ca2+ depletion in cells co-transfected with hRyR2 and the ER-targeted Ca2+ sensor R-CEPIA1er. Surface membrane permeabilization allowed control over cytosolic [Ca2+] ([Ca2+]Cyt) and [CaM]. We found that CaM was bound to hRyR2 at [Ca]Cyt ranging 200 nM to 5 μM, suggesting that CaM remains attached to hRyR2 throughout the entire Ca2+ cycle. In HEK293 cells, we found that CaM-hRyR2 binding caused early CICR termination during spontaneous Ca2+ waves, without affecting resting ER Ca2+ load. Adult rodent ventricular myocyte studies have previously shown that RyR2 oxidation, CaMKII activation, and phosphomimetic S2814D-RyR2 increase Ca2+ leak and decrease CaM-RyR2 affinity. Here, phosphomimetic mutations at PKA and CaMKII hRyR2 sites did not affect CaM-RyR2 binding, but these mutations did abolish CICR inhibition by CaM in HEK293 cells. Furthermore, hRyR2 oxidation caused CaM dissociation and depletion of ER Ca2+ load in nM [Ca2+]Cyt. Since CaM did not affect ER Ca2+ at low (resting) [Ca2+]Cyt, ER Ca2+ leak induced by oxidative stress may be partially mediated by CaM-independent mechanisms. Together, these results suggest that (1) the [Ca2+]Cyt increase during Ca2+ sparks/waves limits CICR via CaM-dependent inhibition of hRyR2; (2) hRyR2 hyper-phosphorylation increases CICR by weakening the negative control by CaM without altering CaM-hRyR2 affinity; (3) hRyR2 oxidation increases CICR via RyR2-CaM dissociation, and ER Ca2+ leak via a combination of CaM-independent and dependent mechanisms.

Identifying Ryanodine Receptor Modulators: from High-Throughput Screening to Single Channel Recording

The ryanodine receptor Ca2+-release channels (RyRs) are central to cytoplasmic Ca2+ signalling in particular in the skeletal muscle of both mammals and insects. Regarding the latter, several insecticides target RyRs but recent studies have shown that specific RyR point mutations can confer insecticide resistance. Consequently, research for new RyR modulators remains crucial for crop protection. The full-length coding sequence of the Plutella xylostella RyR was synthesized and cloned into suitable mammalian expression vector. We engineered HEK-293 expressing the insect target and functionally selected stable clones by the use of Ca2+-sensitive fluorescence. In order to exclude any endogenous non-specific response, a related mock clone was also generated in parallel. Stimulation of the cells to activate the RyR elicited a Ca2+ mobilization, which was detected as a fluorescence increase using a FLIPR-TETRA instrument. This RyR cell-based assay was validated with RyR ligands and putative blockers and further optimized to make it suitable for high throughput screening. Drug discovery programs, which may involve the screening of >105 compounds, requires a high fidelity method of determining the effect of putative hits on the function of the target protein. Since RyR expression is targeted to internal membranes, patch-clamp of the nuclear membrane has been previously shown to be an effective method of isolating them for single-channel recording. We therefore set up this very challenging technique to measure the RyR activity single-channel properties (conductance, open probability -Po- and dwell time distribution) and to show the modulatory effect of Ca2+ on these above-mentioned parameters. From cell cloning to Mode of Action determination, we develop an assay funnel, optimized for hit sensitivity and specificity that is easily scalable to larger compound libraries.

TRPC1 mediates slow excitatory synaptic transmission in hippocampal oriens/alveus interneurons

Hippocampal GABAergic interneurons play key roles in regulating principal cell activity and plasticity. Interneurons located in stratum oriens/alveus (O/A INs) receive excitatory inputs from CA1 pyramidal cells and express a Hebbian form of long-term potentiation (LTP) at their excitatory input synapses. This LTP requires the activation of metabotropic glutamate receptors 1a (mGluR1a) and Ca2+ entry via transient receptor potential (TRP) channels. However, the type of TRP channels involved in synaptic transmission at these synapses remains largely unknown. Using patch-clamp recordings, we show that slow excitatory postsynaptic currents (EPSCs) evoked in O/A INs are dependent on TRP channels but may be independent of phospholipase C. Using reverse transcription polymerase chain reaction (RT-PCR) we found that mRNA for TRPC 1, 3–7 was present in CA1 hippocampus. Using single-cell RT-PCR, we found expression of mRNA for TRPC 1, 4–7, but not TRPC3, in O/A INs. Using co-immunoprecipitation assays in HEK-293 cell expression system, we found that TRPC1 and TRPC4 interacted with mGluR1a. Co-immunoprecipitation in hippocampus showed that TRPC1 interacted with mGluR1a. Using immunofluorescence, we found that TRPC1 co-localized with mGluR1a in O/A IN dendrites, whereas TRPC4 localization appeared limited to O/A IN cell body. Down-regulation of TRPC1, but not TRPC4, expression in O/A INs using small interfering RNAs prevented slow EPSCs, suggesting that TRPC1 is an obligatory TRPC subunit for these EPSCs. Our findings uncover a functional role of TRPC1 in mGluR1a-mediated slow excitatory synaptic transmission onto O/A INs that could be involved in Hebbian LTP at these synapses.

Moringa oleifera leaf extract induces vasorelaxation via endothelium-dependent hyperpolarization and calcium channel blockade in mesenteric arterial beds isolated from L-NAME hypertensive rats

ABSTRACT Background An aqueous extract of Moringa oleifera leaves (MOE) is known to cause relaxation of mesenteric resistance arteries of rats in which hypertension has been induced by the administration of L-NAME, but the mechanism(s) of action of MOE remains unclear. The purpose of this study was to investigate these mechanisms in mesenteric arterial beds isolated from L-NAME induced hypertensive rats. Methods: An investigation of vascular reactivity was conducted on isolated mesenteric arterial beds by measuring the changes in perfusion pressure using an in vitro system. Results MOE (0.001–3 mg in 0.1 ml injection volume) caused a dose-dependent relaxation in methoxamine (5 µM) pre-contracted arterial beds, which was partially abolished by endothelium removal. The endothelium-dependent component of vasorelaxation was insensitive to both L-NAME (100 µM) and indomethacin (10 µM), while completely inhibited in high KCl (45 mM)-induced contraction. MOE (1 and 3 mg/ml) showed a dose-dependent inhibitory effect on CaCl2-induced contractions of denuded preparations in Ca2+-free medium containing a high KCl (60 mM) or methoxamine (10 µM). In Ca2+-free medium, MOE (3 mg/ml) also inhibited phenylephrine-induced contractions of denuded preparations. Conclusion: These findings suggest that MOE relaxes mesenteric arterial beds of L-NAME hypertensive rats via both endothelium-dependent and endothelium-independent mechanisms. The endothelium-dependent action occurred via endothelium-derived hyperpolarizing factor-mediated hyperpolarization. The endothelium-independent action was related to blocking the entry of extracellular Ca2+ via voltage-operated and receptor-operated Ca2+ channels, and inhibiting mobilization of sarcolemmal Ca2+ via inositol trisphosphate receptor Ca2+ channels. MOE may be potentially useful as a natural vasodilator against hypertension.

Super-Resolution Imaging Using a Novel High-Fidelity Antibody Reveals Close Association of the Neuronal Sodium Channel NaV1.6 with Ryanodine Receptors in Cardiac Muscle.

The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (NaV1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for NaV1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal NaV1.6 antibody and undertook super-resolution microscopy studies of NaV1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (NaV1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of NaV1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca2+) cycling protein, in cardiac myocytes. In summary, our novel NaV1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the NaV1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca2+ release channels.

Ca2+ oscillations in rat carotid body type 1 cells in normoxia and hypoxia.

We studied the mechanisms by which carotid body glomus (type 1) cells produce spontaneous Ca2+ oscillations in normoxia and hypoxia. In cells perfused with normoxic solution at 37°C, we observed relatively uniform, low-frequency Ca2+ oscillations in >60% of cells, with each cell showing its own intrinsic frequency and amplitude. The mean frequency and amplitude of Ca2+ oscillations were 0.6 ± 0.1 Hz and 180 ± 42 nM, respectively. The duration of each Ca2+ oscillation ranged from 14 to 26 s (mean of ∼20 s). Inhibition of inositol (1,4,5)-trisphosphate receptor and store-operated Ca2+ entry (SOCE) using 2-APB abolished Ca2+ oscillations. Inhibition of endoplasmic reticulum Ca2+-ATPase (SERCA) using thapsigargin abolished Ca2+ oscillations. ML-9, an inhibitor of STIM1 translocation, also strongly reduced Ca2+ oscillations. Inhibitors of L- and T-type Ca2+ channels (Cav; verapamil>nifedipine>TTA-P2) markedly reduced the frequency of Ca2+ oscillations. Thus, Ca2+ oscillations observed in normoxia were caused by cyclical Ca2+ fluxes at the ER, which was supported by Ca2+ influx via Ca2+ channels. Hypoxia (2-5% O2) increased the frequency and amplitude of Ca2+ oscillations, and Cav inhibitors (verapamil>nifedipine>>TTA-P2) reduced these effects of hypoxia. Our study shows that Ca2+ oscillations represent the basic Ca2+ signaling mechanism in normoxia and hypoxia in CB glomus cells.