Sarco/endoplasmic Reticulum Ca(2+)-ATPase Pump Research Articles

Positive Allosteric Modulators of SERCA Pump Restore Dendritic Spines and Rescue Long-Term Potentiation Defects in Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects memory formation and storage processes. Dysregulated neuronal calcium (Ca2+) has been identified as one of the key pathogenic events in AD, and it has been suggested that pharmacological agents that stabilize Ca2+ neuronal signaling can act as disease-modifying agents in AD. In previous studies, we demonstrated that positive allosteric regulators (PAMs) of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump might act as such Ca2+-stabilizing agents and exhibit neuroprotective properties. In the present study, we evaluated effects of a set of novel SERCA PAM agents on the rate of Ca2+ extraction from the cytoplasm of the HEK293T cell line, on morphometric parameters of dendritic spines of primary hippocampal neurons in normal conditions and in conditions of amyloid toxicity, and on long-term potentiation in slices derived from 5xFAD transgenic mice modeling AD. Several SERCA PAM compounds demonstrated neuroprotective properties, and the compound NDC-9009 showed the best results. The findings in this study support the hypothesis that the SERCA pump is a potential therapeutic target for AD treatment and that NDC-9009 is a promising lead molecule to be used in the development of disease-modifying agents for AD.

Positive Allosteric Modulator of SERCA Pump NDC-1173 Exerts Beneficial Effects in Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease that affects millions of people worldwide. AD does not have a cure and most drug development efforts in the AD field have been focused on targeting the amyloid pathway based on the "amyloid cascade hypothesis". However, in addition to the amyloid pathway, substantial evidence also points to dysregulated neuronal calcium (Ca2+) signaling as one of the key pathogenic events in AD, and it has been proposed that pharmacological agents that stabilize neuronal Ca2+ signaling may act as disease-modifying agents in AD. In previous studies, we demonstrated that positive allosteric regulators (PAMs) of the Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump might act as such Ca2+ stabilizing agents. In the present study, we report the development of a novel SERCA PAM agent, compound NDC-1173. To test the effectiveness of this compound, we performed behavioral studies with the APP/PS1 transgenic AD mouse model. We also evaluated effects of this compound on expression of endoplasmic reticulum (ER) stress genes in the hippocampus of APP/PS1 mice. The results of this study support the hypothesis that the SERCA pump is a potential novel therapeutic drug target and that NDC-1173 is a promising lead molecule for developing disease-modifying agents in AD.

Hypermetabolism in mice carrying a near-complete human chromosome 21.

The consequences of aneuploidy have traditionally been studied in cell and animal models in which the extrachromosomal DNA is from the same species. Here, we explore a fundamental question concerning the impact of aneuploidy on systemic metabolism using a non-mosaic transchromosomic mouse model (TcMAC21) carrying a near-complete human chromosome 21. Independent of diets and housing temperatures, TcMAC21 mice consume more calories, are hyperactive and hypermetabolic, remain consistently lean and profoundly insulin sensitive, and have a higher body temperature. The hypermetabolism and elevated thermogenesis are likely due to a combination of increased activity level and sarcolipin overexpression in the skeletal muscle, resulting in futile sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) activity and energy dissipation. Mitochondrial respiration is also markedly increased in skeletal muscle to meet the high ATP demand created by the futile cycle and hyperactivity. This serendipitous discovery provides proof-of-concept that sarcolipin-mediated thermogenesis via uncoupling of the SERCA pump can be harnessed to promote energy expenditure and metabolic health.

Antidiabetic omarigliptin dilates rabbit aorta by activating voltage-dependent K+ channels and the sarco/endoplasmic reticulum Ca2+ -ATPase pump.

We investigated the vasodilatory effect of omarigliptin, an oral antidiabetic drug in the dipeptidyl peptidase-4 inhibitor class, and its related mechanisms using phenylephrine (Phe)-induced pre-contracted aortic rings. Omarigliptin dilated aortic rings pre-constricted with Phe in a dose-dependent manner. Pretreatment with the voltage-dependent K+ channel inhibitor 4-aminopyridine significantly attenuated the vasodilatory effect of omarigliptin, whereas pretreatment with the inwardly rectifying K+ channel inhibitor Ba2+ , ATP-sensitive K+ channel inhibitor glibenclamide, and large-conductance Ca2+ -activated K+ channel inhibitor paxilline did not alter its vasodilation. Pretreatment with the sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid significantly reduced the vasodilatory effect of omarigliptin. Neither cAMP/PKA-related signaling pathway inhibitors nor cGMP/PKG-related signaling pathway inhibitors modulated the vasodilatory effect of omarigliptin. Removal of endothelium did not diminish the vasodilatory effect of omarigliptin. Furthermore, pretreatment with the nitric oxide synthase inhibitor L-NAME or small-conductance Ca2+ -activated K+ channel inhibitor apamin, together with the intermediate-conductance Ca2+ -activated K+ channel inhibitor TRAM-34, did not influence the vasodilatory effect of omarigliptin. In conclusion, omarigliptin induced vasodilation in rabbit aortic smooth muscle by activating voltage-dependent K+ channels and the SERCA pump independently of other K+ channels, cAMP/PKA- and cGMP/PKG-related signaling pathways, and the endothelium.

The effects of neurogranin knockdown on SERCA pump efficiency in soleus muscles of female mice fed a high fat diet

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is responsible for the transport of Ca2+ from the cytosol into the sarcoplasmic reticulum at the expense of ATP, making it a regulator of both muscle relaxation and muscle-based energy expenditure. Neurogranin (Ng) is a small protein that negatively regulates calcineurin signaling. Calcineurin is Ca2+/calmodulin dependent phosphatase that promotes the oxidative fibre type in skeletal muscle and regulates muscle-based energy expenditure. A recent study has shown that calcineurin activation reduces SERCA Ca2+ transport efficiency, ultimately raising energy expenditure. Since the biomedical view of obesity states that it arises as an imbalance between energy intake and expenditure which favors the former, we questioned whether heterozygous Ng deletion (Ng+/- ) would reduce SERCA efficiency and increase energy expenditure in female mice fed a high-fat diet (HFD). Young (3–4-month-old) female wild type (WT) and Ng+/- mice were fed a HFD for 12 weeks with their metabolic profile being analyzed using metabolic cages and DXA scanning, while soleus SERCA efficiency was measured using SERCA specific Ca2+ uptake and ATPase activity assays. Ng+/- mice showed significantly less cage ambulation compared to WT mice but this did not lead to any added weight gain nor changes in daily energy expenditure, glucose or insulin tolerance despite a similar level of food intake. Furthermore, we observed significant reductions in SERCA’s apparent coupling ratio which were associated with significant reductions in SERCA1 and phospholamban content. Thus, our results show that Ng regulates SERCA pump efficiency, and future studies should further investigate the potential cellular mechanisms.

Natural Polyphenols as SERCA Activators: Role in the Endoplasmic Reticulum Stress-Related Diseases.

Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a key protein responsible for transporting Ca2+ ions from the cytosol into the lumen of the sarco/endoplasmic reticulum (SR/ER), thus maintaining Ca2+ homeostasis within cells. Accumulating evidence suggests that impaired SERCA function is associated with disruption of intracellular Ca2+ homeostasis and induction of ER stress, leading to different chronic pathological conditions. Therefore, appropriate strategies to control Ca2+ homeostasis via modulation of either SERCA pump activity/expression or relevant signaling pathways may represent a useful approach to combat pathological states associated with ER stress. Natural dietary polyphenolic compounds, such as resveratrol, gingerol, ellagic acid, luteolin, or green tea polyphenols, with a number of health-promoting properties, have been described either to increase SERCA activity/expression directly or to affect Ca2+ signaling pathways. In this review, potential Ca2+-mediated effects of the most studied polyphenols on SERCA pumps or related Ca2+ signaling pathways are summarized, and relevant mechanisms of their action on Ca2+ regulation with respect to various ER stress-related states are depicted. All data were collected using scientific search tools (i.e., Science Direct, PubMed, Scopus, and Google Scholar).

Heterozygous SOD2 deletion selectively impairs SERCA function in the soleus of female mice.

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) restores intracellular Ca2+ ([Ca2+]i) to resting levels after muscle contraction, ultimately eliciting relaxation. SERCA pumps are highly susceptible to tyrosine (T)‐nitration, impairing their ability to take up Ca2+ resulting in reduced muscle function and increased [Ca2+]i and cellular damage. The mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2), converts superoxide radicals into less reactive H2O2. Heterozygous deletion of SOD2 (Sod2 +/−) in mice increases mitochondrial oxidative stress; however, the consequences of reduced SOD2 expression in skeletal and cardiac muscle, specifically the effect on SERCA pumps, has yet to be investigated. We obtained soleus, extensor digitorum longus (EDL), and left ventricle (LV) muscles from 6 to 7 month‐old wild‐type (WT) and Sod2 +/− female C57BL/6J mice. Ca2+‐dependent SERCA activity assays were performed to assess SERCA function. Western blotting was conducted to examine the protein content of SERCA, phospholamban, and sarcolipin; and immunoprecipitation experiments were done to assess SERCA2a‐ and SERCA1a‐specific T‐nitration. Heterozygous SOD2 deletion did not alter SERCA1a or SERCA2a expression in the soleus or LV but reduced SERCA2a in the EDL compared with WT, though this was not statistically significant. Soleus muscles from Sod2 +/− mice showed a significant reduction in SERCA’s apparent affinity for Ca2+ when compared to WT, corresponding with significantly elevated SERCA2a T‐nitration in the soleus. No effect was seen in the EDL or the LV. This is the first study to investigate the effects of SOD2 deficiency on muscle SERCA function and shows that it selectively impairs SERCA function in the soleus.

Mechanisms underlying the vasodilatory effects of canagliflozin in the rabbit thoracic aorta: Involvement of the SERCA pump and Kv channels

AimsCanagliflozin is an anti-diabetic agent and sodium glucose co-transporter-2 inhibitor. Despite numerous clinical trials demonstrating its beneficial effects on blood pressure, the cellular mechanisms underlying the effects of canagliflozin on vascular reactivity have yet to be clarified. We investigated the vasodilatory effect of canagliflozin on aortic rings isolated from rabbits. Main methodsWe used rabbit thoracic aortic rings and its arterial tone was tested by using wire myography system. Key findingsCanagliflozin caused concentration-dependent vasodilation in aortic rings pre-constricted with phenylephrine or high K+. However, the degree of canagliflozin-induced vasodilation of the aortic rings pre-constricted with high K+ was less than that of rings pre-constricted with phenylephrine. Application of 4-aminopyridine, a voltage-dependent K+ (Kv) channel inhibitor, reduced canagliflozin-induced vasodilation. However, pre-incubation of an inwardly rectifying K+ channel inhibitor, a large-conductance Ca2+-activated K+ channel inhibitor, and an ATP-sensitive K+ inhibitor did not modulate the vasodilatory effects of canagliflozin. Indeed, canagliflozin increased Kv currents in aortic smooth muscle cells. Pre-treatment with thapsigargin or cyclopiazonic acid, a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors, reduced the vasodilatory effects of canagliflozin. Conversely, pre-treatment with a Ca2+ channel inhibitor, adenylyl cyclase/PKA inhibitors, and guanylyl cyclase/PKG inhibitors did not modulate the vasodilatory effects of canagliflozin. Endothelium removal, and pre-treatment with the nitric oxide synthase inhibitor L-NAME, and small- and intermediate-conductance Ca2+-activated K+ channel inhibitor apamin and TRAM-34, did not diminish the vasodilatory effects of canagliflozin. SignificanceOur results indicate that canagliflozin induces vasodilation, which is dependent on the robust SERCA activity and Kv channel activation.

The anti-diabetic drug trelagliptin induces vasodilation via activation of Kv channels and SERCA pumps

AimsIn this study, we investigated the vasodilatory effects of trelagliptin (a dipeptidyl peptidase-4 inhibitor) and its related mechanisms using rabbit aortic rings. Main methodsArterial tone measurement was performed in rabbit thoracic aortic rings. Key findingsTrelagliptin induced vasodilation in a dose-dependent manner. Pretreatment with the ATP-sensitive K+ channel inhibitor glibenclamide, large-conductance Ca2+-activated K+ channel inhibitor paxilline, and inwardly rectifying K+ channel inhibitor Ba2+ did not affect the vasodilatory effect of trelagliptin. However, pretreatment with the voltage-dependent K+ (Kv) channel inhibitors 4-aminopyridine and tetraethylammonium significantly attenuated the vasodilatory effect of trelagliptin, suggesting that the vasodilatory effect of trelagliptin is associated with Kv channel activation. Although pretreatment with Kv1.5 and Kv2.1 subtype inhibitors did not affect the response to trelagliptin, pretreatment with a Kv7.X subtype inhibitor effectively reduced the vasodilatory effect of trelagliptin. Furthermore, sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors also significantly attenuated the vasodilatory effect of trelagliptin. These effects, however, were not affected by pretreatment with Ca2+ channel inhibitors, adenylyl cyclase/PKA inhibitors, guanylyl cyclase/PKG inhibitors, or removal of the endothelium. SignificanceFrom these results, we concluded that the vasodilatory effect of trelagliptin was associated with the activation of Kv channels (primary the Kv7.X subtype) and SERCA pump regardless of other K+ channels, Ca2+ channels, cAMP/PKA-related or cGMP/PKG-related signaling pathways, and the endothelium. Therefore, caution is required when prescribing trelagliptin to the patients with hypotension and diabetes.

Heterozygous SOD2 deletion impairs SERCA function in soleus and heart muscles from female mice

Background Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pumps actively transport intracellular Ca2+ back into the sarcoplasmic reticulum and are the primary facilitators of muscle relaxation, restoring intracellular [Ca2+] to resting levels. Like many proteins, SERCA pumps are subject to oxidation which may affect their functional capacity. Superoxide dismutase 2 (SOD2) is a critical antioxidant enzyme localised in the mitochondrial matrix which converts superoxide radicals into less reactive H2O2. The present study sought to explore the structural and functional consequences of halved SOD2 expression, particularly to SERCA isoforms, in skeletal and cardiac muscle. Specifically, we examined whether heterozygous SOD2 deletion would result in changes in levels of SERCA tyrosine nitration and S-glutathionylation. Tyrosine nitration is known to impair SERCA function, particularly SERCA2a (slow isoform) but not SERCA1a (fast isoform). Conversely, glutathionylation of SERCA may act as an adaptive modality to preserve function during moderate oxidative stress. Methods We obtained soleus, extensor digitorum longus (EDL), and left ventricle (LV) muscles from wild-type (WT) and SOD2 heterozygote (SOD2+/-) C57BL/6J female mice (n=8 per genotype, aged 6-7 months). Ca2+-dependent SERCA activity assays were performed along with immunoprecipitation experiments to examine SERCA2a and SERCA1a tyrosine nitration and S-glutathionylation. Results Heterozygous SOD2 deletion did not alter SERCA1a or SERCA2a expression in the soleus, EDL or LV compared with WT. However, relative to WT, SOD2+/- soleus muscles showed significant impairments in SERCA function, with a reduction in SERCA's affinity for Ca2+. This corresponded well with Western blot data showing significantly elevated SERCA2a tyrosine nitration and significantly reduced S-glutathionylation in the soleus. In contrast, we observed no change in SERCA function, SERCA1a tyrosine nitration, or S-glutathionylation in the EDL. Furthermore, there were no differences in SERCA1a tyrosine nitration in the soleus, which is consistent with the notion that the SERCA1a isoform is less susceptible to nitrosative modification. Together, these results suggest that the slow SERCA isoform is more susceptible to heterozygous SOD2 deletion, and this is further supported by a significant reduction in SERCA's affinity for Ca2+ in the LV where SERCA2a is the predominant isoform. Conclusions This is the first study showing the physiological effects of halved SOD2 expression on muscle SERCA function. Our results reinforce the idea that SERCA2a is more sensitive to nitrosative modification.

The anti-diabetic drug alogliptin induces vasorelaxation via activation of Kv channels and SERCA pumps

In the present study, we investigated the vasorelaxant effects of alogliptin, an oral antidiabetic drug in the dipeptidyl peptidase-4 (DPP-4) inhibitor class, using phenylephrine (Phe)-induced pre-contracted aortic rings. Alogliptin induced vasorelaxation in a dose-dependent manner. Pre-treatment with the voltage-dependent K+ (Kv) channel inhibitor 4-aminopyridine (4-AP) significantly decreased the vasorelaxant effect of alogliptin, whereas pre-treatment with the inwardly rectifying K+ (Kir) channel inhibitor Ba2+, ATP-sensitive K+ (KATP) channel inhibitor glibenclamide, and large-conductance Ca2+-activated K+ (BKCa) channel inhibitor paxilline did not alter the effects of alogliptin. Although pre-treatment with the Ca2+ channel inhibitor nifedipine did not affect the vasorelaxant effect of alogliptin, pre-treatment with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitors thapsigargin and cyclopiazonic acid effectively attenuated the vasorelaxant response of alogliptin. Neither cGMP/protein kinase G (PKG)-related signaling pathway inhibitors (guanylyl cyclase inhibitor ODQ and PKG inhibitor KT 5823) nor cAMP/protein kinase A (PKA)-related signaling pathway inhibitors (adenylyl cyclase inhibitor SQ 22536 and PKA inhibitor KT 5720) reduced the vasorelaxant effect of alogliptin. Similarly, the vasorelaxant effect of alogliptin was not changed by endothelium removal or pre-treatment with the nitric oxide (NO) synthase inhibitor L-NAME or the small- and intermediate-conductance Ca2+-activated K+ (SKCa and IKCa) channel inhibitors apamin and TRAM-34. Based on these results, we suggest that alogliptin induced vasorelaxation in rabbit aortic smooth muscle by activating Kv channels and the SERCA pump independent of other K+ channels, cGMP/PKG-related or cAMP/PKA-related signaling pathways, and the endothelium.

Neuronatin regulates whole-body metabolism: is thermogenesis involved?

Neuronatin (NNAT) was originally discovered in 1995 and labeled as a brain developmental gene due to its abundant expression in developing brains. Over the past 25 years, researchers have uncovered NNAT in other tissues; notably, the hypothalamus, pancreatic β‐cells, and adipocytes. Recent evidence in these tissues indicates that NNAT plays a significant role in metabolism whereby it regulates food intake, insulin secretion, and adipocyte differentiation. Furthermore, genetic deletion of Nnat in mice lowers whole‐body energy expenditure and increases susceptibility to diet‐induced obesity and glucose intolerance; however, the underlying cellular mechanisms remain unknown. Based on its sequence homology with phospholamban, NNAT has a purported role in regulating the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump. However, NNAT also shares sequence homology with sarcolipin, which has the unique property of uncoupling the SERCA pump, increasing whole‐body energy expenditure and thus promoting adaptive thermogenesis in states of caloric excess or cold exposure. Thus, in this article, we discuss the accumulating evidence suggestive of NNAT’s role in whole‐body metabolic regulation, while highlighting its potential to mediate adaptive thermogenesis via SERCA uncoupling.

The role of phospholamban and GSK3 in regulating rodent cardiac SERCA function.

Cardiac contractile function is largely mediated by the regulation of Ca2+ cycling throughout the lifespan. The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump is paramount to cardiac Ca2+ regulation, and it is well established that SERCA dysfunction pathologically contributes to cardiomyopathy and heart failure. Phospholamban (PLN) is a well-known inhibitor of the SERCA pump and its regulation of SERCA2a-the predominant cardiac SERCA isoform-contributes significantly to proper cardiac function. Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase involved in several metabolic pathways, and we and others have shown that it regulates SERCA function. In this mini-review, we highlight the underlying mechanisms behind GSK3's regulation of SERCA function specifically discussing changes in SERCA2a and PLN expression and its potential protection against oxidative stress. Ultimately, these recent findings that we discuss could have clinical implications in the treatment and prevention of cardiomyopathies and heart failure.

Calcium extrusion mechanisms in dendrites of mouse hippocampal CA1 inhibitory interneurons

Local circuit GABAergic inhibitory interneurons control the integration and transfer of information in many brain regions. Several different forms of plasticity reported at interneuron excitatory synapses are triggered by cell- and synapse-specific postsynaptic calcium (Ca2+) mechanisms. To support this function, the spatiotemporal dynamics of dendritic Ca2+ elevations must be tightly regulated. While the dynamics of postsynaptic Ca2+ signaling through activation of different Ca2+ sources has been explored, the Ca2+ extrusion mechanisms that operate in interneuron dendrites during different patterns of activity remain largely unknown. Using a combination of whole-cell patch-clamp recordings and two-photon Ca2+ imaging in acute mouse hippocampal slices, we characterized the Ca2+ extrusion mechanisms activated by Ca2+ transients (CaTs) associated with backpropagating action potentials (bAPs) in dendrites of hippocampal CA1 stratum radiatum interneurons. Our data showed that Ca2+ clearance increased as a function of activity, pointing to an activity-dependent recruitment of specific Ca2+ extrusion mechanisms. bAP-CaTs were significantly prolonged in the presence of the plasma membrane Ca2+ ATPase (PMCA) and Na+/Ca2+ exchanger (NCX) inhibitors as well as the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) and the mitochondria Ca2+ uniporter (MCU) blockers. While PMCA, NCX and SERCA pumps cooperated in the cytosolic Ca2+ removal at a wide range of concentrations, the MCU was only activated at higher Ca2+ loads produced by repetitive interneuron firing. These results identify a division of labor between distinct Ca2+ extrusion mechanisms shaping dendritic Ca2+ dynamics and possibly contributing to activity-dependent regulation of synaptic inputs in interneurons. In addition, the MCU activated by larger Ca2+ levels may be involved in the activity-dependent ATP production or interneuron-selective vulnerability associated with cytosolic Ca2+ overloads under pathological conditions.

Inhibition of Sarco-Endoplasmic Reticulum Ca2+ ATPase Extends the Lifespan in C. elegans Worms

The sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) refills the endoplasmic reticulum (ER) with Ca2+ up to the millimolar range and is therefore the main controller of the ER [Ca2+] level ([Ca2+]ER), which has a key role in the modulation of cytosolic Ca2+ signaling and ER-mitochondria Ca2+ transfer. Given that both cytosolic and mitochondrial Ca2+ dynamics strongly interplay with energy metabolism and nutrient-sensitive pathways, both of them involved in the aging process, we have studied the effect of SERCA inhibitors on lifespan in C. elegans. We have used thapsigargin and 2,5-Di-tert-butylhydroquinone (2,5-BHQ) as SERCA inhibitors, and the inactive analog 2,6-Di-tert-butylhydroquinone (2,6-BHQ) as a control for 2,5-BHQ. Every drug was administered to the worms either directly in the agar or via an inclusion compound with γ-cyclodextrin. The results show that 2,6-BHQ produced a small but significant increase in survival, perhaps because of its antioxidant properties. However, 2,5-BHQ produced in all the conditions a much higher increase in lifespan, and the potent and specific SERCA inhibitor thapsigargin also extended the lifespan. The effects of 2,5-BHQ and thapsigargin had a bell-shaped concentration dependence, with a maximum effect at a certain dose and smaller or even toxic effects at higher concentrations. Our data show therefore that submaximal inhibition of SERCA pumps has a pro-longevity effect, suggesting that Ca2+ signaling plays an important role in the aging process and that it could be a promising novel target pathway to act on aging.

Abstract 2324: The role of SERCA pump in cell death and autophagy

Abstract The purpose of the study is to elucidate structural and molecular determinants of SERCA inhibition by Thapsigargin (Tg) and related analogs for their effects on intracellular calcium homeostasis, ER stress, cell death and autophagy. Tg specifically binds and blocks the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), which pumps Ca2+ from the cytosol to the ER. Sustained SERCA inhibition leads to calcium depletion from the ER causing ER stress and ultimately cell death. Recently we showed that Tg-induced calcium perturbation potently blocks autophagy (1). Tg is an attractive potential anti-tumor drug because it effectively kills both slow and fast proliferating cancer cells. However, since Tg is toxic also to normal cells, it must be targeted towards the cancer cells. Replacing a side chain with a linker connecting the Tg core to a peptide prevents Tg from entering cells. Two different linker-peptide sequences have been introduced in clinically tested Tg prodrugs; one is cleaved by PSA, secreted by prostate cancer cells, and the other is cleaved by PSMA, which is secreted by neovascular tissues of a broad range of tumors. The Tg analogs unmasked by the cleavage are able to enter cells and exert their toxic effects. Interestingly, however, in vitro experiments indicate that Tg analogs have different potencies and cellular effects depending on the terminal amino acid residue (2). Exploring why this is the case may lead not only to better Tg prodrug formulations, but also to a deeper understanding of the biological functions of SERCA pump activity. We compare a broad panel of Tg analogs in various cell types for their biological effects. Methods used so far are western blotting, real-time RT-PCR, live-cell imaging, flow cytometry, and assays that measure autophagic sequestration and degradation activity. Our unpublished data indicate the ER-stress sensors PERK, ATF4, CHOP, and IRE1 but not XBP1 and ATF6 to be involved in cell death signaling in LNCaP prostate cancer cells. Moreover, Tg-induced cell death required death receptor 5 and caspase-8. All Tg analogs tested displayed exactly the same molecular requirements for induction of cell death, although some required 5 to 10 times higher doses than Tg to evoke the same strength of death signal. Like Tg (1) the analogs inhibited autophagy before the closure of phagophores, but at higher doses and/or with slower kinetics. These results indicate that thapsigargin and its analogs evoke similar anti-autophagic and death signaling pathways. Further investigations are aimed at exploring the causes for differential potencies, and include measurements of cytosolic and compartmentalized calcium, as well as solving crystal structures of selected analog:SERCA complexes complemented with biophysical studies of analog:SERCA interactions.

Widespread control of calcium signaling by a family of SERCA-inhibiting micropeptides.

Micropeptides function as master regulators of calcium-dependent signaling in muscle. Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), the membrane pump that promotes muscle relaxation by taking up Ca2+ into the sarcoplasmic reticulum, is directly inhibited by three muscle-specific micropeptides: myoregulin (MLN), phospholamban (PLN), and sarcolipin (SLN). The widespread and essential function of SERCA across diverse cell types has raised questions as to how SERCA is regulated in cells that lack MLN, PLN, and SLN. We identified two transmembrane micropeptides, endoregulin (ELN) and another-regulin (ALN), that share key amino acids with their muscle-specific counterparts and function as direct inhibitors of SERCA pump activity. The distribution of transcripts encoding ELN and ALN mirrored that of SERCA isoform-encoding transcripts in nonmuscle cell types. Our findings identify additional members of the SERCA-inhibitory micropeptide family, revealing a conserved mechanism for the control of intracellular Ca2+ dynamics in both muscle and nonmuscle cell types.

A Compartment Model to Investigate the Roles of SR Membrane Channels during E-C Coupling

During excitation-contraction (E-C) coupling, efficient Ca2+ release by ryanodine receptor (RyR) channels from the sarcoplasmic reticulum (SR) requires that counterions move into the SR. These counterion fluxes are thought to effectively clamp the SR membrane potential (Vm) near 0 mV, sustaining the trans-SR Ca2+ driving force. Likewise, Ca2+ uptake by sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps is facilitated by the existence of counterion fluxes. The SR also contains TRIC (SR K+ channels) and Cl- channels in addition to RyRs and SERCA pumps. Thus, SR Vm during and between RyR-mediated Ca2+ releases is determined by multiple channels whose function spatially and temporally varies. The contribution of each channel to SR Vm control is unclear and debated. We used a compartment model to examine counterion (K+, Cl- and Mg2+) fluxes across the SR membrane during E-C coupling. The model allowed us to study the roles of the different ion channels, as well as the time evolution of SR Vm and local changes in intra-SR/cytosolic ion concentrations. Our results show that RyR Ca2+ efflux is indeed electrically balanced by K+, Cl- and Mg2+ countercurrents. When RyRs are open, Mg2+ initially moves into the SR and then surprisingly moves back out. Redundant pathways exist to carry K+ countercurrent (RyR and TRIC) during SR Ca2+ release. When RyRs close, the K+ must exit through TRIC channels to maintain resting SR Vm at 0 mV. Our analysis indicates TRIC channels are essential for resting SR K+ re-equilibration that is needed to re-establish the normal trans-SR Ca2+ driving force.

Mapping of Sarcolipin Conformational States along the Enzymatic Pathway of SERCA

Sarcolipin (SLN), a 31 amino acid transmembrane peptide, binds to the Sarco(endo)plasmic Reticulum Ca2+ ATPase (SERCA) decreasing its apparent Ca2+ affinity. SERCA pumps Ca2+ from the cytoplasm of muscle cells into the sarcoplasmic reticulum (SR) using energy derived from ATP hydrolysis. This re-establishes the Ca2+ gradients needed for normal muscle function. Phosphorylation of SLN relieves the inhibition of SERCA and may be involved in the beta-adrenergic response. Although a significant amount is known about SERCA, and many of the conformations throughout its transport cycle have been crystallized, the mechanism of how SLN decreases SERCA's Ca2+ affinity and alters the coupling between ATP hydrolysis and Ca2+ transport is not well understood. Using solid-state nuclear magnetic resonance spectroscopy and functional assays, we investigated the differences in the interaction of phosphorylated SLN with SERCA compared to non-phosphorylated SLN with SERCA. Previous work in our lab demonstrates that the topology of SLN in complex with SERCA changes depending on SERCA's conformation. As such, continuing work involves mapping the structural changes of phosphorylated SLN with the various conformations of SERCA. Overall, gaining a better understanding of how SERCA is regulated will add in the development of therapies for diseases resulting from improper calcium cycling.

Mitochondrial calcium handling within the interstitial cells of Cajal.

The interstitial cells of Cajal (ICC) drive rhythmic pacemaking contractions in the gastrointestinal system. The ICC generate pacemaking signals by membrane depolarizations associated with the release of intracellular calcium (Ca(2+)) in the endoplasmic reticulum (ER) through inositol-trisphosphate (IP3) receptors (IP3R) and uptake by mitochondria (MT). This Ca(2+) dynamic is hypothesized to generate pacemaking signals by calibrating ER Ca(2+) store depletions and membrane depolarization with ER store-operated Ca(2+) entry mechanisms. Using a biophysically based spatio-temporal model of integrated Ca(2+) transport in the ICC, we determined the feasibility of ER depletion timescale correspondence with experimentally observed pacemaking frequencies while considering the impact of IP3R Ca(2+) release and MT uptake on bulk cytosolic Ca(2+) levels because persistent elevations of free intracellular Ca(2+) are toxic to the cell. MT densities and distributions are varied in the model geometry to observe MT influence on free cytosolic Ca(2+) and the resulting frequencies of ER Ca(2+) store depletions, as well as the sarco-endoplasmic reticulum Ca(2+) ATP-ase (SERCA) and IP3 agonist concentrations. Our simulations show that high MT densities observed in the ICC are more relevant to ER establishing Ca(2+) depletion frequencies than protection of the cytosol from elevated free Ca(2+), whereas the SERCA pump is more relevant to containing cytosolic Ca(2+) elevations. Our results further suggest that the level of IP3 agonist stimulating ER Ca(2+) release, subsequent MT uptake, and eventual activation of ER store-operated Ca(2+) entry may determine frequencies of rhythmic pacemaking exhibited by the ICC across species and tissue types.