Specificity Of Thapsigargin Towards Sercas Biology Essay

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Thapsigargin has been used prodigiously as a tool to elucidate the mechanisms of store-operated calcium entry. Critically evaluate how useful thapsigargin has been in furthering our understanding of the regulation of calcium fluxes in mammalian cells.


Thapsigargin, a compound isolated from the Mediterranean plant Thapsia garganica, is a specific inhibitor of SERCA pumps on the endoplasmic reticulum. Over the past 20 years it has been of great importance as a means to deplete the endoplasmic reticulum Ca2+ stores. This process has been fundamental for the previous and recent breakthroughs in Ca2+ signalling with the understanding of mechanisms of store operated and receptor operated calcium entry. Thapsigargin was originally used as an effective probe to study the mechanism of store operated calcium entry and nowadays it is still one the most used. Also, thapsigargin must be taken into account as an effective drug in treating prostate cancer. Here, we give a comprehensive overview either on how thapsigargin has been outstanding in widening our view of the function of intracellular Ca2+ stores and how the development of the mechanisms of SOCE and ROCE have progressed over the time.


The agonist-mediated release of Ca2+ from endoplasmic reticulum (ER) or its specialized subcompartments is one of the central themes of chemical signalling in cells. This mode of signalling is dependent on an active sequestration of Ca2+ within the stores, a task accomplished by a class of transporters termed sarco-endoplasmic reticulum Ca2+-ATPases (SERCAs). Thapsigargin is the most widely used SERCA inhibitor as well as cyclopiazonic acid (CPA). Thapsigargin inhibits the skeletal muscle sarcoplasmic reticulum Ca2+-ATPase with a dissociation constant lower than 1 nM. The formation of the thapsigargin enzyme complex is stoichiometric (1: 1) and inhibits the Ca2+-dependent hydrolysis activity and the formation of the phosphorylated intermediate. Calcium binding to the Ca"-ATPase is also affected by thapsigargin: it has also been reported that only a single calcium ion would bind to the Ca2+-ATPase after inhibition by thapsigargin and that this remaining bound Ca2+ would not be located on the high-affinity binding external sites, but binds to the enzyme with a relatively lower affinity.

Thapsigargin was first shown to increase free cytosolic Ca2+ in platelets in 1985 (Ali et al., 1985), and by 1994, an average of more than one report daily involving thapsigargin was being registered by Medline.

Established its function as a specific inhibitor of SERCA pumps on the endoplasmic reticulum, its application to cells results in passive depletion of intracellular Ca2+ stores and thereby activation of the store-operated channels. (Takemura et al. 1989)

The most significant outcome of the discovery of thapsigargin and other SERCA inhibiting drugs was that it provided a functional and pharmacological diagnosis of capacitative entry (or store operated calcium entry): a thapsigargin-induced sustained elevation of intracellular Ca2+ that is dependent upon extracellular Ca2+ is generally attributed to store-operated or capacitative Ca2+ entry. (Putney, Jr. 2001) Notably, the ability of thapsigargin specifically to activate capacitative Ca2+ entry, while minimizing the roles of other upstream players in the pathway (receptors, G proteins, phospholipase C, etc.), formed the basis for the high throughput assays that led to the discoveries of the functions of both stromal interaction molecule (Stim1) and Orai1.

Thapsigargin was isolated from the Mediterranean plant Thapsia garganica L. (Linnaeus), and its structure was elucidated by a combination of chemical, spectroscopic and X-ray-crystallographic methods. It belongs to a group of related, naturally occurring 6,12- guaianolides with a 1b-disposed hydrogen and a 7b-disposed hydroxy group, found in several species belonging to the genus Thapsia. The high lipid solubility of these compounds accounts for their excellent penetration of biological membranes.(Rasmussen et al., 1978)

Specificity of thapsigargin towards SERCAs

Ca2+-ATPase belongs to the family of P-type ion pumps, all of which use ATP to generate ion gradients across a wide variety of cellular membranes.

The best-studied pumps appear to use the same, basic reaction cycle, the hallmark of which is transient phosphorylation of an aspartate residue in the catalytic site and cycling between two main conformations, dubbed E1 and E2.

More specifically, these conformational changes serve to couple the energy of the aspartyl phosphate with translocation of ions across the membrane.

Today, thapsigargin is widely used by cell biologists wishing to empty the internal Ca2+ stores in eukaryotic cells, a result of total inhibition of the SERCA pumps.

Thapsigargin inhibits Ca2+-ATPase by binding to the E2 conformation and by preventing further cycling of the enzyme. Normally, the E2 reaction intermediate is converted to the E1 intermediate by the binding of cytoplasmic Ca2+ to their high-affinity sites, which in turn activates the ATP-binding site to produce the phosphoenzyme intermediate (E1 _ P).

In the presence of Ca2+, thapsigargin does not inhibit the enzyme immediately, but allows a single cycle of phosphorylation and ion transport, indicating that thapsigargin can bind to only the E2 state. This interaction is of very high affinity (Kd < 1 nM) and full inhibition is obtained with a 1:1 molar stoichiometry in what has been termed a dead-end complex.

Release of Ca2+ from intracellular stores by thapsigargin accounts for most of its popularity in the literature. However, it achieves this effect indirectly, as opposed to

ionophores such as calcimycin or ionomycin. Inhibition of SERCAs by thapsigargin prevents the pumps from counterbalancing the passive Ca2+ leak from the stores to the cytosol. Thus, the Ca2+-releasing action of thapsigargin depends on two factors simultaneously: the presence of thapsigargin-sensitive pumps and an appreciable rate of Ca2+ leak (and thus of Ca2+ turnover). (Young et al., 2001)

Thapsigargin as a drug

The long-standing interest in the potential medical benefits of Thapsia garganica is attested by writings of Theophrastos (c. 372-287 BC) and by its use in traditional Arabian and European medicine for rheumatic pains, up to its listing in the French Pharmacopoeia of 1937. Modern work using purified thapsigargin uncovered its tumour-promoting action on mouse skin. Following an initial rise, the fraction of mice developing tumours unexpectedly dropped before a much slower rise ensued after long-term thapsigargin exposure.(Hakii et al.1986)

More recently, it has become clear that application of thapsigargin is associated with activation of programmed cell death in a number of cell types. This apoptotic aspect of thapsigargin action might account for the transient decline of tumours in the mice assay mentioned above. Importantly, it may also be seen as an opportunity to develop a thapsigargin-derived anti-cancer drug.

Literature evidences have shown that since thapsigargin is able to kill slowly proliferating and non-proliferating cells, prodrugs of thapsigargin, which are currently undergoing preclinical evaluation, have been developed as novel means for anti-cancer therapy, notably for the treatment of prostate cancer. (Denmeade et al., 2003)

However, a foremost problem in any such application will be the selective and efficient targeting of the drug to the malignant cells.

Furthermore, compounds disrupting the Ca2+ homeostasis in endoplasmic reticulum (ER), including thapsigargin, have been shown to possess virustatic activity.

One of the major factors inhibiting replication of a number of viruses is nitric oxide (NO) (Karupiah et al., 1995; Melkova et al.,1995). NO is effective against all poxviridae, herpetoviridae, rhabdoviridae, retroviridae, and parvoviridae, including HCV, HSV and many others, although not all viruses are sensitive to the virucidal activity of NO in vitro. Thapsigargin has been shown to interfere with nitric oxide (NO) biosynthesis. Therefore, work of Kmoníčková et al. (2008) demonstrate unequivocally that thapsigargin on its own very potently induces NO production in macrophages of rats under conditions in vitro.

In conclusion, having described the nature of thapsigargin and its role in Ca2+ signalling, the aim of this dissertation is to elucidate the usefulness of thapsigargin in studying and understanding the difficult mechanisms which underlie store operated and receptor operated calcium entry.

It is likely that thapsigargin will become in the future an effective tool or/and drug in treating several conditions of cancer and diseases wherein calcium fluxes are compromised.

Literature review

Calcium is a crucial regulator of many physiological processes, and a variety of stimuli produce their cellular effects by increasing the cytosolic free calcium concentration ([Ca2+]i). This increase in [Ca2+]i can be due to calcium entry from the extracellular space, through channels in the plasma membrane or from intracellular stores of calcium (mainly endoplasmic reticulum, ER). In a variety of electrically non-excitable cells (for example epithelial cells, blood cells, fibroblasts), and in many instances for excitable cells, agonists of G-protein linked receptors activate phospholipase C (PLC) causing hydrolysis of phosphatidylinositol (4,5) biphosphate (PIP2) to release the signalling molecule inositol-1,4,5-trisphosphate (IP3). The receptor for IP3 (IP3R) is located on the membrane of the internal stores and functions as a ligand-gated channel. Its activation by IP3 leads to a rapid release of calcium in the cytoplasm resulting in an increase in [Ca2+]i. An exciting development came with the discovery that in many non-excitable cells, the depletion of internal calcium stores triggers an influx of calcium across the plasma membrane (Putney et al., 1999). As described above, this form of calcium entry was termed as store-operated calcium entry. Experimentally, this calcium influx can be activated by agents that deplete calcium stores, such as thapsigargin.

Store operated calcium entry

The capacitative model of calcium entry has been confirmed in a large number of cell types and is clearly a widespread mechanism of calcium entry. Work in this area has proceeded mainly along two paths. First, to identify the protein channel that serves this function. Second, to delineate the mechanism by which the signal is transmitted from the depleted stores to the SOCs in the plasma membrane.

Over the last 20 years, several studies have been conducted in order to understand the mechanism of activation of SOCE. Here, we show the first hypothesis and the most recent theories about it.

At the beginning, three main hypotheses had been proposed: (1) a diffusible messenger, (2) conformational coupling and (3) vesicle secretion.

The diffusible messenger hypothesis proposed that a messenger molecule is generated and that activates calcium entry in response to store depletion. Several molecules had been proposed as candidates.

These included small G proteins, ATP, GTP, pertussis toxin-sensitive heterotrimeric G proteins, a product of cytochrome P450 activity, arachidonic acid. Perhaps the most interesting and controversial candidate for second messenger was calcium influx factor (CIF). (Putney et al., 1999)

The conformational coupling hypothesis postulated a direct interaction between the IP3R in the ER membrane with the SOCE in the plasma membrane. According to this model, the IP3R senses the calcium depletion within the store and transduces this signal to SOC by physically coupling with it. (Berridge, 1995)

This theory was further confirmed after the discoveries of Stim1 and Orai.

The third model for explaining SOCE is the vesicle-mediated channel insertion hypothesis in which it is proposed that the decrease in store calcium causes additional preformed SOC proteins to be inserted in the plasma membrane. (Patterson et al., 1999; Yao et al., 1999)

So, after the concept of SOCE had been introduced, much research was focused on the mechanism by which the endoplasmic reticulum (ER) Ca2+ status was communicated to the plasma membrane (PM). The most attractive idea was the 'conformational coupling' model, assumed that a physical contact exists between the PM and peripheral elements of the ER where proteins sensing the ER luminal Ca2+ concentration would transfer the information to a Ca2+ channel in the PM. Because Ins(1,4,5)P3 receptors in the ER have a large flexible N-terminal domain that could, in theory, contact the PM, and, because Ca2+ influx is tightly coupled to Ins(1,4,5)P3-induced Ca2+ release, the Ins(1,4,5)P3 receptor seemed like a good candidate to serve as a coupling molecule. This was later discounted based on clear evidence that Ins(1,4,5)P3-receptor-deficient cells still showed functional SOCE and ICRAC (Sugawara et al.,1997) (Ma et al., 2001)

Concerning the nature of the Ca2+ channel, most of the past 20 years was dominated by studies on a group of nonselective cation channels: the transient receptor potential (TRP) channels. (Parekh et al., 2005)

The real breakthrough, however, was brought about by the large-scale use of small interfering RNA (RNAi) for gene silencing and its adaptation for functional screens targeting SOCE. As we mentioned above, in 2005, the numerous studies regarding this topic have culminated with the discovery of two important molecules: Stim1 and Orai. (Liou et al., 2005) (Zhang et al., 2005)

Two studies simultaneously which used small interfering RNA identified Stim1 as a necessary component of SOCE and noted that this protein and its sister, Stim2, serve as sensors of the ER luminal Ca2+ concentration. (Roos et al., 2005)

Within a year, the protein Orai was identified and RNAi screens discovered the same protein as a molecule required for the Ca2+-Release-Activated Ca2+ current (ICRAC). (Vig et al., 2006) (Zhang et al., 2006)

The truth is that the discoveries of Stim and Orai proteins have revolutionized previous thoughts about capacitative Ca2+ entry and ICRAC.

Several studies have identified novel members that can have a role in SOCE, such as the TRPC1 channel. The interaction between Stim1 and TRPC1 upon Ca2+ store depletion was first presented in human platelets endogenously expressing both proteins.(Lopez et al., 2006)

Recently, Jardin et al. (2011) have shown that impairment in their association might be involved in the pathogenesis of the altered platelet responsiveness observed in diabetic patients.

Recently, studies have shown that another molecule that might be involved in SOCE is the microtubule end tracking protein, EB1. (Sampieri et al., 2009) (Grigoriev et al., 2008)

What they found is that in resting conditions (with calcium stores replenished) Stim1 travels continuously through the ER associated to EB1. Upon depletion of the ER Stim1 dissociates from EB1 and aggregates into macromolecular complexes at the ER, throughout aggregation with Orai at the PM.

These series of events involving the association and dissociation of several protein complexes culminate with the activation of calcium influx upon depletion of the ER.

Therefore, Stim1-Orai assembly does not appear to occur in random areas of the plasma membrane, but rather in highly specialized areas known as lipid raft domains. These results strongly suggest that not only proteins but lipids also may be part or active players in the modulation of SOCE.

Dionisio et al. (2011) showed that lipid raft domains are essential for the inactivation of SOCE by extracellular Ca2+ mediated by the interaction between plasma membrane-located Stim1 and Orai.

To simplify the current theory, following Ca2+ store depletion, Stim1 has been shown to move from locations throughout the membrane of the Ca2+ stores to accumulate in regions close to the plasma membrane. Aggregation of Stim1 underneath the plasma membrane induces Orai clustering at punctae in the plasma membrane directly opposite the Stim1 clusters, resulting in the activation of SOCE.(Putney et al., 2007)

Our understanding of the mechanisms underlying both Ca2+ release and store-operated Ca2+ entry have evolved from experimental approaches that include the use of fluorescent Ca2+ indicators and electrophysiological techniques.(Bird et al., 2008)

Receptor operated calcium entry

Another pathway of Ca2+ entry is taking place in both excitable and non-excitable cells, known as receptor operated calcium entry (ROCE). It depends on the generation of second messengers but do not require the depletion of the ER to be activated. So far, the best characterized ROCE that can be clearly distinguished from SOCE is the arachidonic acid-activated Ca2+-selective current (ARC). This Ca2+ influx depends exclusively on the presence of arachidonic acid and was reported to be the major Ca2+ entry pathway during stimulation by low agonist concentrations in different cell types, such as smooth muscle cells (Broad et al., 1999), HEK293 cells (Holmes et al., 2007), or murine parotid and pancreatic acinar cells. (Mignen et al., 2005)

Beside ARC, additional non-SOCE Ca2+ entry channels, in particular non-selective cation channels, are activated by a variety of second messengers like diacylglycerol, 5_,6_ epoxyeicosatrienoic acid, inositol 1,3,4,5-tetrakisphosphate or Ca2+ itself, and most likely are responsible for the appearance of ROCE.

So far the best candidates for the ROCE channels belong to the family of the transient receptor potential (TRP) channels. As we have mentioned, among them the TRPC subfamily comprises both store-operated and non-store-operated cationic channels.

In Lorenzo et al. (2008) study, it has been shown that TRPV4, a sub member of the TRPC family, takes part in receptor operated calcium entry in mouse airway epithelial cells.

Also, in Brechard et al. (2008), it has been shown that TRPC3 acts in a SOCE-independent pathway, as demonstrated in experiments regarding the production of NAPDH oxidase in neutrophil-like cells.

Despite the numerous reports in this field, it still remains confusing to experimentally discriminate between SOCE and ROCE under physiological conditions as agonist stimulation triggers Ca2+ release from the ER, and, thus, may activate SOCE, while it also activates additional signalling pathways leading to ROCE.

Recently, Salmon & Ahluwalia (2011) have shown further consideration about distinguishing a non-store operated from store operated Ca2+ entry pathway in neutrophils.


As previously discussed, thapsigargin has been useful to understand the mechanism of Ca2+ entry through SOCE; from different studies it seems that Stim1 and Orai1 do not act alone but they are also coupled to TRPC family. (Antigny et al., 2010)

Experiments have shown that after TG-mediated Ca2+ store depletion Stim1 and Orai1 account only for 50% of Ca2+ entry (fig.1 Antigny et al., 2010), while using siRNA against PKC and PLC abolished Ca2+ entry for 85%.(fig.2 Antigny et al., 2010)

These findings strongly suggest that PKC and PLC do not target Orai1 and Stim1, but more likely TRPC3, thereby highlighting an association of TRPC3 with ROCE.

Fig. 1. Effect of Stim1 and Orai knock down on TG-induced Ca2+ entry (B)Cells were transiently transfected with control siRNA (scramble, siControl) ,or siRNA against Stim1 or Orai1. Each trace represents the Ca2+ entry phase following store depletion, and is the mean of are presentative coverslip.(C)Quantification of the effects of siStim1, siOrai1 and siStim1/Orai1 on the initial slope of ratio increases, after Ca2+ re-addition. Bars are mean ± SEM. The number of cells is written on the bar graph.

Fig. 2. Effect of Stim1 and TRPC3 knock down on TG-induced Ca2+ entry. (A) EA.hy926cells were loaded with 2 M Fura-2 to monitor cytosolic Ca2+ changes. Traces are the mean of are presentative coverslip showing the Ca2+ entry phase. Cells were transiently transfected with control siRNA, siRNA against Stim1,or siRNA against Stim1 together with siRNA against TRPC3; siStim1 was transfected 24h before siTRPC3( 72h and 48h after transfection for Stim1 and TRPC3, respectively). (B)The bars represent the slope of the ratio increase after Ca2+re-addition in siControl, siStim1 and siStim1/siTRPC3 silenced cells. Bars are mean ± SEM (n ranges from 46 to 190 cells).

However, in Salmon & Ahluwalia (2011) study, these theories are not confirmed. They performed experiments using Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (Pyr3), which is a specific TRPC channel inhibitor, to show if TRPC3 might have been considered a valid player in receptor operated calcium entry.




Fig. 3.  Effect of Pyr3 on intracellular [Ca2+] in Fura-2AM loaded human neutrophils. About 1 mM EGTA with or without 10 μM Pyr3 was added at time zero and cells were stimulated with 1 nM FMLP (A), LTB4 (B) or PAF (C). Following the Ca2+-release transient, 2 mM CaCl2 was added and the influx followed by measuring fluorescence at 340 and 380 nm. (â-) Shows responses in control cells and (â-‹) indicates responses in Pyr3 treated cells. Each point and bar shows the mean ± SEM (n = 3), but is typical of two other experiments.

Pyr3 appeared to cause Ca2+ release on its own, but had no effect on Ca2+ release in response to agonists. (Fig. 3A, B and C. Salmon & Ahluwalia 2011)

In addition, Pyr3 did not significantly affect Ca2+ influx following agonist stimulation. (Fig. 3A, B and C. Salmon & Ahluwalia 2011)

Fig. 4.  Effect of Pyr3 on intracellular [Ca2+] in thapsigargin stimulated Fura-2AM loaded human neutrophils. About 1 mM EGTA with or without 10 μM Pyr3 was added at time zero and cells were stimulated with 1 μM thapsigargin followed by 2 mM CaCl2 and the influx followed by measuring fluorescence at 340 and 380 nm. (â-) Shows responses in control cells and (â-‹) indicates responses in Pyr3 treated cells. Each point and bar shows the mean ± SEM (n = 3), but is typical of two other experiments.

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Thapsigargin induced Ca2+ influx was far greater in comparison to agonist stimulated neutrophils and Pyr3 nearly abolished Ca2+ influx in neutrophils stimulated with thapsigargin. (Fig. 4 Salmon & Ahluwalia 2011)

The specificity of Pyr3 towards TRPC3 channels makes these findings suggest that this protein is associated with SOCE in human neutrophils.

Recently, several studies have been done on discrete plasma membrane domains known as lipid rafts. These lipid rafts have shown to be resistant to mild detergents, rich in cholesterol and sphingolipids, and to organize the assembly of signalling molecules. (Prieschl et al., 2000)

Different studies have reported that proteins involved in Ca2+ entry are localized in these lipid rafts, which have been suggested to support store-operated Ca2+ entry by facilitating Stim1 clustering in endoplasmic reticulum-plasma membrane junctions as well as the interaction of Stim1 with TRPC1. (Galan et al., 2010)

In Alicia et al. (2008) study, it is explored the possibility that Stim1 may function as a molecular switch for TRPC1. They showed that TRPC1 can form both ROC and SOC in the same cell line, and that Stim1 association to TRPC1 converts the channel from a ROC to a SOC. Furthermore, they showed that Stim1 association to TRPC1 at the ER favours its insertion into lipid rafts, at the plasma membrane. Is at these specialized plasma membrane locations where TRPC1 functions as a SOC in a Stim1-dependent fashion.

Moreover, lots of studies are focused on discovering and testing new compounds that might be useful in better understanding the mechanisms of Ca2+ entry. (Itagaki et al., 2002)

In Salmon & Ahluwalia (2011) work, besides Pyr3, several other compounds such as Gd3+, 2-APB, Sr2+, ML-9 have been used to discriminate between SOCE and ROCE.

In Facemire et al. (2004) study, it has been proposed that norephinefrine primarily activates ROCs rather than SOCs in renal cortical interlobular arteries (ILAs) of rats and that this receptor-operated Ca2+ entry mechanism is regulated by calmodulin.


In summary, in this dissertation it is given an overview about thapsigargin and its usefulness in understanding the process of Ca2+ signalling in cells.

As it has been mentioned, over the last 20 years several progresses have been done.

Thapsigargin, as well as other compounds, have provided us useful means to analyse the mechanisms of store operated calcium entry, and recently, also of receptor operated calcium entry.

However, recent results are still in some way controversial, which means that there is still a long way to go to give a clear explanation of this process of Ca2+ signalling.

As mentioned above, TRPC channels have provided further targets for research in Ca2+ influx, although the controversial results do not allow their fully utility. To finish off, TRPC channels seem to be the best candidate to finally resolve this intricate puzzle, but it is likely that in the forthcoming years new molecular players will be discovered, thereby opening new insights in the mechanisms that underlie the store operated and receptor operated calcium entry.