Cell Rapid Transient Influx And Depletion Of Calcium Biology Essay

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Many critical processes in the cell require rapid transient influx of calcium and then quick depletion. This influx of calcium is bought about by inter-cell signalling, which causes calcium to be released from the endoplasmic reticulum. Then there is communication of the endoplasmic reticulum with calcium channels in the outer membrane which open up and allow influx of calcium to replenish the intracellular stores. This process is known as capacitative calcium entry or store operated calcium entry (SOCE). There are three different opinions in the field of SOCE as to what are the key molecular components involved in the inter cell signalling. One possible route of SOCE is the Stim/Orai mechanism, which has been researched extensively and in my opinion is the most likely method. On the other side of the field transient receptor potential (TRP) channels are thought to be the key components involved in Ca2+ entry into the cells and finally a number of scientists seem to think that calcium influx factor (CIF) is the component of the cell that is allowing SOCE to occur.


Calcium, the fifth most abundant element in the earths crust, is an important ion in the human body and its passage from outside the cell into the cytoplasm is essential in many signalling processes e.g. muscle contraction, neurotransmitter release. The amount of calcium in the cell is tightly regulated and if it goes above or below the critical value it can lead to severe dysfunction in the cell and ultimately lead to apoptosis of the cell[12][14].

In 1986 James Putney Jr proposed a model for receptor regulated calcium entry [1], which he named capacitative calcium entry. The foundation of this model is described below.

G-protein-coupled receptors are present on plasma membranes and are used to sense molecules outside the cell which bring about changes in signal transduction pathways inside the cell. One type of G-protein-coupled receptors (Gαq/11) can be activated by certain ligands and results in activation of phospholipase C, which cleaves phosphatidyinositol 4, 5-biphosphate (PIP2) into diacylglycerol (DAG) and inositol 1, 4, 5-triphosphate (IP3). IP3 diffuses across the membrane into the cytosol where it binds IP3 receptors on the endoplasmic reticulum. Activation of IP3 receptors results in release of Ca2+ from the intracellular stores in the ER into the cytosol, the Ca2+ is then used up in various processes around the cell. Upon depletion of the intracellular stores of calcium in the endoplasmic reticulum there is communication of the ER with components on the plasma membrane resulting in opening of certain channels and influx of calcium. This process by which the intracellular calcium stores are replenished is known as capacitative calcium entry [1] or store operated calcium entry (SOCE). This mechanism of calcium entry only occurs in non-excitable cells, excitable cells have voltage gated Ca2+ pump which are used in movement of Ca2+ across membranes.

The membrane channel involved in the influx of calcium is known as a CRAC channel (calcium-release-activated-calcium). Hoth and Penner[7] were the first to characterise this channel and were able to determine the electrophysiological current involved in capacitative calcium entry which they termed ICRAC. They found this channel to have some properties similar to previously known Ca2+ selective voltage activated channels and some properties different. The main difference between ICRAC and other known currents was the conductance of ICRAC, which was too small to be measured directly, so it was determined indirectly by noise analysis. Due to there being a difference in properties of ICRAC's and other Ca2+ selective channels it is widely accepted that the molecular structure of the CRAC channels is also different to that of other known Ca2+ selective channels. Another difference between ICRAC and other Ca2+ selective channels was that ICRAC showed unusual regulation compared to other Ca2+ selective channels. These results helped scientists gain a better understanding of the molecular nature of CRAC channels[7].

1989 was a big year for SOCE, after the discovery of the effect of the tumour promoter Thapsigargin on parotid acinar cells and the role they play in SOCE the theory of capacitative calcium entry became a concept[19]. Thapsigargin was able to inhibit SERCA and release intracellular Ca2+ by a mechanism that does not involve any upstream components in the pathway (G-proteins, phospholipase C, IP3 etc). This provided an excellent model to test the capacitative calcium entry hypothesis and was also used in assays that led to the discovery of Stim and Orai.

Stim (stromal interaction proteins) were first discovered by Roos et al [16] using RNAI screens for thapsigargin activated Ca2+ entry in Drosophila S2 cells. Liou et al [8] also discovered Stim cells in mammalian HeLa cells in the same year. In mammalian cells there are known to be two types of Stim's; Stim1 and Stim 2, whereas in Drosophila S2 cells there is only one type of Stim present; Stim1. Stim 1 and 2 are both single pass transmembrane proteins, Stim1 is located on both the ER and the plasma membrane whereas Stim 2 is only located on the ER. They both consist of an EF hand motif in their N-termini which extend towards the lumen of the ER. Roos et al showed that knock out experiments involving Stim1 but not Stim2 show a significant decrease in Ca2+ entry implying that Stim1's play an essential role in SOCE. Wedel et al found no effect on SOCE of knocking down Stim2, however Soboloff et al showed that Stim2 may act as an inhibitor of SOCE. At this moment in time there is still a lot of work to be done on determining the true function of Stim2.

Stim1 has numerous significant functions in the process of SOCE. One of them is by acting as a sensor, sensing depletion of calcium in the ER (29). Liou et al has demonstrated that mutating the EF hand region of Stim1 results in continuous activation of SOCE. This demonstrates that the EF hand of Stim1 which extends into the lumen of the ER is used as a sensor, to detect low levels of Ca2+. It then activates other components in the cell which leads to opening of channels in the plasma membrane resulting in influx of calcium into the cell. Stim1 has also been found to be a microtubule plus end tracking protein (30). It associates with the microtubule end binding protein EB1 via its proline rich region. This interaction allows Stim1's to travel around different parts of the ER membrane; upon depletion of Ca2+ Stim1's undergo rapid rearrangement. This occurs by disassociation of Stim1's from EB1 and then translocation towards the plasma membrane where Stim1's redistributes into punctate structures. This reorganisation has indicated to scientists that there is some sort of interaction at the plasma membrane involving Stim1, Orai proteins and TRP's.

Orai proteins are part of the pore forming subunits of the Ca2+ channels in the plasma membrane. They were first discovered by Feske et al [5] using gene mapping and RNAi screening in a family with an immune deficiency due to a mutation in the Orai proteins (5). Orai proteins are present on the plasma membrane of cells and consist of four transmembrane domains. These proteins are also known to redistribute into punctate structure after activation of SOCE. Orai proteins in the inactive form are present as dimers. Upon activation of SOCE, Orai proteins reorganise into active tetramers(31).

Two major observations led to the conclusion that Orai proteins are essential members of the SOCE process[10]. Firstly when Orai1 was co-expressed with Stim, there was over expression of Ca2+ entry by 10-100 folds as opposed to little increase in activity when Stim1 or Orai are over expressed independently. The second observation was the complete loss of function of SOCE by a single amino acid mutation of a glutamate to alanine at position 106 in the Orai protein. After the results from these two experiments were published there was strong evidence showing that Orai is part of the pore forming subunit of the CRAC channel.

Most recent evidence presented by Liao et al shows Orai to function as a regulatory subunit of the store operated channel. It can not function as a calcium channel due to it not resembling any of the previously identified channels.

The final question to answer for this mechanism is; how do Stim1's convey messages to Orai channels. Many papers have attempted to discover this process but have failed to find any strong evidence. Luik et al (2006) showed that communication between Stim1 and Orai occur over very short distances, this compliments previous observations of Stim1 rearranging in the ER and migrating closer to Orai channel in the plasma membrane. The question is still unanswered but the most logical way for these two components to communicate would be through a direct mechanism; however there is still possibility that there may be a second messenger involved.

A possible identity of store operated channels is thought to involve transient receptor potentials (TRP). TRP's are a family of about 20 ion channels that are made up of 6 transmembrane domains which assemble into distinct structures to allow for calcium influx. The most studied TRP's are those involved in responses to pain stimuli. TRP's are thought to act as cationic channels in the refilling of depleted calcium stores. TRP's were initially studied in Drosophila, and early experiments on their function as being the calcium channel proved to be very successful. An observation by Harde and Minke (1992) showed that TRP, a photoreceptor calcium channel was activated downstream of phospholipase C showing that it may play a role in capacitative calcium entry. When human TRP's were cloned, early experimental findings were encouraging (Zhu et al 1996, Zitt et al 1996). However other laboratories failed to reproduce the early findings and the idea of TRP's acting as store operated channels became highly unlikely. Some laboratories still pursued the idea of TRP's acting as a SOC channel, however they have not been able to come up with strong evidence which has passed the intense scrutiny of other researchers in the field, for example Mori et al (2002) found that after eliminating TRPC expression, store operated entry was significantly reduced, it is also clear that under certain conditions TRPC channels can exhibit store operated activity. Many reports have been published claiming that TRPC1 is a SOC channel which contains a calcium-permeable channel that opens up when there are low levels of calcium in the ER. However with every report published claiming that TRPC channels are involved in SOCE(32,33) there are also contradictory reports published claiming that TRPC channels are not involved in store operated calcium entry but are involved in receptor operated calcium entry(34). Recently TRPC1 has been shown to be present on the membrane as part of the SOCIC (35).

These findings show that there may be a link between TRP channels and SOCE but they do not give definite conclusions.

The main problem with accepting TRPC channels as store operated channels is that they do not display properties similar to those of CRAC channels e.g. TRP's show slight Ca2+ selectivity whereas ICRAC are completely Ca2+ selective.

There is still a lot of work being done on the possible role of TRP's as store operated calcium channels. This area is proving to be the one of most controversial area in calcium entry and hopefully in the near future we will have a better understanding of the exact role of TRP's.

Recent studies show that the process of allowing calcium influx into the cell requires all components involved in the process to aggregate into a macromolecular complex known as the store operated calcium influx complex (SOCIC) (27). The aggregation of key components of store operated entry such as Stim, Orai, TRPC1, SERCA and the microtubule end tracking protein EB1 is seen to occur in specific areas of the plasma membrane called lipid rafts (28). This discovery showed that lipids are also required to modulate the process of SOCE as well as proteins.

The store operated calcium influx complex has two conformations, the active form and the inactive form. At this moment not much is known of the conformations but a model has been described which fits in all the components.

In the inactive form of SOCIC there can be two possible configurations, firstly the TRPC1-Orai complex may be assembled but in an inactive part of the membrane i.e. outside the lipid raft (Figure? A Left). The second possible configuration also occurs outside of the lipid rafts but in this form the TRPC1 and Orai proteins are not assembled (Figure? A right).

Upon depletion of the ER's calcium store, Stim1's dissociate from EB1 proteins and translocate to the plasma membrane to form punctuate structures. This activates SERCA, a microsomal calcium ATPase which associates to Stim1 forming an outer ring (37). Calmodulin is also present; it has recently been shown to modulate the activity of both Orai proteins and the TRPC1 channel (36).


Fig? A possible model of the store operated calcium influx complex (SOCIC).

This diagram shows the two possible conformations of the inactive form of the complex. On the left side the Orai and TRPC complex are present outside the lipid raft. On the right side, Orai and TRPC are disassembled and again are outside the lipid raft.

This diagram shows the possible active forms of the SOCIC complex. On the left Orai/TRPC1 complex are associated to Stim1 and form the calcium channel.

In the middle only Orai is associated to Stim1 and it forms the calcium channel without TRPC1.

On the right TRPC1 that is not associated to any Orai is able to act as a receptor operated channel while associated with the IP3R.

In all cases above in the active form Calmodulin is used to modulate the activity of SOCE and ROCE.

Lipid Rafts are

Another area of controversy in calcium entry is activation mechanisms. Many activation mechanisms have been proposed, but only two of these have survived the test of time whereas other ideas that may have once seemed to be correct have been disregarded due to not enough scientific backing. The two major mechanisms that have been studied for the past 15 years are:

A diffusible messenger

Conformational coupling

Putney and colleagues proposed that when intracellular calcium levels are low a messenger of some sort is released into the cytoplasm and diffuses to the plasma membrane where it activates the entry of calcium into the cell via store operated calcium channels. Irvine and Berridge proposed a principally different mechanism in which they said that IP3 receptors on the ER would extend out and form a bridge like structure with the channels in the plasma membrane. The bridge would allow direct communication between the empty calcium stores and the channels in the plasma membrane allowing calcium entry into the cell.

Both these mechanisms have been studied rigorously over the past 15 years and in light of recent evidence it seems that the diffusible messenger theory is more likely to be the activation mechanism.

Randriamampita and Tsien (1993) were the first to discover this diffusible messenger which they named CIF (Calcium Influx Factor). CIF is released by intracellular stores on depletion of calcium; it then diffuses to the plasma membrane and activates the SOC channels to allow calcium to enter the cell. This was a revolutionary and exciting discovery which laid down the foundations for other researchers who exploited this idea and managed to solve the mystery of how SOCE is activated. It is now believed that CIF displaces calmodulin from a membrane bound Ca2+-independent phospholipase A2 (iPLA2) leading to activation of iPLA2. Activated iPLA2 generates lysophospholipids which activate SOC channels in the membrane allowing influx of calcium.

In recent years there has been many exciting discoveries leading to the determination of the store operated calcium entry pathway, however there are still many unknowns in this puzzle like the structure of the key molecular players; SOC channels and CIF.

Observing the pharmacological effects of certain agents on the store operated calcium entry pathway can provide a meaningful insight into the molecular mechanism of how it works.

One of the most extensively studied pharmacological agents is 2-Aminoethyldiphenyl Borate. 2-APB has been shown to inhibit SOCE by preventing the re-organisation of Stim1 into discrete punctuate structures.