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E-Conference: Defining Calcium Entry Signals

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Calcium signaling and microdomains

4 June 2004

Indu S. Ambudkar

Is store-operated calcium entry organized in functionally and structurally distinct microdomains?

Since other sections of this forum address more didactic issues relating to the actual definition and measurements of these channels, modes of regulation and their molecular composition, let's start this discussion by agreeing on the fact that calcium influx is activated in cells upon stimulation by agonists coupled to the stimulation of PIP2 hydrolysis. For now, I will set aside the question of whether this calcium influx is comprised of one type of channel or several, and examine the evidence that agonist-stimulated calcium influx occurs within specific spatially restricted microdomains.

Early studies using fluorescence measurements showed that Ca2+ entry continues to occur after removal of an agonist and this leads to store refill without any significant change in cytosolic calcium levels, other than the small overshoot observed upon re-addition of calcium to cells treated with agonists. This lead to the proposal that Ca2+ that enters the cell is rapidly taken up into the store.

Electrophysiological studies using Ca2+-activated Cl- and K+ channels as readouts also showed that there is minimal diffusion of Ca2+ in the plasma membrane region. Ca2+ entry during refill did not activate these channels, unless SERCA was inhibited. Thus, SERCA and Ca2+ entry channels can be suggested to be present in close proximity to each other, which also then suggests that the ER membrane has to be juxtaposed with the PM.

If the Ca2+ status within the ER regulates activation of this channel, then apposition of the two membranes not only determines the route for Ca2+ once it enters the cell, but could also regulate channel activation. Subsequently, it was shown that not only SERCA, but also PMCA and mitochondria affect calcium entry by regulating the calcium concentration in the vicinity of the channel. These organelles together act as a buffer to keep calcium concentration near the channel low and prevent Ca2+-dependent inactivation of the channel inactivation, a regulation that has been well documented. SERCA of course also enables refill of the internal calcium stores, i.e. its activity leads to channel inactivation.

Questions that can be raised for further discussion during this forum are:

(i) Does [Ca2+] in this region change during the refill process and whether this directly affects gating of calcium entry (distinct from the Ca2+-dependent inactivation, which is reversible)?

(ii) Whether this microdomain organization is universally found in all cell types or whether it is dictated by the architecture of the cell and the localization of the channel?

Compartmentalization of calcium signaling and calcium entry:

Biochemical and morphological data support the suggestion that Ca2+ signaling proteins are assembled in multiprotein complexes that are localized in distinct regions of the cells and that Ca2+ signaling events occur is spatially segregated domains of the cell. The best data available in this regard come from studies with polarized epithelial cells (see reports from the Petersen and Muallem labs). Exocrine gland acinar and ductal cells are polarized epithelial cells that also demonstrate a functional polarity. Thus, the cellular location of the Ca2+ signal has important physiological implications in the regulation of function.

A number of calcium signaling proteins have been shown to be localized in the apical region of these cells. Further, initial Ca2+ signals are generated in this region and then spread to the basal region. Importantly, at low concentrations of agonist, calcium spikes are observed in the apical region and do not spread to the basolateral region. These Ca2+ spikes are sufficient for functional regulation of ion channels that are present in the apical membrane. It was also recently reported by Muallem and co-workers that distinct RGS proteins regulate Ca2+ signaling events at different cellular locale. Thus, each Ca2+ signaling complex has a specific function within a cell. Its activity and location are strategically designed in order to facilitate and optimally enable the cellular function it regulates.

Our knowledge about localization of calcium influx is rather sparse. In nonpolarized cells, which most of us use for our studies, we do not take into consideration the locale of the channel. When working with a more physiological system, such as primary cultures or freshly dispersed cells, this is crucial for understanding function and regulation of calcium entry. I think such understanding ultimately depends on knowing

(i)the site of calcium influx,

(ii) the function carried out by the calcium entering the cell, and

(iii) the calcium signaling cascade it is associated with.

Studies reported with acinar cells present an interesting problem. Low concentrations of agonists that produce apical calcium spikes, have not thus far been associated with calcium entry. Higher agonist concentrations or thapsigargin that induce substantial depletion of Ca2+ stores appear to stimulate Ca2+ entry via the basolateral membrane. An interesting point to consider is whether in fact apical Ca2+ entry does occur in response to apical Ca2+ store depletion, and, if so, whether it is "store-operated." Alternatively, signals within the ER might transmit this apical Ca2+ depletion to the basal region for SOCE activation. Receptors that activate Ca2+ signaling have been detected in the apical region of cells. PIP2 hydrolysis resulting from activation of such receptors produces local signals, which, in addition to inducing internal release, could activate calcium channels in the same signaling domain. Such channels might not only provide calcium for refilling the local stores, but could also modify local Ca2+ signals and recruit other signaling proteins. Thus, agonist-activated Ca2+ channels might serve functions in cells other than refilling of internal Ca2+ stores. As we give some thought to these calcium channels in the coming weeks, it will be important to consider and discuss some of these other possibilities. Ultimate understanding of this will depend on the identification of the channels and defining their location in cells.

Physiological relevance of SOCE:

Since we are defining cellular signals for activation of calcium entry, I think it is important to consider the function and regulation of SOCE in the context of what we have discussed above. The first point that needs to be addressed is whether SOCE serves any other purpose other than to replenish internal Ca2+ stores. Although this will require further studies and our definition of this calcium influx pathway might change as we learn more, we should consider the conditions under which SOCE is activated. Typically, high levels of stimulus are required to deplete the stores. In acinar cells, local Ca2+ signals with low levels of agonists do not appear to activate it. This is also true for ICRAC, where relatively high agonist concentrations are needed for activation. Thus, it is quite relevant to ask whether SOCE in fact has any role under normal physiological conditions of cell stimulation or whether it is a "response" to more extreme conditions that induce substantial depletion of stores, which might put the cells in a "stress mode". This suggestion does not in any way detract from the concept of SOCE but might give us another perspective in understanding its mode of regulation.

Assembly and trafficking of Ca2+ signaling proteins:

It is becoming increasingly clear that Ca2+ signaling proteins are associated physically to form multiprotein complexes. Specific complexes exist that are coupled to different receptors. Functional distinctions will be dictated by the location of these proteins, the intensity of the signals they perceive, and functional cross-talk between the various signaling pathways. Both protein and lipid components are structurally involved in the assembly of these signaling complexes.

PDZ-domain containing and several other proteins, e.g. RACK1, have been shown to act as scaffolds for the assembly of receptor associated signaling complexes in the plasma membrane. These scaffolding proteins provide a framework that brings functionally related proteins in close proximity to each other.

In addition, lipid raft domains (LRD) in membranes might also have a role in assembly of these complexes. Caveolae are a specialized form of LRD that contain caveolin-1, a cholesterol binding protein. Key protein and non-protein molecules involved in the Ca2+ signaling cascade, such as phosphatidylinositol -(4,5)-bisphosphate (PIP2), Galphaq/11, muscarinic receptor, PMCA, and IP3R-like protein, and Ca2+ signaling events such as receptor-mediated turnover of PIP2 have been localized to caveolar microdomains in the plasma membrane. An interesting study showed that agonist-stimulated Ca2+ signal in endothelial cells originates in specific areas of the plasma membrane that are enriched in caveolin-1 (Issihiki et al., 1998). Furthermore, we and others have reported that intact lipid rafts are required for activation of SOCE (Lockwich et al 2000, Issihiki et al 2002, Kunzelmann-Marche et al 2002). Thus, caveolae might regulate the spatial organization of calcium signaling by contributing to the localization of Ca2+ signaling complex as well as the site of Ca2+ entry.

However, exactly how caveolae regulate SOCE is not yet known. Added to the problem is the observation that cells that lack caveolin appear to be functionally intact. However, possible compensatory mechanisms have not yet been assessed in this case.

Nevertheless, it is interesting to propose, that at least in some cases, caveolar LRD might facilitate and coordinate the signals that lead to activation of SOCE via two possible mechanims.

(1) Since Ca2+ signaling proteins that lead to the activation of SOCE are colocalized in the same microdomain, caveolae could mediate interactions between the SOC channels and proposed regulatory proteins such as IP3R, or PLC-gamma and PLC-beta. The invaginated morphology of caveolae would uniquely enable the plasma membrane in this region to have access to regulatory components located further inside the cells, such as the ER.

(2) Since caveolae are also found as subplasma membrane vesicles, it is interesting to speculate that they might act as holding platforms for pre-assembled Ca2+ signaling complexes, or key components of this complexes, which upon stimulation of the cell are recruited to the plasma membrane via vesicle fusion. It is important to note that proteins involved in docking and membrane fusion are enriched in caveolae (Issihiki and Anderson, 1999). Such regulation of SOCE would be consistent with the secretion-coupling model. Finally, caveolae could also function as regulators of SOCE inactivation. Caveolin-1 is known to act as a tonic inhibitor of a number of signaling proteins. Additionally, caveolae have been shown to undergo dynamic internalization and the internalized vesicles have been shown to fuse with the ER. Thus, during prolonged activation of calcium influx, channels could be down-regulated via internalization.

An interesting idea proposed by Andersen is that this process would allow the external Ca2+ to be trapped in the vesicles and delivered to the ER. Thus, recycling of plasma membrane calcium channels would both limit the number of "active" channels and provide a route for the refill of internal Ca2+ stores with external Ca2+.

Another possible mechanism for inactivation of SOCE would be exit from caveolae and internalization via clathrin-coated pits. Channels internalized this way would be routed to endosomes for degradation.

There is almost no information regarding trafficking and assembly of calcium entry channels. Since these mechanisms can determine their surface expression as well as regulation, it is important to examine this is in our future studies. One interesting observation in MDCK cells is that caveolar-lipid rafts are found basolaterally while non-caveolar LRDs are found apically. This can then form the basis for functional segregation of proteins in such cells.

What can we learn from TRPC channels?

In our quest for the identity of the store-operated calcium channels, many of us have focused our attention on TRPC channels. While it might be possible that none of the TRPCs might in fact be the SOC channel (further discussed in sections 1 and 2 of this forum), our studies have demonstrated that TRPCs are the only presently available candidate proteins for calcium influx channels activated by agonist-stimulation of calcium signaling.

1. Such studies have identified at least two possible types of channels that can be activated in response to agonist-stimulation of PIP2 hydrolysis. One set of TRPC channels appears to be independent of internal Ca2+ store depletion, but requires IP3 generation and likely involvement of IP3R. Another set of channels can be activated by conditions used to deplete intracellular Ca2+ stores.

2. Knock-down studies suggest that some TRPCs might be components for calcium entry stimulated in response to PIP2 hydrolysis or internal Ca2+ store depletion (TRPC1, TRPC4). Thus, TRPCs can be used to identify regulatory mechanisms that are initiated by Ca2+ signaling events.

3. TRPCs appear to form complexes with Ca2+ signaling proteins and be localized within the same microdomains as Ca2+ signaling and SOCE. Thus, determining the localization of TRPC channels, their assembly into protein complexes, and their trafficking in cells will provide us valuable information.

4. TRPC channels have been proposed to exist as heteromers, based on studies, most of them done with heterologous expression systems. This has been used to account for the distinct characteristics of SOCE in different cell types. However, this is yet to be supported by data. For example, are the TRPCs that form heteromers in fact co-localized in cells? More importantly, we need to examine this with endogenous proteins and see which TRPCs are spatially segregated, which Ca2+ signaling complexes they interact with, and whether Ca2+ entry mediated by them has any physiological role in the cell.

5. TRPC proteins can be used to fish out novel proteins; e.g. the/an as yet unknown calcium channel or key scaffolding and regulatory proteins.

I am sure we will continue to discuss these issues vociferously in the coming weeks and beyond.

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