Cellular Domains That Contribute to Ca2+ Entry Events

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Science's STKE  27 Jul 2004:
Vol. 2004, Issue 243, pp. pe32
DOI: 10.1126/stke.2432004pe32


Stimulation of cell surface receptors that increase phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis leads to intracellular Ca2+ release and activation of plasma membrane Ca2+ entry channels. Ca2+ entry via these channels regulates a wide array of physiological functions. The molecular composition of these channels and the mechanisms that activate or inactivate them have not yet been elucidated. Members of the TRPC subfamily of the TRP (transient receptor potential) family of proteins have been recently suggested as molecular components of these channels. In addition, Ca2+ signaling proteins and the signals they generate are compartmentalized and spatiotemporally regulated. Thus, the mechanisms involved in the assembly and trafficking of Ca2+ signaling proteins, including TRPC channels, will determine the regulation of Ca2+ entry and its effect on cellular function.

Agonist-stimulated PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis leads to activation of two major types of Ca2+ entry pathways. Capacitative or store-operated Ca2+ entry (SOCE) is suggested to be regulated by the depletion of Ca2+ from the internal Ca2+ store per se (13), not by PIP2 hydrolysis. Understanding the mechanism(s) involved in this process continues to be a major challenge. Three major models have been proposed to explain the endoplasmic reticulum (ER) to plasma membrane (PM) signaling that is involved in the activation of SOCE. In these models, (i) a PM channel senses ER status through interaction with the inositol 1,4,5-trisphosphate receptor (IP3R); (ii) a diffusible factor is released during, or generated in response to, depletion of internal Ca2+ stores and relays the signal; and (iii) a signal triggers regulated recruitment of channels or movement of ER to the PM. To add to this complexity, there is now convincing evidence that distinct store-operated Ca2+ influx channels (SOCs) are activated in different cell types. Thus, it is important to consider whether the same signal activates different channels (that is, they have a common sensor) or whether depletion of internal Ca2+ stores (either by agonist or thapsigargin, an agent that releases Ca2+ from intracellular stores) induces multiple intracellular signals that act on different channels.

Second messenger–operated channels (SMOCs) are also activated by the same initial signals that trigger SOCs (4). Studies with the TRPC subfamily of the TRP (transient receptor potential) family of proteins, which have been suggested as components of agonist-activated Ca2+ channels (5, 6), have shown that some agonist-stimulated Ca2+ entry channels (for example, TRPC3 and TRPC6) are probably activated by direct local action of diacylglycerol (DAG) or by PIP2 hydrolysis, but not by store depletion. In addition, other ion channels, such as the ARC (arachidonate-regulated, Ca2+-selective) channel, are activated by the same agonist, but via distinct intracellular signals (7). This leads us to the intriguing problem of how cells coordinate the spatiotemporal constraints that are involved in the generation of these discreet intracellular signals, activation of specific Ca2+ channels, and decoding of the respective Ca2+ signals that are generated. A possible way to achieve compartmentalization of intracellular signals is by assembly of proteins in specific microdomains. Thus, understanding the microenvironment of agonist-stimulated Ca2+ channels will be critical for resolving their physiological function and regulation.

Regulation of Ca2+ Entry and Microdomains

Regardless of the ongoing debates about the identity and mechanism of agonist-stimulated Ca2+ entry channels, there is general consensus that Ca2+ signaling, including Ca2+ entry, occurs in specific spatially restricted microdomains (8). Biochemical and morphological data demonstrate that Ca2+ signaling proteins are assembled in multiprotein complexes that are localized in distinct regions of the cells and that Ca2+ signaling events occur in these spatially segregated domains (Fig. 1). In polarized epithelial cells, initiation of internal Ca2+ release occurs in the apical region, consistent with the presence of a high concentration of Ca2+ signaling proteins [including phospholipase C (PLC), heterotrimeric guanine nucleotide-binding protein (G protein) α subunits Gαq and Gα11, and IP3Rs] in this region of the cell (9, 10). Furthermore, plasma membrane calcium ATPases (PMCAs), mitochondria, and sarcoplasmic reticulum or endoplasmic reticulum Ca2+-ATPases (SERCAs) have been functionally localized to the microdomain where SOCE occurs. These components contribute to fast removal of Ca2+ from this region and prevent Ca2+-dependent feedback inhibition of Ca2+ entry (11, 12). Thus, we proposed that Ca2+ entry channels may form a complex with these Ca2+ signaling proteins. The Drosophila Trp channel provides a well-characterized prototype of such a signaling complex (5, 6). We have demonstrated that mammalian TRPC channels are also assembled in a complex with key Ca2+ signaling proteins (1316). The architecture of such Ca2+ signaling microdomains may facilitate direct physical, or functional, coupling between molecular components that are involved in the activation or inactivation, or both, of Ca2+ entry channels (17, 18). Further, proteins that decode the Ca2+ signal to enable regulation of cellular function are also likely to be associated with this complex, although currently we know very little about these downstream events.

Fig. 1.

Ca2+ entry mechanisms and Ca2+ signaling microdomains. A Ca2+ signaling complex assembled in plasma membrane lipid raft domains (LRD, orange region in PM, green) is illustrated. Ca2+ signaling proteins [receptors, G protein, PLC, as well as SOC (TRPC) channels] are localized in this region and assembled in a complex (scaffolding proteins, not shown, contribute to the assembly of this complex). PM proteins such as TRPCs physically and functionally interact with ER proteins (such as SERCA and IP3R). Proposed IP3R-SOC (red oval) interaction is shown by the solid blue arrow (dashed blue arrow shows potential interaction between IP3R and SMOC, purple oval). Additional scaffolding proteins (not shown) might regulate these interactions. The IP3R interacting with SOC (light blue cylinder) is distinct from IP3Rs that mediate Ca2+ release (dark blue cylinder). Receptor stimulation (brown arrow) decreases ER Ca2+ concentration (by IP3-induced Ca2+ release), which is detected and conveyed to the PM SOC channel by currently unknown mechanisms (dashed red line). SMOC channels are also activated by the same initial stimulus. The activation could involve recruitment by regulated exocytosis or local action of metabolites such as DAG. Other SMOCs (e.g., ARC) could be activated by the same agonist, but via different intracellular signals (for example, arachidonate). SMOCs can contribute to the amplitude and duration of the local Ca2+ signals.

In characterizing these plasma membrane microdomains, it is important to consider contributions of both protein and lipid components. The available data indicate that PDZ domain–containing proteins [for example, HOMER and NHERF (Na+/H+-exchanger regulatory factor)] as well as others [RACK1 (receptor for activated C kinase 1)] might not only act as scaffolds to regulate assembly of the complex, but also affect protein-protein interactions, and thus channel function. However, the type of cell as well as the subcellular localization of the signaling complexes will likely determine what type of scaffold is present. For example, distinct scaffolds are present in the basal and apical membrane domains of polarized epithelia. Furthermore, the pathways involved in trafficking proteins to these two regions are also different. Several accessory proteins that function in scaffolding, trafficking, or regulation (exocyst proteins, kinases, and phosphatases, for example) have been identified that interact directly or indirectly with IP3Rs, PMCAs, SERCAs, PLCs, and TRPCs. However, further studies are required to determine exactly how these proteins affect Ca2+ entry.

Plasma membrane lipids also have a critical impact on Ca2+ signaling. For example, PIP2, apart from being a source for DAG, can also directly regulate PMCA as well as Ca2+ channel function and trafficking. Furthermore, functionally and biochemically distinct lipid domains in the plasma membrane (lipid raft domains, LRDs) or caveolar LRDs (LRDs that contain caveolin-1) may provide a platform for the assembly of signaling complexes and determine the specificity and rate of interaction(s) between proteins. Key protein and nonprotein molecules associated with Ca2+ signaling—such as PIP2, Gαq and Gα11, muscarinic receptors, PMCAs, IP3Rs, and TRPCs—as well as Ca2+ signaling events (such as receptor-mediated turnover of PIP2) have been localized to plasma membrane caveolar microdomains. Several Ca2+ influx–regulated processes such as generation of cAMP (adenosine 3′,5′-monophosphate) and activation of endothelial nitric oxide synthase (eNOS) have also been localized to caveolar LRDs. Fatty-acylated proteins can also associate with noncaveolar LRDs. LRDs are dynamic and appear to move within the membrane as well as fuse with each other. However, plasma membrane proteins that are localized in distinct cellular regions, such as Ca2+ channels, might be more stably anchored. The key question, of course, is how LRDs affect protein and channel function. Do LRDs facilitate protein movement? Clearly, some proteins do move in LRDs. Alternatively, LRD movement could change the lipid milieu of an immobile channel (for example, by increasing the local concentration of PIP2) or bring LRD-associated proteins [for example, glycosylphosphatidylinositol (GPI)–anchored proteins] into the vicinity of the channel.

Intact lipid rafts appear to be required for SOCE, and caveolin-1 interacts with several Ca2+ signaling proteins, such as G proteins and TRPCs. Although it is not yet known exactly how caveolae regulate SOCE, caveolar LRDs might facilitate and coordinate the signals that lead to activation of SOCE. The invaginated morphology of caveolae might enable the PM to come into contact with organelles and proteins located further inside the cells, such as the ER (analogous to a t-tubule). Additionally, because caveolae are found as vesicles beneath the plasma membrane, it is possible that they might act as holding platforms for preassembled Ca2+ signaling complexes or key regulators of Ca2+ entry, which could be recruited to the PM through vesicle fusion in stimulated cells. Indeed, proteins involved in docking and membrane fusion are enriched in caveolae. Such regulation of SOCE would be consistent with the secretion-coupling model according to which channel activation involves recruitment from intracellular compartments and is mediated by a vesicle fusion event. Further, because caveolin-1 acts as a tonic inhibitor of a number of signaling proteins, it could suppress the channel activity in resting cells.

Trafficking of Ca2+ Entry Channels

Ca2+ entry into cells can be increased by increasing the number of channels in the PM. This can be achieved by (i) recruitment of channels (by increasing exocytosis or trafficking of channels to the PM), or (ii) decreasing internalization of channels already present in the membrane (by decreasing endocytosis per se or increasing channel interaction with scaffolding molecules that promote retention in the PM). Thus, understanding agonist regulation of Ca2+ entry channels will depend on determining the mechanisms involved in their trafficking and assembly. TRPC1 interaction with caveolin-1 determines its surface localization (16). However, we do not know whether caveolin-1 regulates TRPC1 trafficking to the PM or its retention in the signaling complex. Studies using recombinant fluorescent proteins have demonstrated the presence of IP3R in mobile ER compartments (19). Similarly, several TRPs, including agonist-sensitive TRPC1, TRPC6, and TRPC3, are recruited to the PM in stimulated cells (2023). These data provide a very different, and more dynamic, perspective of the Ca2+ signaling process that might necessitate rethinking some of the earlier views. We believe that local changes in the cytoskeleton or microtubules might also contribute to the trafficking of these channels to their cellular locations. Determining the proteins that regulate vesicle trafficking and channel recruitment will be key to understanding their regulation. At a physiological level, increasing or decreasing the number of channels at strategic locations in the cell provides a way to modulate local Ca2+ signals and to recruit downstream signaling proteins such as kinases and PLCγ.

In conclusion, distinct Ca2+ entry pathways can be activated by the same external stimuli. Decoding these Ca2+ entry signals for regulation of distinct cellular function is coordinated through compartmentalization of the function and localization of the proteins that mediate and regulate Ca2+ entry. Understanding the molecular mechanism of agonist-stimulated Ca2+ entry will ultimately depend on identifying the Ca2+ channel(s) as well as their regulatory proteins and determining how they are trafficked and assembled.

Editor’s Note: This Perspective is part of a series related to an E-Conference held at Science’s STKE. Invited participants were asked to provide Perspectives that summarized the ideas, conclusions, areas of controversy, and challenges for future work that emerged in the online discussion at;14.


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