Perspective

Toward a Consensus on the Operation of Receptor-Induced Calcium Entry Signals

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Science's STKE  27 Jul 2004:
Vol. 2004, Issue 243, pp. pe39
DOI: 10.1126/stke.2432004pe39

Abstract

Receptor-induced Ca2+ signals involve both Ca2+ release from intracellular stores and extracellular Ca2+ entry across the plasma membrane. The channels mediating Ca2+ entry and the mechanisms controlling their function remain largely a mystery. Here we critically assess current views on the Ca2+ entry process and consider certain modifications to the widely held hypothesis that Ca2+ store emptying is the fundamental trigger for receptor-induced Ca2+ entry channels. Under physiological conditions, receptor-induced store depletion may be quite limited. A number of distinct channel activities appear to mediate receptor-induced Ca2+ entry, and their activation is observed to occur through quite diverse coupling processes.

Receptor-induced Ca2+ signals control a vast array of cellular processes in virtually all cells. Ca2+ signals in response to receptors for hormones, growth factors, or neurotransmitters are effected through a combination of Ca2+ release from endoplamic reticulum (ER) stores and extracellular Ca2+ entry across the plasma membrane. The channels that mediate release from stores [inositol 1,4,5-trisphosphate (IP3) receptors and ryanodine receptors] are well characterized and their activation is generally understood. The channels mediating Ca2+ entry are not known, and the mechanisms controlling their function remain largely a mystery (1). Understanding the nature and function of these Ca2+ entry channels is crucial, yet there is a remarkable diversity of approach, information, and opinions on their operation. The recent Science’s STKE "E-Conference: Defining Calcium Entry Signals" allowed these issues to be discussed with the broader scientific community and offered an opportunity to reach some consensus on the approaches, leading to a more definitive understanding of the Ca2+ entry process (2). This Perspective contains a critical assessment and attempts to draw together the proceedings of the E-Conference and the views presented in a related set of STKE Perspectives contributed by the invited participants of the conference (310).

The concept of a "store-operated" Ca2+ entry mechanism has been a central paradigm on which understanding of the process of Ca2+ entry after receptor activation is based. The original concept of Putney (11) was that depletion of stores per se is sufficient to trigger Ca2+ entry. The observation that store emptying with a simple SERCA (sarcoplasmic reticulum or endoplasmic reticulum Ca2+ adenosine triphosphatases) pump blocker (for example, thapsigargin) could trigger Ca2+ entry appeared to rule out the necessity for a role of receptors or subsequent phospholipase C (PLC) activation in triggering the entry of Ca2+ (12). Although this original observation remains unchallenged, current thinking deviates in a number of important ways from this simplistic model. The E-Conference was originally centered on three distinct parameters defining the operation of receptor-induced Ca2+ entry: (i) the mechanisms of coupling to activate entry channels, (ii) the cellular domains wherein such coupling operates, and (iii) the nature of the channels that actually mediate entry. Clearly, most participants perceived these parameters as being intimately related and sought to cross the rather arbitrary demarcations to bring together a number of formerly unrelated aspects. Many questions remain unanswered, but this cross-fertilization of ideas has resulted in remarkable consensus on certain key parameters. Furthermore, objective insights into the approaches emerged that may provide more useful information in the future.

The Physiology of Store Depletion

A recurrent theme throughout the E-Conference proceedings was reflection on the "physiological reality" of store emptying. Most participants agreed that whereas it is possible to artificially "empty" stores in cells with pump blockers or ionophores, the activation of receptors under physiological conditions results in only a rather modest depletion of Ca2+ from stores through release channels. Quoting the earlier studies of Petersen (13), Berridge (4) reminded us that measurements of intraluminal Ca2+ in pancreatic acinar cells undergoing repetitive oscillations in response to physiological agonist activation results in only a very small reduction in the concentration of free Ca2+ within the ER lumen. Putney (9) was not alone in pointing out that in smaller nucleated cells such as lymphocytes, the limited volume of the ER may result in more complete emptying and a more robust and sustained entry of Ca2+. This may be a special case, and it appears that the entry of Ca2+ through store-activated calcium release–activated calcium (CRAC) channels plays a major role in the physiological Ca2+ signals in lymphocytes (14). Penner (8) suggested that the relation between ICRAC (Ca2+ release–activated Ca2+ current) activation and IP3 production indicates that only a fraction of stores (perhaps too small a fraction to be measured) may be coupled to entry activation. Berridge (4) pondered at length the question of whether the ER undergoes transient local depletion in the vicinity of the plasma membrane in response to local IP3 production, but thinks that ER is probably one continuous luminal entity. Indeed, "store emptying" may be a misnomer, at least in larger cells. Physiologically, the high micromolar concentrations of free Ca2+ in the bulk of the ER may not deviate very significantly even during prolonged physiological Ca2+ signal generation. Clearly, there is a need to examine luminal Ca2+ changes with greater spatial and temporal precision and to be able to observe changes in hitherto "invisible" stores that may be subjacent to the plasma membrane. With the development of new generations of ER-directed Ca2+ probes, as well as those to accurately measure cytosolic Ca2+ in the vicinity of the plasma membrane, such studies may become feasible.

Physiological Activation of Ca2+ Entry Channels

The conference participants debated whether the only defined store-operated channel activity (ICRAC) (14) is activated by physiological agonist concentrations (that is, under conditions of more limited Ca2+ release from the stores), an issue that becomes crucial. The CRAC channel carries only a very small current, and it is difficult to measure the operation of this channel in response to more modest agonist conditions. Shuttleworth (10) pointed out that one receptor-induced channel activity that is reported to be activated by physiological agonist levels is the arachidonic acid–regulated Ca2+-selective (ARC) channel (15), which is present in various cell types. Although activation of this channel requires neither store emptying nor the lipase activity of PLC, the ARC channel is an interesting candidate for mediating receptor-induced Ca2+ entry. However, this activity has largely been studied by only one group of collaborators, so further understanding of its function, control, and impact on Ca2+ entry will benefit greatly from comparative studies in other labs. We hope that the E-Conference discussions will spur a greater focus on the ARC channel activity and assess its relevance and universality in receptor-mediated Ca2+ entry signals.

The Role of Ca2+ Entry

An important question is the purpose of receptor-induced Ca2+ entry: whether it is to generate Ca2+ signals, to accomplish cellular Ca2+ homeostasis, or a combination of both functions. Considering that physiological Ca2+ signals are likely to involve regenerative Ca2+ spikes (transient increases in the intracellular concentration of free Ca2+ [Ca2+]i), the frequency of which is modulated by receptor occupancy (16), there was agreement that even small amounts of Ca2+ entry into cells appear to be crucial for both maintaining the occurrence of spikes and for modulating the frequency of spikes. This means that receptor- or store-operated channels have a crucial but subtle role in allowing physiological Ca2+ signals to operate. Except in the case of small cells such as lymphocytes, the concept that long-term sustained Ca2+ entry occurs through the store-operated mode seems to be losing favor as we consider that in many larger cells, such depletion may not occur. However, there seems to be little disagreement that the entry channels mediate replenishment and maintenance of luminal Ca2+, which is crucial to cell survival. Loss of Ca2+ from stores through repeated signaling would necessarily be deleterious to cells because protein translation, translocation, and folding all appear to depend on high intraluminal Ca2+ concentrations (17). Thus, Ca2+ entry appears to have a dual role: (i) modulating Ca2+ release signals that contribute to oscillations in intracellular Ca2+, and (ii) maintaining continued ER function. These two processes are essentially inseparable. The highly Ca2+-selective currents such as CRAC and ARC provide promising candidates for the process of Ca2+ replenishment, because they economize on the otherwise wasteful charge-carrying activity of nonselective cation channels. However, this is certainly not to say that these are the only such channels activated in response to receptors or store depletion. ARC is a receptor-dependent channel activity, but it is likely that additional channels are involved in physiological receptor-induced Ca2+ signals. Moreover, there was much agreement within the E-Conference that nonselective channels activated under conditions of receptor activation or store emptying could have a quite different role in cellular signaling.

The Nature of Coupling

Although there has been debate in the field on whether coupling to activate Ca2+ entry channels is through direct conformational coupling between the ER and plasma membranes or involves a chemical messenger, discussion in the E-Conference was relatively subdued and deemphasized the mutual exclusivity of these two models. Most now accept the view that there may be multiple activation mechanisms, probably involving both coupling paradigms. Bolotina (5) emphasized findings from her lab on the role of products from iPLA2 (Ca2+-independent phospholipase A2), which may be chemical mediators of Ca2+ entry (18) . However, even she agrees that distinct subgroups of Ca2+ entry channels, perhaps in the same cell, may be activated conformationally by coupling with activated IP3 receptors (IP3R’s).

The IP3R has been a major focus within the conformational coupling model, and Berridge (4) provided new perceptions on how entry channels and IP3R’s may be coordinated. Originally, Irvine (19) and Berridge (20) predicted that IP3R’s in the ER that took part in direct conformational coupling to plasma membrane entry channels might be nonconducting. Store-operated channels (SOCs) appear to be exquisitely sensitive to increased concentrations of cytosolic Ca2+ (14), and thus their operation in the immediate vicinity of Ca2+ release appeared paradoxical. However, recent data from IP3R knockout cells indicate that nonconducting IP3R mutants can indeed mediate coupling to promote Ca2+ entry in response to activated receptors (21). However, because thapsigargin-induced Ca2+ entry or CRAC channel activation both appear to be independent of IP3R’s (22, 23), this suggests a significant functional dichotomy between receptor-operated as opposed to purely store-operated Ca2+ entry. In an assessment of functional coupling to activate entry, Berridge (4) presents compelling arguments that Ca2+ entry during receptor-induced spike events should precede Ca2+ release from stores, militating against the long-held principle that depletion of stores triggers the coupling process to activate entry. Although theories abound, in reality the coupling process and the proteins involved in this mechanism remain perhaps the weakest area in our understanding of Ca2+ entry. There is a clear need to broaden the search for the entities involved (both channels and possible coupling moieties) and to more objectively apply approaches such as expression cloning, microarray technology, the use of small interfering RNA libraries for reverse expression cloning, and mass spectrometry–based proteomic analysis to screen for and identify binding partners involved in the coupling process. Thus, the way forward likely depends on expanding the repertoire of molecular search parameters.

Coupling Domains

It has been widely held that the coupling process, be it conformational or chemical, takes place within spatially restricted domains at the plasma membrane. Ambudkar (3) supports the view that Ca2+ entry channels [in particular, transient receptor protein C (TRPC) channels] exist in a multimolecular complex with other functional proteins [including heterotrimeric guanine nucleotide–binding proteins (G proteins), PLC, and Ca2+ pumps] and adaptor or scaffold proteins. Analogies are drawn with the better-characterized organization of TRP channels in the Drosophila retina (24). However, the highly specialized and organized phototransduction system in flies appears to function independently of Ca2+ stores or Ca2+-release channels (25), and we should be wary of drawing such close analogies. Ambudkar (3) also notes recent observations on the trafficking of entry channels and plausible evidence that this process may modulate Ca2+ entry (26). Berridge’s hypothesis on coupling (4) rests heavily on the existence of coupling domains wherein portions of the ER containing IP3R’s may be juxtaposed with the plasma membrane. There have been few recent EM studies characterizing the occurrence of ER–plasma membrane densities, and there is little direct information to place the IP3R in such locations. Such entities would seem to be sparse in most cells. Therefore, coupling domains where sensing of ER Ca2+ content is transmitted to the plasma membrane or where entering Ca2+ might be directed efficiently into the ER may be very few in cells. Much study in the Ca2+ signaling field uses imaging at the light level, which is largely inappropriate for resolving the putative coupling domains described here. Hence, it is probable that useful characterization of these entities will only come from reapplying EM analyses and trying to characterize the dimensions and constituents of such domains with high resolution.

The Entry Channels Themselves

Detailed considerations by Nilius (7), Penner (8), and Shuttleworth (10) indicate that members of the now quite diverse TRP family of channels do not yet fulfill the criteria of being either authentic CRAC or ARC channels. Their closest characteristics to those expected of CRAC channels may be those of the Ca2+-selective TRPV5 and TRPV6 channels (27). Nevertheless, it is clear that there are a number of distinctions between the properties of these channels and those of CRAC channels (28). Whether exogenously expressed TRP channels operate in a store-dependent mode is controversial, with some studies claiming that a number of TRPC, TRPM, and TRPV channels are activated when stores are emptied, whereas many other studies suggest that they are not [reviewed in (1)]. Considering the above criticisms about the physiological relevance of emptying stores artificially, we should remember that overexpressing channels and then subjecting them to such a severe stress conditions is likely to result in spurious observations. Nilius (7) provides a critical assessment of whether, using knockout or knockdown approaches, there has been a definitive identification of the function of natively expressed TRP channels. In some cases, Ca2+ signaling has been affected by such deletion approaches, but curiously, the lost currents do not correspond to those attributed to the overexpression of the corresponding channel. The properties of such channels may be influenced not only by the variety of their subunit constitution, but also by the presence of any one of many proteins suggested to be in tight proximity with the channels. When overexpressed, particularly by transient overexpression, which yields expression levels up to 50 times higher than those of endogenous expression, it is very likely that channels are not assembled and organized as they are in native cells.

Gudermann (6), Bolotina (5), Nilius (7), and Putney (9), all raise awareness of the importance of other classes of receptor-activated or store-dependent channels that, unlike CRAC, are not selective for Ca2+ ions. This is an increasingly voiced perspective, and it is likely that nonselective cation channels play crucial roles in mediating depolarization responses and hence in contributing to the modification of a number of other voltage-dependent channels or electrogenic transporters (for example, K+ channels, Cl channels, or the Na-Ca exchanger). Indeed, Penner (8) and Nilius (7) consider the possibility that with conductance in the subpicosecond range, CRAC currents may represent the function of one of many transporters rather than of channels per se. Which brings us finally to one of the more contentious issues raised during the conference: the relative merits of Ca2+ imaging techniques as opposed to electrophysiological measurements applied to studying the function of receptor-induced Ca2+ entry signals. Clearly, electrophysiological recordings have great functional utility for measuring the biophysical properties of channel activity. On the other hand, Ca2+ imaging is a less invasive and extremely sensitive method for measuring Ca2+ entry, capable of sensing perhaps a few hundred Ca2+ ions entering the cell. Given the small changes in Ca2+ and the low conductances of implicated channels, there is no question that the most meaningful results derive from a careful integration of both approaches.

Concluding Comments

The E-Conference has promoted a highly objective introspection on the mechanisms of receptor-induced Ca2+ signals involving the entry of Ca2+ and perhaps other ions across the cell membrane. The whole store-operated concept has been reframed as an operplasma membrane– and ER-derived signals. In many cells, stores are unlikely to really be emptied, but a signal probably derives from a modest diminution of luminal Ca2+ resulting in a change in the steady-state entry of Ca2+. It is also clear that physiological receptor activation can induce Ca2+ entry that does not require store depletion, even though it may be usual that some, albeit modest or restricted, release of Ca2+ from stores does accompany receptor activation. A number of different channel molecules are likely to be involved. Certain channels may be responsive to both the receptor- and store-induced signals, whereas the two channel activities so far defined, ARC and CRAC, appear to be more exclusive in their responses to these signals. Although we sometimes consider receptor- and store-derived signals as distinct, in reality they may be closely integrated. Moreover, the spectrum of responses, ranging from activation in response to low concentrations of agonists to global store depletion, may provide cells with the ability to generate a broad and dynamic range of Ca2+ signal responses. Interpreting this fascinating signaling problem requires us to address challenging aspects of physical interactions and functional coupling between proteins organized within at least two distinct membranes. It will be solved only by the application of broader screening approaches, integration of complementary physiological measurements, and dedication of creative and open minds.

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