Conformational Coupling: A Physiological Calcium Entry Mechanism

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Science's STKE  27 Jul 2004:
Vol. 2004, Issue 243, pp. pe33
DOI: 10.1126/stke.2432004pe33


The entry of external Ca2+ that is activated by inositol 1,4,5-trisphosphate (IP3)may occur through a conformation coupling mechanism. IP3 receptors in the endoplasmic reticulum located in a junctional zone make contact with entry channels in the plasma membrane. IP3 may act directly to stimulate this coupling complex or IP3 could act indirectly by stimulating uncoupled IP3Rs in the vicinity of the junctional zone to induce a localized depletion of the ER store to switch on a store-operated mechanism. At physiological agonist concentrations, the earliest Ca2+ response to receptor activation may be the stimulation of entry, which is then responsible for charging up the internal store to prime the IP3Rs for the large-scale regenerative release of Ca2+ that occurs during each spike.

The way in which Ca2+ entry is controlled by receptor activation remains a mystery. A number of receptors with intrinsic channel properties, such as the N-methyl-d-aspartate (NMDA) receptor or the nicotinic receptor, gate Ca2+ upon binding the appropriate ligand. However, there are many other examples where receptors act indirectly to promote Ca2+ influx by activating a Ca2+ entry signal. The nature of these entry signals is the subject of considerable debate. Most attention has focused on those receptors that act through phospholipase C (PLC) to generate both diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Both of these second messengers are putative Ca2+ entry signals. Release of Ca2+ from the endoplasmic reticulum (ER) by IP3 can lead to store depletion, and it was proposed that this might activate a process of capacitative Ca2+ entry (CCE) (1).

The conformational coupling hypothesis (2, 3) was originally put forward to explain this process of capacitative Ca2+ entry (CCE) (1). Later, the original hypothesis was extended to include new information to indicate that this mechanism may also be activated by IP3 (4). Here, I delineate the main features of this coupling hypothesis before attempting to describe how it might function to control entry at the low agonist concentrations that drive physiologically meaningful Ca2+ signals.

The Conformational Coupling Hypothesis

The basic idea, as originally proposed by Robin Irvine, is that stimulation of the IP3 receptor (IP3R) by store emptying induces a conformation change that is transferred to the channel in the plasma membrane through a direct protein-protein interaction (3). The nature of the store-operated channel (SOC) has yet to be characterized, and current contenders are the channel mediating ICRAC (Ca2+ release-activated Ca2+ current) and some of the Trp (transient receptor potential) channels. The interaction between the IP3R and the entry channel is thought to occur in junctional zones where portions of the ER containing the coupled IP3Rs lie close to the plasma membrane (Fig. 1A). Just how this coupling complex is activated is a critical issue. An increase in IP3 may activate conformational coupling in two ways. As illustrated in Fig. 2, the formation of IP3 at low physiological agonist concentrations may be highly localized in the vicinity of activated receptors. If this punctate domain of IP3 overlaps with a junctional zone, conformational coupling could be activated through two mechanisms (4). (i) IP3 may act directly to stimulate the IP3Rs that are coupled to the entry channels (Fig. 1B). It will be argued later that the direct activation by IP3 may be particularly important at low agonist concentrations to induce entry before store emptying occurs. (ii) IP3 could act indirectly by stimulating uncoupled IP3Rs in the vicinity of the junctional zone to induce a localized depletion of the ER store to switch on a store-operated mechanism (Fig. 1C). The latter case could explain how a highly localized depletion in the junctional zone could control entry while the bulk of the Ca2+ is kept within the main body of the ER, where it is available for the generation of Ca2+ spikes (Fig. 1C).

Fig. 1.

Conformational coupling hypothesis. (A) In the resting state, the coupling between the IP3R and the SOCs occurs in a junctional zone where a finger of the tubular ER network makes close contact with the plasma membrane. SERCA (sarcoplasmic or endoplasmic reticulum Ca2+-ATPase) pumps on the ER ensure that the store is kept full of Ca2+. (B) Agonist activation of receptors induces a high concentration of IP3 immediately below the membrane, where it activates the coupled IP3Rs to induce a conformational change that is transmitted to the SOCs to activate channel opening. It is proposed that these coupled IP3Rs are nonconducting, so there is no release of stored Ca2+ when they are operating in this mode. (C) If there are uncoupled IP3Rs near the junctional zone, IP3 will release Ca2+, and this local emptying of the store will activate a conformational change in the IP3R that then activates entry through the SOCs. For both (B) and (C), the Ca2+ that enters the cell can either be pumped back out by the PMCA (plasma membrane calmodulin-dependent Ca2+-ATPase) pumps or it can be taken up into the ER by the SERCA pumps to increase the luminal concentration of Ca2+ that tunnels through the ER (yellow arrows) to sensitize the uncoupled IP3Rs. IP3 diffusing in from the plasma membrane will also sensitize these uncoupled IP3Rs.

Fig. 2.

Functional organization of the putative conformational coupling mechanism. The tubular ER network (shown in red) has periodic connections to the plasma membrane to form junctional zones. At low agonist (yellow dots) concentrations, when relatively few of the receptors (green) are occupied, IP3 formation is likely to be punctate (yellow domains). If these local domains occur in the vicinity of a junctional zone, they may activate Ca2+ entry either through a direct action on the coupled IP3Rs (region 1, see Fig. 1B) or by a local depletion of Ca2+ from the ER (region 2, see Fig. 1C). The localized generation of IP3 results in the autonomous activation of individual junctional zones. If the local IP3 domain does not impinge on the junctional zone (region 3), it may cause a local depletion of ER Ca2+ without stimulating entry.

Another prediction of the original model was that, when the IP3R is coupled to the entry channel, it is nonconducting and thus not able to release Ca2+ (4). A recent study has supported this idea that the IP3R can undergo a conformational change, leading to the opening of an entry channel without itself conducting Ca2+. DT40 cells that lack IP3Rs are unable to induce Ca2+ entry or release after agonist stimulation. However, Ca2+ entry was restored following expression of a wild-type IP3R or a mutated IP3R that had a C-terminal truncation preventing it from releasing Ca2+ (5). In the latter case, the mutated IP3R was able to respond to IP3 to induce the conformational change necessary to promote an entry of Ca2+ even though it was unable to release Ca2+ from the store. It may be important for the coupled IP3Rs to be nonconducting; otherwise, they would release large amounts of Ca2+ into the junctional zone that would inhibit the SOCs, which are inactivated by Ca2+.

Although both mechanisms can activate Ca2+ entry and may operate together, it is possible that their primary functions are different: The direct IP3 activation pathway (Fig. 1B) may be dedicated to coupling receptor activation to Ca2+ entry, whereas the store-operated pathway (Fig. 1C) may be a homeostatic mechanism that ensures that the internal stores remain filled. Having such a dual activation mechanism may also explain how entry can be activated by mechanisms that deplete stores independently of IP3, as occurs after activation of ryanodine receptors.

Organization of the ER and Junctional Zones

The organization and distribution of the junctional zones described earlier (Fig. 1) may help to explain how the conformational coupling mechanism might operate to regulate entry at such low agonist concentrations (Fig. 2). Small fingers of the tubular ER network form flattened sacks that make close contact with the plasma membrane to form the specialized junctional zones where the conformational coupling units are located. The 10-nm gap separating the ER from the plasma membrane has periodic densities that are thought to be the large cytoplasmic heads of the IP3R, which communicate information to the entry channels. These junctional zones appear to be few in number, which may explain why the rate of entry at physiological concentrations is often so low.

An important issue arises as to whether these junctional zones are coupled to the bulk of the ER (as illustrated in Fig. 2) or whether there is a separate store associated with the plasma membrane that is dedicated to controlling entry. Compelling evidence for a continuous network has come from studies on endothelial cells where receptor-dependent depletion of ER Ca2+ in one location of the cell was able to induce entry at a site 100 μm away, even though the second site failed to display any IP3-induced Ca2+ release (6). The local activation at one site was able to drain the bulk of the ER sufficiently to activate a store-operated entry mechanism much further afield. This experiment supports the homeostatic function of store-operated entry, which is to ensure that the store remains filled irrespective of how it has been emptied. I would argue that a small store might fill up too quickly, and entry would cease, even when the bulk of the ER was still empty and required further Ca2+.

Operation of rhe Conformational Coupling Mechanism at Physiological Agonist Concentrations

The essential requirement for any Ca2+ entry signal is that it should operate in a dose-dependent manner at physiological agonist concentrations. Because many cells have spare receptors, a maximal cellular response is often obtained with minimal receptor occupancy when the rate of second messenger production is very low. This is clearly apparent in the insect salivary gland, where the EC50 (median effective concentration) value for 5-HT(5-hydroxytryptamine)–induced fluid secretion is 3 × 10−9 M, whereas the transepithelial flux of Ca2+ (an indirect measure of Ca2+ entry) has a lower value (1.5 × 10−8 M), and this was lower still for inositol efflux (an indirect monitor of IP3 formation)(5 × 10−7 M) (7). The curves for Ca2+ entry and IP3 formation were displaced to the right of the physiological response such that a 50% activation of secretion was seen at a 15% activation of entry and a 2.5% activation of IP3 formation. The point of this example is to stress that a very small activation of IP3 formation is sufficient to induce the small activation of Ca2+ entry necessary to fully activate a downstream physiological response. A similar conclusion emerges if one considers the agonist concentrations responsible for setting up Ca2+ oscillations.

Ca2+ Entry Drives Ca2+ Oscillations

For some cell types, the low agonist concentrations that activate physiological responses often induce Ca2+ oscillations. It has been proposed that it is the entry of external Ca2+ that functions both to maintain such oscillations and to change their frequency by recharging the internal store following each spike (8). It is relevant to ask, therefore, what the rate of Ca2+ entry is and how much of the store is released during the course of a spike. When the ER luminal level of Ca2+ concentration was monitored in a pancreatic cell spiking regularly in response to a low dose of acetylcholine, it declined by about 5% during each spike (9). This small loss was then gradually replenished during the next interspike interval. In many cells, this refilling of the partially depleted store occurs during the interspike interval, even though the cytosolic concentration of Ca2+ is close to its resting level. This implies that the rate of Ca2+ entry is very low and is rapidly taken up by the ER to set the stage for the next spike. Indeed, I consider that it is the rate of Ca2+ entry that is the timing mechanism for the frequency-modulated (FM) signaling mechanism seen in many cell types. This consideration of oscillations further enhances the view that the rate of Ca2+ entry that operates during normal physiological responses is very low and is activated by occupation of a limited number of the available cell surface receptors.

Function of IP3 in Integrating the Processes of Ca2+ Entry and Release

Another important issue concerns the way IP3 functions to initiate a Ca2+ transient (that is, a transient increase in the concentration of free intracellular Ca2+). IP3 is likely to have two roles. (i) It can act locally at the membrane to promote entry through a conformational coupling mechanism activated through either one or both of the mechanisms described earlier (Fig. 1, B and C). The Ca2+ that enters the cell is then taken up by the bulk of the ER, and this increases the luminal concentration of Ca2+ to sensitize the IP3Rs (10, 11). (ii) Subsequent diffusion of IP3 into the cell will raise the ambient level of IP3, which will also heighten the sensitivity of the IP3Rs distributed throughout the ER. The combination of luminal loading of the ER and a small increase in the cytosolic concentration of IP3 will raise the sensitivity of the uncoupled IP3Rs sufficiently for them to participate in the regenerative release of Ca2+.

Whatever the entry mechanism turns out to be, the critical point is that entry is responsible for bringing Ca2+ into the cell before the onset of the Ca2+ spike either at the beginning or during an oscillation. In the case of the insect salivary gland, I obtained some indirect evidence that 5-HT was capable of stimulating entry during the latent period before the onset of the first spike (12). When the stores were depleted, the latency was greatly prolonged, presumably because the entry mechanism required a longer period to refill the store. At low agonist concentrations, therefore, the earliest Ca2+ response to receptor activation may be the stimulation of entry, which is then responsible for charging up the internal store to prime the I3Rs for the large-scale regenerative release of Ca2+ that occurs during each spike.

Is it possible that the long-held assumption that entry occurs after release might in fact represent the opposite of what actually occurs? Perhaps the earliest Ca2+ response at physiological agonist concentrations is an IP3-induced increase in entry that then sets the stage for Ca2+ release.

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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