PerspectiveSignal Transduction

The Calcium Entry Pas de Deux

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Science  03 Mar 2000:
Vol. 287, Issue 5458, pp. 1604-1605
DOI: 10.1126/science.287.5458.1604

Calcium ions (Ca2+) are universal secondary messengers that are key players in many cellular signal transduction pathways (1). There are two sources of these signaling cations in the cell: internal stores that release Ca2+, and channels in the plasma membrane that open to allow external Ca2+ to flow into the cell. Internal Ca2+ stores—located in the sarcoplasmic reticulum of muscle cells and the endoplasmic reticulum (ER) of other cells—have a limited capacity for Ca2+ storage, and so they have to be replenished through entry of Ca2+ from the external environment. Putney (2) first recognized that the processes of emptying and replenishing internal Ca2+ stores must be linked. Somehow, empty Ca2+ stores activate store-operated channels (SOCs) in the plasma membrane that then allow Ca2+ ions to enter the cell. Putney termed this mechanism “capacitative calcium entry” (CCE) because the stores behave like a capacitor in an electrical circuit. When Ca2+ stores are replete the SOCs are closed, but once the stores discharge their contents, the SOCs open and Ca2+ ions enter the cell.

Since the first observations of CCE, there has been intense debate about the identity of SOCs and the way in which the ER Ca2+ stores communicate with them. Evidence is emerging in support of the popular conformational-coupling hypothesis (3, 4), which proposes that information is transferred through a direct interaction between the inositol 1,4,5-trisphosphate receptor (InsP3R) in the ER and SOCs in the plasma membrane (see the figure). On page 1647 of this issue, Ma et al. (5) now present evidence showing that CCE depends on the close proximity of the ER and plasma membranes and that InsP3Rs partner with SOCs to control Ca2+ entry through the plasma membrane.

A happy coupling.

The conformational-coupling mechanism for the regulation of Ca2+ entry into cells. (A) The endoplasmic reticulum (ER) contains the inositol trisphosphate receptor (InsP3R) (red), which interacts with store-operated channels (SOCs) in the plasma membrane (blue). (B) After addition of the phosphatase inhibitor calyculin A, a dense cytoskeletal matrix forms just below the plasma membrane, which displaces the ER and disrupts the molecular interaction between SOCs and InsP3Rs. (C) Emptying of internal Ca2+ stores in the ER results in a conformational change (CC) in those InsP3Rs that are coupled to SOCs. In the case of endogenous SOCs (possibly TRPs 1, 2, 4, and 5), this CC is sufficient to activate opening of SOCs and Ca2+ entry from outside the cell. Store emptying, together with InsP3 activation and diacylglycerol (DAG) production, may be necessary for activation of TRPs 3, 6, and 7.


The InsP3R, embedded in the ER membrane, is a good candidate for this molecular coupling job. Its amino-terminal domain is large enough to span the 10-nm gap that separates the ER and the plasma membrane. Meanwhile, its carboxyl-terminal region forms a channel in the ER membrane, out through which flow Ca2+ ions in response to the signaling molecule inositol trisphosphate (InsP3). It is this InsP3-induced release of Ca2+ that normally depletes internal stores of the cation and results in activation of CCE.

Detection of light by photoreceptor cells in the compound eye of Drosophila activates a Ca2+-entry channel known as TRP (transient receptor potential) in the photoreceptor cell membrane. Much excitement has surrounded the realization that mammalian cells express homologs of TRP and there has been speculation that one or more of these TRP homologs may be the elusive SOC. To date, seven TRP isoforms have been cloned that vary markedly in their channel conductance and mode of activation.

The human TRP3 channel (hTRP3) has been extensively characterized. It is clear that this channel is not activated solely by depletion of Ca2+ stores but rather also requires a direct physical connection to InsP3R (5, 6). Excised membrane patches containing hTRP3 channels can be activated by addition of vesicles containing the InsP3R bound to its ligand (6). Activation of hTRP3 can be blocked by rearranging the actin cytoskeleton; this displaces the ER membrane from sites of close apposition with the plasma membrane (6, 7) (see the figure). More significant still is the finding that hTRP3 and InsP3R form a signaling complex (they can be coimmunoprecipitated from membranes of human embryonic kidney cells overexpressing hTRP3) (8). Furthermore, high-affinity binding assays have been used to map the sites of interaction between the carboxyl-terminus of hTRP3 and the amino-terminus of InsP3R (8). Finally, engineering cells to express peptides that encode the interacting domains of either hTRP3 or InsP3R modulates the extent of Ca2+ entry (8, 9).

These studies clearly indicate that hTRP3 is activated by a conformational-coupling mechanism. But what about the identity of SOCs in mammalian cells? Perhaps the most exciting recent development has been the observation that experimental maneuvers that inhibit hTRP3 activity also prevent activation of endogenous SOCs. In their study, Ma et al. (5) show that displacement of actin in human embryonic kidney cells inhibits activation of both hTRP3 and SOCs (although, interestingly, the SOC response was not completely attenuated). In addition, both hTRP3 and SOC activity can be blocked with pharmacological agents—such as 2-aminoethoxydiphenyl borate (2-APB) and xestospongin C—that inhibit InsP3R activation (see the figure).

This new evidence thus implicates the InsP3R in the coupling of both hTRP3 and endogenous SOCs to Ca2+ entry after depletion of internal Ca2+ stores. The InsP3R is a central player in CCE by virtue of its ability to sense both cytosolic InsP3 and empty Ca2+ stores. Although the mechanism of CCE is becoming clearer, we still do not know the identity of endogenous SOCs. In the strictest sense, TRP3 is not a SOC because it is not activated by Ca2+-store depletion alone. The functional homology between TRP3, TRP6, and TRP7 suggests that they are activated in a similar manner, thus TRP6 and TRP7 are also unlikely to be SOCs. Furthermore, these TRP isoforms can be activated by diacylglycerol in an apparently store-independent manner (5, 7, 10). TRPs 1, 2, 4, and 5 are presently the best candidates for endogenous SOCs because, when expressed in cells, they are activated solely by Ca2+ store depletion.

Although cells may use subtly different conformational-coupling systems to activate various TRP isoforms, it seems that we are closing in on both the mechanisms and molecular players involved in CCE. But there are plenty of problems left to solve. For example, the expression pattern of mammalian TRPs is puzzling. TRPs are ubiquitously expressed yet are most abundant in excitable tissues, such as muscle and nerve, where CCE is least apparent. Another unresolved issue is the nature of the channels formed by TRPs when heterologously expressed (that is, when expressed in cells that normally have very low levels of the protein). The flow of Ca2+ ions through SOCs has been defined electrophysiologically as a Ca2+ release-activated current (ICRAC) (11), which preferentially selects Ca2+ over monovalent ions. By contrast, cells expressing TRPs have Ca2+ channels that are not nearly as selective. Of all the isoforms, TRP4 comes the closest to resembling ICRAC. On the positive side, however, antisense oligonucleotides directed against TRPs can prevent Ca2+ entry, supporting the notion that these proteins form endogenous CCE channels. It is plausible that the heterologous expression systems simply do not yet faithfully mimic the endogenous conformational-coupling complexes, and that other accessory proteins are required. Clearly, there is still much to learn about the enigmatic mechanism that determines how and when Ca2+ enters the cell.


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