TRP, inositol 1,4,5-trisphosphate receptors, and capacitative calcium entry.

C calcium serves as a ubiquitous signal for acute cellular activation and for regulation of important cellular processes such as cell growth, division, differentiation, and even cell death and apoptosis. Increases in cytoplasmic calcium or calcium signals can be generated either by release of calcium from intracellular stores or by influx of calcium across the plasma membrane, but commonly by both means. The release of intracellular calcium comes from the endoplasmic reticulum or its specialized counterpart in muscle, the sarcoplasmic reticulum, and is generally signaled by the formation or influx of small mediators such as inositol 1,4,5-trisphosphate (IP3) (1), cyclic ADP ribose (2), and even calcium itself (3). Mechanisms for controlling calcium influx are somewhat more varied (4). Calcium influx can be more directly controlled by the membrane potential (5) or by the binding of extracellular neurotransmitters directly to the channels (6). Calcium influx can be signaled by second messengers such as cyclic nucleotides (7); however, in the case of receptors coupled to the phospholipase C pathway, the classical messengers of this pathway, IP3 and diacylglycerol, do not appear to be the primary mediators of activated calcium entry. Rather, in most instances, the signal for entry is somehow derived from the IP3-mediated depletion of calcium from intracellular stores, a process called “capacitative calcium entry” or “storeoperated calcium entry” (8, 9). Two key issues regarding the mechanism of capacitative calcium entry have been (i) the nature of the signal from the endoplasmic reticulum that activates the channel, and (ii) the molecular identity of the channels themselves. It has been generally thought that the answer to the second question, the molecular identity of the channels, would likely lead to knowledge of the mechanism of signaling. Recent findings suggest that this may indeed be the case. In a report appearing in this issue of PNAS, Boulay et al. (10) provide compelling evidence for the involvement of a member of the TRP family of channel proteins in this signaling pathway, and for signaling activation of the TRP channels through interaction with IP3 receptors. Mammalian TRP proteins are homologues of the Drosophila photoreceptor mutants TRP and TRPL. There are at least seven mammalian genes, designated TRP1 through TRP7 (11, 12). Expression of these genes in various cell lines has resulted in varying patterns of response, including constitutive activity, augmentation of capacitative calcium entry, apparent direct activation by receptors, activation by IP3, and activation by diacylglycerol (13). Thus, despite considerable speculation regarding the function of these proteins in capacitative calcium entry, none of the available evidence directly implicates one or more of these gene products in this pathway in nontransfected cells. A number of mechanisms of activation of capacitative calcium entry have been proposed. The two major categories of mechanisms include those involving a diffusible messenger (14, 15) and a more direct communication with the endoplasmic reticulum involving interaction of the plasma membrane channels with closely underlying IP3 receptors (16, 17). The latter, conformational coupling model was originally proposed by Irvine (16) based on a suggested homology with the coupling that occurs in skeletal muscle between the ryanodine receptor calcium release channels in the sarcoplasmic reticulum and surface membrane (t-tubule) dihydropyridine receptor calcium channels. In support of the conformational coupling model, recent evidence indicates that the activation of capacitative calcium entry channels requires close physical interactions between endoplasmic reticulum and plasma membrane constituents (18, 19). There is also recent evidence that TRP3, when exogenously expressed in cells, can be activated by an IP3 receptordependent conformational coupling mechanism. Kiselyov et al. (20) demonstrated that depletion of intracellular Ca21 stores activated single channels in the plasma membrane of cells stably transfected with the gene for human TRP3. On excision, channel activity was lost but could be restored by IP3. With more extensive washing, restoration of channel activity required addition of both IP3 and the IP3 receptor. These results suggest that expressed TRP3 is gated by IP3-liganded IP3 receptors, consistent with the conformation coupling model. In a more recent study, Kiselyov et al. (21) demonstrated that TRP3 activation depended on a limited region of the IP3 receptor, spanning the IP3 binding domain. In the study by Boulay et al. (10), evidence was first obtained for direct protein–protein interaction of TRP3 and IP3 receptors. In cells stably expressing epitope-tagged TRP3 or its homolog TRP6, IP3 receptor could be detected in immunoprecipitates of TRP, and, reciprocally, TRPs could be detected in immunoprecipitates of the IP3 receptor. So next, Boulay et al. sought to determine the sites of interaction of these proteins and to determine the functional significance of these interactions. To accomplish this, they generated glutathione S-transferase fusions of peptides coded by cDNA fragments of the human type 3 IP3 receptor and human TRP3 and used a series of glutathione S-transferase pull-down experiments to map minimal interacting domains on the two proteins. A sequence in the C-terminal domain of TRP3 consisting of 54 amino acids (designated C7, from position 742 to position 795) was identified, as well as a shorter sequence of 21 amino acids (C8, 777 to 797), which strongly bound specific sequences within the IP3 receptor. A 289-aa sequence just downstream from the IP3 binding domain of the IP3 receptor, designated F2r (positions 638 through 926), strongly bound TRP3, and within this sequence, a number of smaller interacting sequences were identified [see Boulay et al. (10)]. Boulay et al. next transfected HEK293T cells with fluorescent protein fusions of three of these peptides and examined the effects of the potential TRP3 and IP3