We investigated the putative roles of phospholipase C, polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP 3 ) in capacitative calcium entry and calcium releaseactivated calcium current (I crac ) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of phospholipase C with U73122 blocked calcium entry and I crac activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and I crac . The membrane-permeant IP 3 receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and I crac . However, it is likely that 2-aminoethoxydiphenyl borane does not inhibit through an action on the IP 3 receptor because the drug was equally effective in wildtype DT40 B-cells and in DT40 B-cells whose genes for all three IP 3 receptors had been disrupted. Intracellular application of another potent IP 3 receptor antagonist, heparin, failed to inhibit activation of I crac . Finally, the inhibition of I crac activation by U73122 or wortmannin was not reversed or prevented by direct intracellular application of IP 3 . These findings indicate a requirement for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP 3 and IP 3 receptor in this mechanism, at least in these particular cell types.

Mammalian Trp proteins are candidates for plasma membrane calcium channels regulated by receptor activation or by intracellular calcium store depletion [capacitative calcium entry (CCE)]. One extensively investigated member of the Trp family, the human Trp3 (hTrp3), behaves as a receptor-activated, calcium-permeable, nonselective cation channel when expressed in cell lines and does not appear to be activated by store depletion. Nonetheless, there is good evidence that Trp3 can be regulated by interacting with inositol trisphosphate receptors (IP3Rs), reminiscent of the conformational coupling mode of CCE. To investigate the role of Trp3 in CCE, and its regulation by IP3R, we transiently expressed hTrp3 in the wild-type DT40 chicken B lymphocyte cell line and its variant lacking IP3R. Expression of hTrp3 in either wild-type or IP3R-knockout cells did not increase basal membrane permeability, but resulted in a substantially greater divalent cation entry after thapsigargin-induced store depletion. This hTrp3-dependent divalent cation entry was significantly greater in the wild type than in IP3R-knockout cells. Thus, it appears that in this cell line, hTrp3 forms channels that are store-operated by both IP3R-dependent and IP 3R-independent mechanisms. Trp3, or one of its structural relatives, is a candidate for the store-operated, nonselective cation channels observed in smooth muscle cells and other cell types.

. Canonical transient receptor potential TRPC7 can function as both receptor-and store-operated channel in HEK-293 cells.

We investigated the actions of the organoborane, 2-aminoethoxydiphenylborane (2APB), on Ca 2ϩ signaling in wild-type human embryonic kidney (HEK) 293 cells and in HEK293 cells stably expressing canonical transient receptor potential (TRPC) channels. Previous reports have suggested that 2APB inhibits agonist activation of TRPC channels because of its ability to act as a membrane-permeant inhibitor of inositol 1,4,5-trisphosphate (IP 3 ) receptors. 2APB was specifically said to inhibit TRPC3 channels when activated through a phospholipase Clinked receptor but not when activated more directly by a synthetic diacylglycerol, oleyl-acetyl-glycerol (OAG) [Science (Wash DC) 287:1647-1651, 2000]. However, we subsequently reported that IP 3 does not activate TRPC3; rather the mechanism of activation by phospholipase C-linked receptors seemed to result from diacylglycerol [J Biol Chem 278: 16244 -16252, 2003]. Thus, the current study was carried out to address the mechanism of action of 2APB in inhibiting TRPC channels. We found that, although the release of Ca 2ϩ by a muscarinic agonist was reduced by high concentrations of 2APB, this effect was indistinguishable from that seen when stores were discharged by thapsigargin, which does not involve IP 3 receptors. This indicates that 2APB is incapable of significant inhibition of IP 3 receptors when applied to intact cells. We found that 2APB partially inhibits divalent cation entry in cells expressing TRPC3, TRPC6, or TRPC7 and that this partial inhibition was observed whether the channels were activated by a muscarinic agonist or by OAG. Thus, as concluded for store-operated channels, 2APB seems to inhibit TRPC channels by a direct mechanism not involving IP 3 receptors.

Inositol 1,4,5-trisphosphate (InsP3) is involved in the mobilization of Ca2+ from intracellular non-mitochondrial stores. In rat liver, it has been shown that the InsP3-binding site co-purifies with the plasma membrane. This suggests that in the liver the InsP3 receptor (InsP3R) associates with plasma membrane. We studied the subcellular distribution of the liver InsP3R by measuring the maximal binding capacity of [3H]InsP3 and using antibodies against the 14 C-terminal residues of the type 1 InsP3R. The antibodies recognized a large amount of an InsP3R protein of 260 kDa in a membrane fraction which is also enriched with [3H]InsP3-binding sites and with markers of the basal, the lateral and the bile-canalicular membrane and the plasma-membrane Ca2+ pump (PMCA). The fractions enriched in markers of the endoplasmic reticulum (ER) and the Ca2+ pump of the ER (SERCA2b) contained low levels of InsP3 receptors. The immunofluorescent labelling of cultured hepatocytes with anti-InsP3R antibodies indicated that the receptor is concentrated in the perinuclear area and in some regions near the plasma membrane. The fraction enriched with InsP3R is also contaminated with markers of the ER and with SERCA2b. It was exposed to alkaline medium (pH 10.5) to extract endogenous actin and membrane-associated proteins before being subfractionated by Percoll-gradient centrifugation. The alkaline treatment allowed partial separation of the markers of the ER from the markers of the plasma membrane. The InsP3R was recovered in the heavy subfraction, which was also enriched with markers for the ER and with the SERCA2b and contained low levels of markers of the plasma membrane. These data indicate that the InsP3R is neither localized on the plasma membrane itself nor homogeneously distributed on the ER membrane. This supports the view that part of the receptor is localized on a specialized sub-region of the ER which interacts with the plasma membrane.

Mammalian homologues of the Drosophila transient receptor potential (TRP) protein have been proposed to function as ion channels, and in some cases as store-operated or capacitative calcium entry channels. However, for each of the mammalian TRP proteins, different laboratories have reported distinct modes of cellular regulation. In the present study we describe the cloning and functional expression of the human form of TRP4 (hTRP4), and compare its activity with another well studied protein, hTRP3. When hTRP4 was transiently expressed in human embryonic kidney (HEK)-293 cells, basal bivalent cation permeability (barium) was increased. Whole-cell patch-clamp studies of hTRP4 expressed in Chinese hamster ovary cells revealed a constitutively active non-selective cation current which probably underlies the increased bivalent cation entry. Barium entry into hTRP4-transfected HEK-293 cells was not further increased by phospholipase C (PLC)-linked receptor activation, by intracellular calcium store depletion with thapsigargin, or by a synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In contrast, transient expression of hTRP3 resulted in a bivalent cation influx that was markedly increased by PLC-linked receptor activation and by OAG, but not by thapsigargin. Despite the apparent differences in regulation of these two putative channel proteins, green fluorescent protein fusions of both molecules localized similarly to the plasma-membrane, notably in discrete punctate regions suggestive of specialized signalling complexes. Our findings indicate that while both hTRP4 and hTRP3 can apparently function as cation channels, their putative roles as components of capacitative calcium entry channels are not readily demonstrable by examining their behaviour when exogenously expressed in cells.

Capacitative Ca2+ entry involves the regulation of plasma membrane Ca2+ channels by the filling state of intracellular Ca2+ stores in the endoplasmic reticulum (ER). Several theories have been advanced regarding the mechanism by which the stores communicate with the plasma membrane. One such mechanism, supported by recent findings, is conformational coupling: inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptors in the ER may sense the fall in Ca2+ levels through Ca2+-binding sites on their lumenal domains, and convey this conformational information directly by physically interacting with Ca2+ channels in the plasma membrane. In support of this idea, in some cell types, store-operated channels in excised membrane patches appear to depend on the presence of both Ins(1,4,5)P3 and Ins(1,4,5)P3 receptors for activity; in addition, inhibitors of Ins(1,4,5)P3 production that either block phospholipase C or inhibit phosphatidylinositol 4-kinase can block capacitative Ca2+ entry. However, the electrophysiological current underlying capacitative Ca2+ entry is not blocked by an Ins(1,4,5)P3 receptor antagonist, and the blocking effects of a phospholipase C inhibitor are not reversed by the intracellular application of Ins(1,4,5)P3. Furthermore, cells whose Ins(1,4,5)P3 receptor genes have been disrupted can nevertheless maintain their capability to activate capacitative Ca2+ entry channels in response to store depletion. A tentative conclusion is that multiple mechanisms for signaling capacitative Ca2+ entry may exist, and involve conformational coupling in some cell types and perhaps a diffusible signal in others.

The D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor was localized by immunofluorescence experiments in situ in liver cryosections. Two anti-Ins(1,4,5)P3 receptor antibodies (against the 14 C-terminal residues of the type 1 receptor or against the entire cerebellar receptor) weakly decorated the whole cytoplasm, and a more intense labelling was observed at the periphery of the hepatocytes, particularly beneath the canalicular and the sinusoidal domains of the plasma membrane (PM). Antibodies against calreticulin, the Ca2+ pump (SERCA2b) or endoplasmic reticulum (ER) membranes homogeneously labelled the cytoplasm and the subplasmalemmal area. These data indicate that the ER can be divided into at least two specialized subregions: one is located throughout most of the cytoplasm and contains markers of the rough ER (RER), calreticulin, SERCA2b and a low density of Ins(1,4,5)P3 receptor, and the other is confined to the periphery of the cells and contains calreticulin, Ca2+ pump, RER markers and a high density of Ins(1,4,5)P3 receptor. A membrane fraction enriched in Ins(1,4,5)P3 receptor and in markers of the PM was immuno-adsorbed with the antibody against the C-terminal end of the Ins(1,4,5)P3 receptor and pelleted with Sepharose protein A. The immuno-isolated material was enriched in Ins(1,4,5)P3 receptor, but none of the markers of the ER or of the PM could be detected. This suggests that the Ins(1,4,5)P3 receptor is localized on discrete domains of the ER membrane beneath the canalicular and the sinusoidal membranes, where it was found at higher densities than the other markers.