In the phospholipase C signaling system, Ca2+ is mobilized from intracellular stores by an action of inositol 1,4,5‐trisphosphate. The depletion of intracellular calcium stores activates a calcium entry mechanism at the plasma membrane called capacitative calcium entry. The signal for activating the entry is unknown but likely involves either the generation or release, or both, from the endoplasmic reticulum of some diffusible signal. Recent research has focused on mammalian homologues of the Drosophila TRP protein as potential candidates for capacitative calcium entry channels.. This review summarizes current knowledge about the nature of capacitative calcium entry signals, as well as the potential role of mammalian TRP proteins as capacitative calcium entry channel molecules. BioEssays 21:38–46, 1999. Published 1999 John Wiley & Sons, Inc.
Canonical transient receptor potential 3 (TRPC3) is a receptor-activated, calcium permeant, non-selective cation channel. TRPC3 has been shown to interact physically with the N-terminal domain of the inositol 1,4,5-trisphosphate receptor, consistent with a "conformational coupling" mechanism for its activation. Here we show that low concentrations of agonists that fail to produce levels of inositol 1,4,5-trisphosphate sufficient to induce Ca 2؉ release from intracellular stores substantially activate TRPC3. By several experimental approaches, we demonstrate that neither inositol 1,4,5-trisphosphate nor G proteins are required for TRPC3 activation. However, diacylglycerols were sufficient to activate TRPC3 in a protein kinase C-independent manner. Surface receptor agonists and exogenously applied diacylglycerols were not additive in activating TRPC3. In addition, inhibition of metabolism of diacylglycerol slowed the reversal of receptor-dependent TRPC3 activation. We conclude that receptor-mediated activation of phospholipase C in intact cells activates TRPC3 via diacylglycerol production, independently of G proteins, protein kinase C, or inositol 1,4,5-trisphosphate.In non-excitable cells, depletion of internal Ca 2ϩ stores activates store-operated channels (SOCs) 1 mediating calcium entry across the plasma membrane, a process known as capacitative calcium entry (CCE) (1-3). Despite considerable attention, the molecular identity of SOCs and their gating mechanism(s) remain unknown. Two major hypotheses for the mechanism of activation of CCE have been proposed. The first involves the release of a soluble factor from the endoplasmic reticulum (4, 5). The second, the "conformational coupling" model, proposes a direct interaction of inositol 1,4,5-trisphosphate (IP 3 ) receptors in the endoplasmic reticulum with SOCs in the plasma membrane (2, 6). The latter hypothesis has gained widespread support in the last few years.TRPC3 is a member of the canonical transient receptor potential (TRPC) family of Ca 2ϩ -permeant, non-selective cation channels (7) whose members have been hypothesized to form, or contribute to the formation of the elusive SOC channel (7-9). TRPC3 and its close structural homologs, TRPC6 and TRPC7, in many expression systems, including HEK293 cells, behave as receptor-operated cation channels (10 -12) that can be activated by exogenous application of diacylglycerols (DAG) independently of store-depletion (13, 14). However, Kiselyov et al. (15,16) reported that TRPC3 channels in excised patches from TRPC3-expressing HEK293 cells could be stimulated with IP 3 and IP 3 receptors. These authors suggested that TRPC3 activation involves interaction with IP 3 receptors in their ligandbound state, consistent with the conformational coupling hypothesis. Ma et al. (17) also proposed a requirement of IP 3 receptors for native SOC and TRPC3 channel activation based on experiments with the membrane-permeant IP 3 receptor antagonist, 2-aminoethoxydiphenyl borane (2-APB). Finally, Boulay et al. (18) provided bi...
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.
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