Capacitative calcium entry (CCE) describes CA2+ influx into cells that replenishes CA2+ stores emptied through the action of IP3 and other agents. It is an essential component of cellular responses to many hormones and growth factors. The molecular basis of this form of Ca2+ entry is complex and may involve more than one type of channel. Studies on visual signal transduction in Drosophila led to the hypothesis that a protein encoded in trp may be a component of CCE channels. We reported the existence of six trp-related genes in the mouse genome. Expression in L cells of small portions of these genes in antisense orientation suppressed CCE. Expression in COS cells of two full-length cDNAs encoding human trp homologs, Htrp1 and Htrp3, increased CCE. This identifies mammalian gene products that participate in CCE. We propose that trp homologs are subunits of CCE channels, not unlike those of classical voltage-gated ion channels.

Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (I(crac)). A critical question in cell signalling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac).

During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca 2؉ homeostasis and the physiological roles of CCE.The two primary second messengers mediating rapid responses of cells to hormones, autacoids, and neurotransmitters are cyclic nucleotides and Ca 2ϩ . Cyclic nucleotides act, for the most part, by activating protein kinases. The actions of Ca 2ϩ are more complex, in that this cation acts in two ways: directly, by binding to effector proteins, and indirectly, by first binding to regulatory proteins such as calmodulin, troponin C, and recoverin, which in turn associate and modulate effector proteins. Effector proteins regulated in these manners by Ca 2ϩ include not only protein kinases and protein phosphatases but also phospholipases and adenylyl cyclases, which are signaling enzymes in their own right, and an array of proteins involved in cellular responses that range from muscle contraction to glycogenolysis, endo-, exo-, and neurosecretion, cell differentiation, and programmed cell death. A common mechanism used by hormones and growth factors to signal through cytosolic Ca 2ϩ ([Ca 2ϩ ] i ) is activation of a rather complex reaction cascade that begins with stimulation of phosphoinositide-specific phospholipase C (PLC) enzymes, PLC␤ and PLC␥, and is followed sequentially by formation of diacylglycerol plus inositol 1,4,5-trisphosphate (IP3), liberation of Ca 2ϩ from intracellular stores, and finally, entry of Ca 2ϩ from the external milieu. The basic mechanisms used to signal through [Ca 2ϩ ] i are determined by the fact that the resting level of cytosolic Ca 2ϩ is very low, in the neighborhood of 100 nM, while that in intracellular stores and in the surrounding extracellular milieu is in the neighborhood of 2 mM, that is, Ϸ10,000-fold higher. As a result, [Ca 2ϩ ] i is set by the balance of two opposing forces. One is passive influx into the cytoplasm. It is driven by the electrochemical gradient and causes cytosolic [Ca 2ϩ ] i to rise without expenditure of energy. This influx is carefully controlled both at the level of the plasma membrane and at the level of the membranes, which delimit the internal storage compartment. Entry of Ca 2ϩ from the extracellular space occurs through three classes of Ca 2ϩ permeable gates: voltagedependent Ca 2ϩ...

, and Gd 3؉ . The Trp3-mediated Ca 2؉ influx activated by agonists was inhibited by a phospholipase C inhibitor, U73122. We propose that expression of hTrp3 in these cells forms a nonselective cation channel that opens after the activation of phospholipase C but not after store depletion. In addition, a subpopulation of the expressed hTrp3 may form heteromultimeric channels with endogenous proteins that are sensitive to store depletion.

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