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.

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...

G proteins are involved in cellular signalling and regulate a variety of biological processes including differentiation and development. We have generated mice deficient for the G protein subunit alpha i2 (G alpha i2) by homologous recombination in embryonic stem cells. G alpha i2-deficient mice display growth retardation and develop a lethal diffuse colitis with clinical and histopathological features closely resembling ulcerative colitis in humans, including the development of adenocarcinoma of the colon. Prior to clinical symptoms, the mice show profound alterations in thymocyte maturation and function. The study of these animals should provide important insights into the pathogenesis of ulcerative colitis as well as carcinogenesis.

Homologues ofS timulation of cells that elevates inositol 1,4,5-trisphosphate (IP3) causes the release of Ca 2ϩ from internal stores and its entry from the external milieu (for reviews see refs. 1-3). The release from internal stores occurs through channels formed by IP3 receptors (IP3Rs), and entry is mediated by a set of functionally heterogeneous but ubiquitous channels that are activated by the store depletion event per se. Transient receptor potential (TRP) proteins have been hypothesized to be structural components of Ca 2ϩ entry channels (4, 5) and to be activated by IP3R in response to IP3 or store depletion. However, neither has the presence of TRP in Ca 2ϩ entry channels been proven nor has the mechanism(s) by which the channels are activated been clearly elucidated. Indeed, the mechanism by which Ca 2ϩ entry channels are activated has received considerable attention, and arguments have been set forth (i) for activation by second messengers or mediators that include cGMP, IP3, diacylglycerol, a G protein, arachidonic acid derivatives, and a complex termed CIF (6-14), (ii) for translocation from internal pools with involvement of an exocytotic event (15, 16), and (iii) for shortrange physical coupling between the membrane delimiting the store and the plasma membrane (17, 18). The short-range physical-coupling model proposed that membrane Ca 2ϩ entry channels may be activated by the same protein that is responsible for store depletion, i.e., the IP3R (for details see ref. 1).The first functional evidence for a direct role of IP3R in Ca 2ϩ entry was obtained by Kiselyov et al. (19), who showed that Ca 2ϩ entry channels found in HEK cells expressing transfected TRP3 in stable form can be activated in inside-out membrane patches by addition of either IP3R-rich cerebellar microsomes or liposomes carrying recombinant IP3R protein truncated at its C terminus to inactivate its channel-forming capacity. However, this study did not determine whether the protein with which IP3R interacted was TRP3. Indeed, TRP was shown to cause changes in protein expression other than TRP, e.g., upregulation of IP3Rs, and attempts to coimmunoprecipitate IP3R and TRP3 failed (19). We now show that IP3R and TRP can be coimmunoprecipitated. We thus sought to identify interacting domains using in vitro protein:protein interaction tests and, if we found them, to test for their function. Such domains were identified and, upon expression in cells whose TRP and IP3R complement had not been manipulated, were found to modulate natural Ca 2ϩ entry stimulated by either a G protein-coupled pathway or store depletion. The data support a model in which store depletion-activated Ca 2ϩ entry is mediated by TRP-based channels that are activated by the IP3R. Materials and Methods