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(Circulation Research. 2002;91:1.)
© 2002 American Heart Association, Inc.

Editorials

TRPC4 Knockout Mice

The Coming of Age of TRP Channels as Gates of Calcium Entry Responsible for Cellular Responses

Lutz Birnbaumer

From the Transmembrane Signaling Group, Laboratory of Signal Transduction, and Division of Intramural Research, National Institute of Environmental Health Sciences, Research Triangle Park, NC.

Correspondence to Lutz Birnbaumer, PhD, Bldg 101, Room A-214, 111 T.W. Alexander Dr, Research Triangle Park, NC 27709. E-mail lutzb{at}niehs.nih.gov

Key Words: transient receptor potential • I_CRAC • calcium signaling • phospholipase • endothelial cell function

In this issue of Circulation Research, Tiruppathi and collaborators present a second report on the phenotypic changes that develop in mice lacking the TRPC4 channels developed by the Flockerzi group at the Institute for Pharmacology and Toxicology of the University of Saarland, Germany.¹ This and the earlier report² show unequivocally that TRPC channels and the Ca²⁺ whose entry they mediate are central players in cellular and organismic homeostasis.

Increases in cytosolic Ca²⁺ drive a myriad of cellular responses to extracellular stimuli, either alone or in conjunction with other signaling pathways. Examples are skeletal and smooth muscle contraction, neuronal and endocrine secretions, and activation of B and T lymphocytes, of neutrophil chemotaxis, of endothelial cell NO production, and of platelet aggregation. With the exception of skeletal muscle contraction, which is mediated exclusively by Ca²⁺ released from the sarcoplasmic reticulum in response to sarcolemmal depolarization, all other cellular responses enabled by Ca²⁺ depend on Ca²⁺ entering from the extracellular space through Ca²⁺-permeable ion channels. True to this adage, cardiac and smooth muscle contraction and neuro-, endo-, and exocrine secretions fail upon removal of extracellular Ca²⁺. Cells are negatively affected by sustained high cytosolic Ca²⁺ ([Ca²⁺]_i) and expend significant amounts of energy to keep [Ca²⁺]_i at low, 100 nmol/L levels—10⁴ times lower than present in the extracellular milieu. Entry of Ca²⁺ into cells is thus carefully gated. The gating is performed by just three classes of ion channels: (1) voltage-gated Ca²⁺ channels, structural relatives of voltage-gated sodium channels, expressed in neuronal, endocrine, and muscle cells; (2) ligand-gated ion channels, including cyclic nucleotide- and excitatory amino acid-activated ion channels, expressed in various sensory and neuronal cells; and (3) an ubiquitous set of voltage-independent, Ca²⁺-permeable cation channels activated for the most part by maneuvers that include activation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP₃). The molecular nature of these channels is just now coming to light.

Agents that lead to IP₃ formation are those that act through receptors that are coupled to intracellular responses by either PLCß activated by G proteins of the Gq and Gi/o classes, or PLC activated by tyrosine kinases, thus reflecting the distinct responsiveness of these two types of PLC. PLCßs respond to Gq-derived GTP-Gq and to Gi/o-derived Gß; PLCs are activated by tyrosine phosphorylation. Stimulation of dedifferentiated fibroblast cell lines that lack voltage- and ligand-gated ion channels, in the presence of extracellular Ca²⁺, with an agonist that acts through a Gq-coupled receptor, typically shows an initial, rapid (within seconds) rise of [Ca²⁺]_i to peak levels that can be as high as 100- to a 1000-fold basal, followed by a decline to a plateau level that averages between 2- and 4-fold the prestimulation level and persists until the stimulating agent is removed. The same experiment without extracellular Ca²⁺ shows the initial peak to be little affected but the plateau to be abolished. Readmission of Ca²⁺ to the medium after [Ca²⁺]_i has returned to baseline—agonist continuously present—now shows an immediate influx of Ca²⁺ and establishment of the plateau phase of elevated [Ca²⁺]_i that persists until the causative agonist is removed. With small variations, this type of experiment has been repeated with the same outcome by researchers across the world with almost all cells that have been isolated and tested for their [Ca²⁺]_i changes in response to agonists, chemokines, cytokines, neurotrophic peptides, etc. Cells with this type of [Ca²⁺]_i response pattern are not only of the nonexcitable type but also include excitable cells such as neurons and myocytes, visualized after blocking their voltage- and/or ligand-gated channels. This underscores the ubiquitous nature of the [Ca²⁺]_i response to maneuvers that elevate IP₃ levels in cells, and by extension, the ubiquitous nature of the Ca²⁺ entry pathway activated in conjunction with increases in cytosolic IP₃.

What are the molecular components of this Ca²⁺ entry pathway? The hunt for channels through which the Ca²⁺ enters began 10 years ago with clues coming from studies with mammalian cells and insect photoreceptor cells. Mammalian cells were found to activate a similar Ca²⁺ entry pathway without prior stimulation of IP₃ formation by simply doing what IP₃ does, ie, by depleting the internal stores with the drug thapsigargin (TG). IP₃ does this by binding to its intracellular receptor, the IP₃R, which is a Ca²⁺ release channel located in the endoplasmic reticulum membrane that delimits the internal store. TG instead acts by inhibiting sarcoplasmic-endoplasmic reticulum ATPases (SERCAs), the Ca²⁺ pumps responsible for transporting Ca²⁺ from cytosol to the store.³ Ca²⁺ entry promoted by mere store depletion without overt activation of a PLC system was referred to as capacitative Ca²⁺ entry, for it eventually restored the capacity of the store to release Ca²⁺.⁴ Experimental maneuvers could now differentiate between agonist and IP₃-mediated Ca²⁺ entry from mere store depletion-induced Ca²⁺ entry.

Although susceptible to blockade by nonspecific agents that block all Ca²⁺-permeable ion channels, such as the lanthanides, curiously—or strangely—this universal form of Ca²⁺ influx knows of no specific inhibitors. Electrophysiological characterization of the channel(s) through which Ca²⁺ enters cells after store depletion was elusive, until in 1992, Hoth and Penner⁵ characterized a store depletion-activated current in mast cells. Noise analysis indicated that single-channel currents had to be in the sub-picosiemen (pS) range. Ion permeation studies showed the channel to be exquisitely selective for Ca²⁺ over other divalent cations, including Mg²⁺. The term I_CRAC for Ca²⁺ release-activated current was coined. In 1993, Penner’s group⁶ reported the existence in the same cells of receptor-activated nonspecific (Ca²⁺-permeable) cation channels with much larger single-channel conductances than those responsible for I_CRAC, 50 pS, and thus laid the foundation for the fact that Ca²⁺ entry activated by the agonist-PLC-IP₃ pathway, which includes store-activated capacitative Ca²⁺ entry channels, were likely to be heterogeneous. Numerous other examples confirmed this assumption.

TRP channels entered the scene at about the same time, when in 1992 Hardie and Minke⁷ reported that a light-activated Ca²⁺ current responsible for the sustained phase of the fly’s compound eye electroretinogram was absent in a Drosophila mutant termed transient receptor potential, TRP. In invertebrates, unlike in vertebrates, light activation of rhodopsin, instead of hyperpolarizing the photoreceptor cell, causes its depolarization through a pathway formed of rhodopsin, a Gq-like G protein, and PLC, akin to the one that elicits in mammalian cells the IP₃- and store depletion-activated Ca²⁺ entry. Interestingly, the trp gene product predicted a structure with some characteristics of an ion channel with limited sequence homology to voltage-gated Na⁺ and Ca²⁺ channels.⁸ Could it be that mammalian homologues existed that are responsible for store depletion- and/or agonist-activated Ca²⁺ entries? By 1996, six mammalian TRP homologues (now referred to as TRPC channels) had been identified.⁹ Full-length cDNAs were available for several of them by 1997, including TRPC4, originally referred to as Trp4 and CCE1.¹⁰

Absence of cells lacking capacitative and/or PLC-IP₃-activated Ca²⁺ entry made it difficult to establish physiological role(s) for the proteins encoded in TRPC cDNAs. Seven TRPC genes were eventually identified. Importantly, expression of none recapitulated I_CRAC in terms of ion selectivity. Although TRPC1, TRPC2, and TRPC4, when expressed in model cells, were susceptible to activation by mere store depletion (TG treatment), channels appearing in TRPC3- and TRPC6-transfected cells seem insensitive to store depletion and require a receptor-PLC-IP₃-IP₃R pathway for their activation.

Formation of hybrid TRPC channels composed of more than one TRPC is at present the best explanation for the elusiveness of I_CRAC properties of expressed TRPC cDNAs and the relative paucity of data from normal cells that predict the electrophysiological characteristics of the channels that appear upon transfection of TRPC cDNAs. In their quest for unequivocal roles, matters turned out still worse for TRPCs, the close homologues to Drosophila TRP, for they have been found to form part of a superfamily of TRP-related proteins. TRP-related proteins include the vanilloid and vanilloid-related receptors (VR1 and VRL1) and the cold and menthol receptor (CMR1), responsible for heat and cold sensing, the presumed anti-oncogene melastatin-1 (MLSN1), the polycystic kidney disease 2 gene product (PKD2), the epithelial calcium channels ECaC1 and ECaC2 (also CaT2 and CaT1), and channels with unrelated protein folds in their C-terminal domains such as an atypical kinase that interacts with PLC (TRP-PLIK) or a NuDix domain able to sense reactive oxygen species and ADP-ribose (see recent reviews^11,12; see nomenclature rules¹³).

Although not addressing specifically the question of subunit complexity of TRP channels—this may have to be solved by applying proteomics approaches—targeted inactivation studies are beginning to shed light on the functional significance of individual TRP channels. So, for example, an antibody approach showed an essential role of TRPC2 in sperm’s acrosome reaction triggered by oocyte zona pellucida protein,¹⁴ and ablation studies showed that TRPM7 (TRP-PLIK/LTRPC7) is essential for cell viability¹⁵ and that TRPC4 (Trp4/CCE1) contributes to endothelium-mediated vascular smooth muscle relaxation.²

Tiruppathi et al expand the studies of Freichel and collaborators with TRPC4^-/- mice² by studying properties of lung endothelial cells both in isolation and in situ.¹ Thrombin, or a thrombin receptor-derived peptide with agonist activity, was used as a trigger of Gq activation to regulate endothelial cell functions such as Ca²⁺ entry, stress fiber formation, and cellular retraction. Further, in a model that assesses endothelial cell-dependent microvessel liquid permeability, they measured thrombin-induced permeability increases. In all instances, loss of TRPC4 correlates with a loss of endothelial cell responses, showing participation of TRPC4 in the response of endothelial cells to thrombin or its surrogate PAR1 receptor-derived agonist. Reduction of Ca²⁺ influx was expected from previous results with TRPC4^-/- aortic endothelial cells,² but the strict dependence on agonist-activated Ca²⁺ entry for stress fiber formation, and its function in retraction and relaxation of cell-cell interaction as seen in the microvascular permeability studies, was not.

It is noteworthy that impairment of thrombin’s responses was not always total. In aortic strips, endothelium-dependent Gq-activated relaxation was lost by only 50%.² Likewise, endothelial cell retraction and microvasculature permeability increases were only reduced by 50% in TRPC4^-/- mice. On the other hand, stress fiber formation was fully eliminated, and Ca²⁺ influx assessed by the Ca²⁺ readdition assay was reduced by 80% to 90%. These results very likely indicate that other TRPCs or one or more of the TRP-related molecules—some known, others just suspected of having the ability to form ion channels—may cover for the loss of TRPC4 in endothelial cell function. It is for future research to answer these questions. In the meantime, it is clear that TRPC4, originally called CCE1, is indeed a critical component of capacitative Ca²⁺ influx, thus validating the efforts of many in the field who, like me, betted on TRPCs being important in agonist and capacitative Ca²⁺ entry.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

References

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2. Freichel M, Suh SH, Pfeifer A, Schweig U, Trost CP, Weissgerber P, Biel M, Philipp S, Freise D, Droogmans G, Hofmann F, Flockerzi V, Nilius B. Lack of an endothelial store-operated Ca²⁺ current impairs agonist dependent vasorelaxation in Trp4-/- mice. Nat Cell Biol. 2001; 3: 121–127.[CrossRef][Medline] [Order article via Infotrieve]

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11. Montell C. Physiology, phylogeny, and functions of the TRP superfamily of cation channels. Sci STKE. 2001. Available at: http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/90/re1. Accessed June 10, 2002.

12. Montell C, Birnbaumer L, Flockerzi V. The TRP channels: a remarkably functional family. Cell. 2002; 108: 595–598.[CrossRef][Medline] [Order article via Infotrieve]

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