© 2001 Macmillan Magazines Ltd articles NATURE CELL BIOLOGY VOL 3 FEBRUARY 2001 http://cellbio.nature.com 121 Lack of an endothelial store-operated Ca 2+ current impairs agonist-dependent vasorelaxation in TRP4 –/– mice Marc Freichel*, Suk Hyo Suh†, Alexander Pfeifer‡, Ulli Schweig*, Claudia Trost*, Petra Weißgerber*, Martin Biel‡, Stephan Philipp*, Doris Freise*, Guy Droogmans†, Franz Hofmann‡, Veit Flockerzi*§ and Bernd Nilius†¶ *Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, D-66421 Homburg, Germany †Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, B-3000 Leuven, Belgium ‡Institut für Pharmakologie und Toxikologie, TU München, Biedersteinerstrasse 29, D-80808 München, Germany §e-mail: veit.flockerzi@med-rz.uni-saarland.de ¶e-mail: bernd.nilius@med.kuleuven.ac.be Agonist-induced Ca 2+ entry into cells by both store-operated channels and channels activated independently of Ca 2+ - store depletion has been described in various cell types. The molecular structures of these channels are unknown as is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca 2+ current in vascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this cur- rent. As a consequence, agonist-induced Ca 2+ entry and vasorelaxation is reduced markedly, showing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca 2+ -entry pathway essentially contributing to the regulation of blood vessel tone. M any different agonists, such as hormones, neurotransmit- ters, growth factors and other physiological and pathologi- cal stimuli, initiate cellular responses through an increase in cytosolic Ca 2+ . This increase is a consequence of Ca 2+ release from intracellular stores, Ca 2+ influx through Ca 2+ permeable cation channels in the plasma membrane or both processes. Activation of Ca 2+ entry through plasma-membrane channels is caused by the depletion of intracellular Ca 2+ stores by inositol 1,4,5-trisphosphate (InsP 3 ) or inhibition of Ca 2+ uptake, or by as yet unknown mecha- nisms downstream of receptor activation 1–3 . Ca 2+ entry due to store depletion is often called capacitative Ca 2+ entry 3 and is mediated by store-operated cation channels or ‘SOCs’. The best-studied store-operated Ca 2+ channels in terms of biophysical properties are Ca 2+ -release-activated Ca 2+ (CRAC) channels, which have been found in mast cells, T lymphocytes and rat basophilic leukaemia (RBL) cells and which are characterized by their inward rectifying currents and by their high selectivity for Ca 2+ (refs 4–8). Other, less Ca 2+ -selective store-operated channels have been described in many tissues 9–12 . The molecular structures of SOCs are unknown and are probably not identical for the channels in the various cell types. In endothelial cells, a sustained influx of extracellular Ca 2+ into the cell 13–15 contributes to the increase of the cytosolic Ca 2+ con- centration, [Ca 2+ ] i , which is necessary for synthesis and release of vasoactive compounds such as nitric oxide or prostaglandins 16–18 . Accordingly, agonist-induced Ca 2+ entry pathways including SOCs have been described in various endothelial cell lines 6,7,12,15,19–21 . These endothelial SOCs can be distinguished from CRACs in that they reveal less inward rectification 20 and give rise to extremely small currents (current densities < 1pA pF –1 at –50 mV) 20 . Several members of the mammalian trp gene family, including trp4, have been implicated in store-operated Ca 2+ entry in various cells, and trp4 messenger RNA is expressed in mouse 22 , human 23 and bovine vascular endothelial cells 24 . These findings prompted us to study the store-operated Ca 2+ currents and agonist-induced Ca 2+ entry in native endothelium, and the link, if there is one, between these processes and the trp4 gene product by deleting the trp4 gene in the mouse. We show here that primary cultured mouse vascular endothelial cells (MAECs) express trp4 transcripts and the TRP4 protein, and that Ca 2+ permeable channels can be activated by store-depletion protocols in these cells. Accordingly, we refer to these channels as store-operated channels or SOCs. By deleting the trp4 gene in the mouse we obtained TRP4 –/– MAECs. These TRP –/– MAECs lack store-operated Ca 2+ currents, indicating that the TRP4 protein is functionally expressed in wild-type MAECs and is responsible for the expression of the store-operated channels in these cells. As a consequence of TRP4 deletion and lack of SOC, agonist-induced Ca 2+ entry is reduced markedly, resulting in a significant decrease of endothelium-dependent vasorelaxation of blood vessels. Therefore, TRP4 is an indispensable component of store-operated channels in native endothelial cells, and TRP4-mediated SOC provides a direct Ca 2+ -entry pathway of physiological relevance, that is, it is essen- tially contributing to the regulation of blood vessel tone. Results Macrovascular endothelial cells display store-operated Ca 2+ cur- rents. Agonist-induced Ca 2+ entry and store-operated Ca 2+ currents have been described in a number of endothelium cell lines and pri- mary cultures cells 12,19–21,24,25 . To characterize store-operated Ca 2+ cur- rents in primary cultured mouse aortic endothelial cells (MAECs), we dialysed MAEC with 30 μM inositol 1,4,5-trisphosphate (InsP 3 ) through the patch and added 20 μM tert-butyl-benzohydrochinone (tBHQ) to the bath. Within about 1 min (depending on the cell size) of rupturing the patch, a sustained inward current developed at –50 mV (Fig. 1a), which reached a stationary level within about two min. Figure 1a (top right) shows currents obtained during inter- mittently applied short (50-ms) voltage ramps from –100 mV to +100 mV. Typically, the first two to four ramps were used as back- ground currents and subtracted from the remaining records. The currents measured from voltage ramps in 5 mM external Ca 2+ ([Ca 2+ ] e ) were found to be inward rectifying (Fig. 1 top right), had