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NATURE CELL BIOLOGY VOL 3 FEBRUARY 2001 http://cellbio.nature.com 121

Lack of an endothelial store-operatedCa2+ current impairs agonist-dependentvasorelaxation in TRP4–/– mice

Marc Freichel*, Suk Hyo Suh†, Alexander Pfeifer‡, Ulli Schweig*, Claudia Trost*, Petra Weißgerber*, MartinBiel‡, 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: [email protected] ¶e-mail: [email protected]

Agonist-induced Ca2+ entry into cells by both store-operated channels and channels activated independently of Ca2+-store depletion has been described in various cell types. The molecular structures of these channels are unknownas is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca2+ current invascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this cur-rent. As a consequence, agonist-induced Ca2+ entry and vasorelaxation is reduced markedly, showing that TRP4 isan indispensable component of store-operated channels in native endothelial cells and that these channels directlyprovide an Ca2+-entry pathway essentially contributing to the regulation of blood vessel tone.

Many different agonists, such as hormones, neurotransmit-ters, growth factors and other physiological and pathologi-cal stimuli, initiate cellular responses through an increase in

cytosolic Ca2+. This increase is a consequence of Ca2+ release fromintracellular stores, Ca2+ influx through Ca2+ permeable cationchannels in the plasma membrane or both processes. Activation ofCa2+ entry through plasma-membrane channels is caused by thedepletion of intracellular Ca2+ stores by inositol 1,4,5-trisphosphate(InsP3) or inhibition of Ca2+ uptake, or by as yet unknown mecha-nisms downstream of receptor activation1–3.

Ca2+ entry due to store depletion is often called capacitativeCa2+ entry3 and is mediated by store-operated cation channels or‘SOCs’. The best-studied store-operated Ca2+ channels in terms ofbiophysical properties are Ca2+-release-activated Ca2+ (CRAC)channels, which have been found in mast cells, T lymphocytes andrat basophilic leukaemia (RBL) cells and which are characterizedby their inward rectifying currents and by their high selectivity forCa2+ (refs 4–8). Other, less Ca2+-selective store-operated channelshave been described in many tissues9–12. The molecular structuresof SOCs are unknown and are probably not identical for thechannels in the various cell types.

In endothelial cells, a sustained influx of extracellular Ca2+ intothe cell13–15 contributes to the increase of the cytosolic Ca2+ con-centration, [Ca2+]i, which is necessary for synthesis and release ofvasoactive compounds such as nitric oxide or prostaglandins16–18.Accordingly, agonist-induced Ca2+ entry pathways includingSOCs have been described in various endothelial celllines6,7,12,15,19–21. These endothelial SOCs can be distinguished fromCRACs in that they reveal less inward rectification20 and give riseto extremely small currents (current densities < 1pA pF–1 at–50 mV)20.

Several members of the mammalian trp gene family, includingtrp4, have been implicated in store-operated Ca2+ entry in variouscells, and trp4 messenger RNA is expressed in mouse22, human23

and bovine vascular endothelial cells24. These findings promptedus to study the store-operated Ca2+ currents and agonist-inducedCa2+ entry in native endothelium, and the link, if there is one,

between these processes and the trp4 gene product by deleting thetrp4 gene in the mouse.

We show here that primary cultured mouse vascular endothelialcells (MAECs) express trp4 transcripts and the TRP4 protein, andthat Ca2+ permeable channels can be activated by store-depletionprotocols in these cells. Accordingly, we refer to these channels asstore-operated channels or SOCs. By deleting the trp4 gene in themouse we obtained TRP4–/– MAECs. These TRP–/– MAECs lackstore-operated Ca2+ currents, indicating that the TRP4 protein isfunctionally expressed in wild-type MAECs and is responsible forthe expression of the store-operated channels in these cells. As aconsequence of TRP4 deletion and lack of SOC, agonist-inducedCa2+ entry is reduced markedly, resulting in a significant decrease ofendothelium-dependent vasorelaxation of blood vessels. Therefore,TRP4 is an indispensable component of store-operated channels innative endothelial cells, and TRP4-mediated SOC provides a directCa2+-entry pathway of physiological relevance, that is, it is essen-tially contributing to the regulation of blood vessel tone.

ResultsMacrovascular endothelial cells display store-operated Ca2+ cur-rents. Agonist-induced Ca2+ entry and store-operated Ca2+ currentshave been described in a number of endothelium cell lines and pri-mary cultures cells12,19–21,24,25. To characterize store-operated Ca2+ cur-rents in primary cultured mouse aortic endothelial cells (MAECs),we dialysed MAEC with 30 µM inositol 1,4,5-trisphosphate (InsP3)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 twomin. 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. Thecurrents measured from voltage ramps in 5 mM external Ca2+

([Ca2+]e) were found to be inward rectifying (Fig. 1 top right), had


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a reversal potential at +43.2 ± 1.6 mV (n = 28, mean ± s.e., correct-ed for liquid junction potentials) and inactivated at negative poten-tials (Fig. 1, bottom right). They were abolished by 5 mM Mg2+

added to a Ca2+-free solution (Fig. 1b) and fully recovered after read-dition of 5 mM Ca2+ and reduction of extracellular Mg2+ to 1 mM.

La3+ (1 µM) almost completely blocked this current (Fig. 1b,89 ± 5% inhibition at –50 mV, n = 26). The current depended onextracellular Ca2+, as it was reduced from –0.89 ± 0.09 pA pF–1

(n = 28) at 5 mM Ca2+ to –0.46 ± 0.06 pA pF–1 (n = 6) at1.5 mM [Ca2+]e and increased to –1.42 ± 0.33 pA pF–1 (n = 4) at20 mM [Ca2+]e (see below). After removal of Mg2+ and Ca2+ from thebath, we observed Na+ currents (Fig. 1c) that were at least 10 timesbigger than the Ca2+ currents in 5 mM Ca2+ (-12.5 ± 2.5 pA/pF,n = 6; Fig. 3e, right panel). The inward component of this current in

divalent-free solutions disappeared when Na+ was substituted byN-methyl-D-glucamine (NMDG; Fig. 1c). Therefore, a large Na+

permeability in the complete absence of divalent cations is anothertypical feature of this current in MAECs. The reversal potential of

NATURE CELL BIOLOGY VOL 3 FEBRUARY 2001 http://cellbio.nature.com122

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Figure 2 Targeted disruption of the mtrp4 gene. a, Restriction maps of the mtrp4target locus, targeting construct and targeted allele. Exons and introns are shown asfilled boxes and lines, respectively. The targeting vector contains 7.7 kb of mtrp4genomic sequence that flank pgk-neo (neo) cloned in the opposite transcriptional ori-entation. Exon size is not to scale. S, SpeI; A, AflIII; B, BglII; E, EcoRI; wt, wild-type;mt, mutant. b, Identification of TRP4+/+, TRP4+/– and TRP4–/– mice by Southern blotanalysis. Wild-type (13 and 12.5 kb) and/or mutant (7 and 10.5 kb) -specific bandswere obtained with the 5’ probe (SpeI cut) and 3’ probe (BglII cut), respectively. c, d,Northern blot analysis of poly(A)+ RNA isolated from brain (c) or MAECs (d) of TRP4+/+

and TRP4–/– mice hybridized with mtrp4-specific probes. Transcripts were identified inwild-type brain (3.9 and 7 kb) and MAECs (3.9 kb). e, Northern blot analysis ofexpression of trp4 homologues trp1, trp3, trp5 and trp6 in brain. GAPDH cDNA wasused as a control. f, Immunoblot analysis of TRP4 expression in HEK (293) cells. Theantibody recognizes the TRP4 protein in 293 cells transiently transfected with mtrp4cDNA (mtrp4; GenBank accession no. U50922), but not in the presence of the anti-genic peptide or in non-transfected cells (non-trans). g, Immunoblot analysis of mousebrain (left) and MAECs (right). In brain and MAECs of wild-type mice, proteins of Mr

102K (TRP4) and Mr 94K (TRP4∆781-864) were identified. The antibody recognizes anadditional protein of unknown identity that runs slightly above TRP4∆781–864.


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the current in the absence of [Ca2+]e shifted to –10.3 ± 5.8 mV(n = 6). From the reversal potentials in Ca2+-free solutions and in5 mM Ca2+, we calculated a permeability of PCs/PNa = 0.74 ± 0.13(n = 6) and of PCa/PNa = 159.7 ± 21.3 (n = 28)26.

In conclusion, a highly Ca2+ permeable cation channel inMAEC is activated by a store-depletion protocol. This channel isnon-selective for monovalents in the absence of Ca2+ and isblocked by Mg2+ and La3+. We henceforth refer to this channel asa store-operated Ca2+-permeable cation channel (SOC).Macrovascular endothelial cells express TRP4. The TRP4 proteincontributes to the formation of cation channels that are activatedby protocols that deplete Ca2+ stores27–31 or by other mechanismsdownstream of receptor activation32. Previously, we showed thattrp4 transcripts can be amplified from MAEC mRNA, and, inagreement with these results, we found here that trp4 transcriptsand the TRP4 protein are expressed in mouse aorta endothelialcells by using northern and immunoblot analysis (Fig. 2d, g).

Endothelial cells from wild-type mice and from mice wherethe trp4 gene has been inactivated provide a system in which tostudy the relation of trp4 expression and the presence of the store-operated currents shown in Fig. 1. This system may also allow tostudy the physiological role of SOCs in endothelium.Targeted disruption of the murine trp4 gene. The trp4 gene wasinactivated by gene targeting in murine embryonic stem cells bydeleting the exon encoding transmembrane segments 4 and 5 andpart of the linker between S5 and S6, which is assumed to contribute

to the pore forming region of the trp4 protein27,28 (Fig. 2). Usingthis strategy, compensative upregulation of truncated splice vari-ants of trp4 such as bCCE1∆514 from bovine adrenal gland33,which might be able to form ion conducting proteins, should beexcluded. Deletion of the trp4 gene and lack of expression wasconfirmed by Southern, northern and western blot analysis (Fig.2b, c, d, g). In northern blot experiments, transcripts of 3.9 kilo-bases (kb) (brain, MAEC) and 7.0 kb (brain) were identified inwild-type but not in TRP4-deficient mice (Fig. 2c, d). The TRP4antibody (Fig. 2f) recognizes both trp4 splice variants identifiedin the mouse (GenBank accession nos U50922, U50921), TRP4(relative molecular mass (Mr) ~102,000 (120K)) and a slightlyshorter version termed TRP4∆781-864 (~94K). Both proteins arepresent in microsomes (brain) or homogenates (MAEC) from

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entry in a wild-type cell. Note the inhibition of the [Ca2+]i increase during Ca2+ reap-plication in the presence of 1 µM La3+ (grey bar). Ca2+ (black bar) concentration inmM; tBHQ (arrow) in µM. b, Comparison of Ca2+ release phase in TRP4+/+ andTRP4–/– MAECs after application of tBHQ in the absence of extracellular Ca2+. c,Comparison of the increase in [Ca2+]i after reapplication of 1.5 mM Ca2+ in TRP4+/+

(left) and TRP4–/– (right) cells; 1 µM La3+, grey bar. d, Summary of the experimentsfor [Ca2+]i owing to Ca2+ release (left bars; P = 0.034) and during Ca2+ reapplica-tion (right bars; P = 0.006). ∆[Ca2+]i describes the differences between peak [Ca2+]iafter tBHQ application or during Ca2+ reapplication and [Ca2+]i before tBHQ (leftbars) or Ca2+ reapplication (right bars). e, Decrease of [Ca2+]i by 1 µM La3+ appliedduring Ca2+ reapplication (P < 0.001). f, Comparison of the agonist-inducedincrease in [Ca2+]i in the absence of [Ca2+]e by the physiological vasodilator ACh(10 µM, TRP4+/+, solid line; TRP4–/–, dotted line). These Ca2+ transients reflect therelease from intracellular stores. g, The Ca2+ release induced by the agonists AChand ATP (in µM) is not significantly changed in TRP4+/+ versus TRP4–/– cells.


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wild-type mice but not in microsomes or homogenates fromTRP4–/– mice (Fig. 2g).

Expression levels of trp1, trp3, trp5 and trp6 are not altered inwild-type, TRP4+/– and TRP4–/– mice (Fig. 2e), indicating that TRP4deletion does not affect the expression of other TRP proteins.Heterozygous (TRP4+/–) and homozygous mutant (TRP4–/–) pupsobtained from heterozygous matings are viable, fertile, exhibit nogross abnormalities and are segregated in the mendelian frequency(93 TRP4+/+, 191 TRP4+/–, and 99 TRP4–/– progeny were obtainedfrom 105 inbred litters of heterozygous crosses), suggesting thatembryonic and fetal development of homozygous mutant micewere not impaired.Lack of store-operated Ca2+ currents in TRP4-deficient mouseaorta endothelial cells. TRP4 is expressed in wild-type aorticendothelial cells but not in MAECs prepared from TRP4–/– mice(Fig. 2 d, g, right panel). To test the hypothesis that TRP4 is part ofthe store-operated channel in MAECs, we recorded currents asdescribed in Fig. 1. An example from wild-type MAECs is shown as

time course at –50 mV (Fig. 3a) with the respective I–V relation-ship (Fig. 3b). At 5mM [Ca2+]e the current density at –50 mV inwild-type MAECs was –0.89 ± 0.09 pA pF–1 (n = 28, Fig. 3e) versus–0.05 ± 0.03 pA pF–1 (n = 27) in TRP4–/– MAECs, indicating thatInsP3/tBHQ-activated SOC currents are almost completely abolishedin TRP4 –/– MAEC. Such a current developed in 28 out of 55 wild-type cells but not in 27 out of 27 TRP4–/– cells (Fig. 3c, d). This 51%success rate for activation of SOC currents might reflect the difficul-ties in perfusion of the extremely flat endothelial cells. No such cur-rent was activated without the store-depletion protocol. With Na+ ascharge carrier the current density was –12.5 ± 2.5 pA pF–1 (n = 6) inwild-type but only –2.5 ± 0.65 pA pF–1 (n = 11) in TRP4-deficientMAECs (Fig. 3e).

In addition to the measurement of store-operated Ca2+ currents,we studied Ca2+ entry by a store-depletion/Ca2+-readdition protocolusing fura-2 fluorescence ratio measurements (Fig. 4a shows a rep-resentative experiment). We applied 20 µM tBHQ to empty intra-cellular Ca2+ stores in the absence of extracellular Ca2+, and thenreapplied extracellular Ca2+. Application of tBHQ in the absence ofextracellular Ca2+ induced a transient increase in [Ca2+]i owing torelease of Ca2+ from intracellular stores. The subsequent reapplica-tion of 1.5 mM extracellular Ca2+ causes an additional increase in[Ca2+]i . This increase depends on [Ca2+]e (Fig. 4a), proceeds mainlythrough plasmalemmal store-operated channels activated duringthis protocol and is reduced in the presence of 1 µM La3+.

Figure 4b and c show examples of the changes of [Ca2+]i fromwild-type (left) and TRP4–/– (right) MAECs. The first peak in Ca2+-free solution represents releasable Ca2+ from intracellular stores.This initial release (Fig. 4b) was hardly decreased in TRP4–/– mice(Fig. 4d, left gray bar) indicating that the filling state of the storeswas only slightly reduced in TRP4–/– cells. Similar results wereobtained when store depletion was evoked by agonists such asacetylcholine (ACh) or ATP in the absence of extracellular Ca2+

(Fig. 4f). In the presence of either agonist Ca2+ release is identical inwild-type and TRP4–/– cells (Fig. 4g).

Reapplication of 1.5 mM Ca2+ after the perfusion period in theabsence of extracellular Ca2+ resulted in an increase in [Ca2+]i (Fig.4c) which was reduced significantly in TRP4–/– cells (Fig. 4c, right),by 57% compared with the increase observed in wild-type cells(Fig. 4d, right, black and grey bars). The remaining Ca2+ influx inTRP4–/– cells is probably caused by non-selective cation currentsthat are present in MAECs34 and in an endothelial cell line derivedfrom human umbilical vein35. In addition, Ca2+-dependent mecha-nisms that carry Ca2+ out of the cell might be differentially activat-ed in TRP4+/+ and TRP4–/– cells36.

In Fig. 1a, we showed that the store-operated Ca2+ current wasalmost completely blocked in the presence of 1 µM La3+.Accordingly, 1 µM La3+ should effect the increase of [Ca2+]i due toCa2+ entry predominantly in TRP4+/+ cells but not in TRP4–/– cellswhere this store-operated Ca2+ current is almost completely absentand Ca2+ entry is reduced markedly. As shown in Fig. 4c and e, theincrease in [Ca2+]i during reapplication of Ca2+ could be also inhib-ited by 1 µM La3+ in TRP4+/+ cells by 72 ± 5% (n = 6). In contrast, inTRP4–/– cells addition of 1 µM La3+ exerts only a minor effect(reduction by 12.8 ± 7.0%, n = 10, P < 0.001). Obviously, in TRP4–/–

cells the inhibitory effect of La3+ was nearly absent (Fig. 4c, right,and e), in agreement with the lack of SOCs in these cells.Agonist-induced Ca2+ entry is reduced in TRP4–/– MAECs. In vivo,store-operated Ca2+ entry in endothelium is due to activation ofreceptors by agonists like ACh or ATP. Therefore, Ca2+ entry wasassessed in MAECs of either genotype with ACh (10 µM; Fig. 5a, b) orATP (30 µM; Fig. 5c, d) in current-clamp mode. The Ca2+ transientduring agonist stimulation of wild-type MAECs with either agonistwas accompanied by a hyperpolarization of the cell membrane whichclosely mirrored the changes in [Ca2+]i, and is mainly mediated byactivation of Ca2+-dependent channels (Fig. 5a, c)34. The plateauphase of the Ca2+ signal was much less pronounced in TRP4-deficientMAECs (Fig. 5b, d), and the hyperpolarization was more transient.


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(f) cells clamped at a holding potential of 0 mV during stimulation with 30 µM ATP.The time course of the simultaneously measured whole-cell current at –50 mV and+50 mV was obtained from repetitively applied voltage ramps and is shown by opensquares and circles, respectively. g–i, Summary of Ca2+ responses from all cellsusing the protocols shown in a–f. The normalized Ca2+ integral (ACa,norm) representsthe ratio of the Ca2+ integral 30–90 s after agonist application to the correspondingvalue 1 min before stimulation. All observed differences are significant (P < 0.01).


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To exclude the possibility that these differences in the sustainedrise in [Ca2+]i are mainly due to differences in driving force for Ca2+

ions, we also compared the Ca2+ transients in wild-type and TRP4-deficient cells in voltage-clamped mode (Fig. 5e, f). As is the case-under non-clamped conditions, the Ca2+ signal in TRP4–/– MAECsis smaller and more transient than in wild-type MAECs. The timecourse of the currents at –50 and +50 mV was reconstructed fromrepetitively applied short voltage ramps, and mirrored the changesin [Ca2+]i. These currents therefore represent mainly Ca2+-activatedcurrents with the main contribution from BKCa channels, Ca2+-acti-vated Cl– currents and Ca2+-activated non-selective cation currents,as described previously34. Under these voltage-clamp conditions,however, the changes in [Ca2+]i in the TRP4–/– MAECs are obvious-ly still smaller than in wild type.

To quantify these Ca2+ responses under the different conditions,we calculated the time integrals of [Ca2+]i from 30 to 90 s afteradministration of the agonist to avoid overlap with the initial releasecomponent of the Ca2+ signal. The summarized data (Fig. 5g, h, i)clearly show that this parameter is significantly reduced in TRP4–/–

MAECs compared with in wild-type cells. Application of 1 µM La3+

roughly 1 min after agonist administration decreased [Ca2+]i by70 ± 4% (n = 8) in wild-type MAECs, but only by 17.3 ± 9.2% inTRP4–/– cells (n = 7, P < 0.001; data not shown). These results agreewith the inhibition of the Ca2+ entry by La3+ after reapplication ofCa2+ following tBHQ-induced store depletion (Fig. 4e).Endothelium-dependent relaxation of aorta rings is impaired inTRP4–/– mice. Stimulation of endothelial cells with vasoactive ago-nists leads to a Ca2+-dependent formation of nitric oxide (NO) andvasorelaxation. We therefore assessed the relevance of the reducedendothelial Ca2+ entry observed in the explanted endothelial cells forthe agonist-induced relaxation of pre-contracted aortic rings.Representative examples of the ACh-induced endothelial-dependent

relaxation of TRP4+/+ and TRP4–/– aortic rings are shown in Fig. 6aand b. Relaxation occurred shortly after ACh application. ACh-induced vasorelaxation depends on the presence of intactendothelium because it was not observed when the endotheliumwas manually removed (Fig. 6c) or when nitric oxide synthase wasblocked (Fig. 6d and e, first column, relaxation 1 ± 0.9%, n = 6),indicating that synthesis of NO is necessary for ACh-inducedrelaxation of these vessel segments. ACh at a concentration of3 µM reduced the tension in aortic rings of wild-type mice by89 ± 4.5% (n = 20, Fig. 6a, e). In contrast, relaxation of aortic ringsfrom TRP4–/– mice were significantly smaller (37 ± 8%, n = 26,P = 0.001, Fig. 6b, e). Relaxation in TRP4–/– rings was also signifi-cantly reduced at 1 µM and 0.1 µM ACh.

Because La3+ had a pronounced effect on store-operated cur-rents, and store-operated and agonist-induced Ca2+ entry, weassumed that La3+ would have a similar effect on endothelium-dependent vessel relaxation. In this series of experiments, 3 µMACh induced a relaxation of 90.2 ± 1.4% (n = 13; Fig. 6f, j) whichwas indeed reduced to 39.5 ± 5.4% (n = 8) by pre-incubation with1 µM La3+ (Fig, 6 g, j). In contrast, La3+ had no inhibitory effect inTRP4-deficient rings (Fig.6 h, i, j).

DiscussionWe have shown that mouse aorta endothelial cells display store-operated Ca2+ currents that contribute to agonist-induced Ca2+

entry and that these currents are almost completely absent in vas-cular endothelial cells lacking the TRP4 protein. The reduction ofthese currents led to a marked decrease of the agonist-induced ele-vation of cytosolic Ca2+, which is accompanied by an impaired ago-nist-induced relaxation of precontracted aortic rings. Therefore, wehave obtained the first direct evidence, to our knowledge, for the

+/+ +/+

+/+ –/–

+/+ +/+–/–

–/–

–/–

Control 1 La3+ Control 1 La3+

n=6

n=20

n=26

n=13

n=8

n=11 n=7

100

50

0

Rel

axat

ion

(%)

100

50

0R

elax

atio

n (%

)

EC removed nitro-L-arg

3 ACh3 ACh

3 ACh3 ACh3 ACh

1 La3+

1 La3+

3 min

3 µM PGF2α

1 m

N

a b f g

c d h i

e j

Figure 6 Impaired agonist-induced vasorelaxation of TRP4-deficient aorticrings. a, b, Application of 3 µM ACh (indicated by black bars) to aortic rings pre-contracted with 3 µM PGF2α (indicated by bottom black line, which also indicatesthe zero tension level) prepared from wild-type (a) and TRP4–/– (b) mice. c, d,Agonist stimulation (3 µM ACh) of pre-contracted rings in which endothelium wasmanually removed by rubbing the inner side of the ring with a piece of cotton wool

(c) or that was pre-incubated for 30 min with 30 µM nitro-L-arginine (d). Calibrationis the same in all panels. e, Summary of vasorelaxation induced by 3 µM ACh (left,TRP4+/+ rings pre-incubated for 30 min in the presence of nitro-L-arginine; middle,TRP4+/+; right, TRP–/–). f–i, Inhibition of ACh-induced vasorelaxation in rings fromwild-type (f, g) or TRP4-deficient (h, i) mice by La3+. j, Summary of La3+ inhibition ofvasorelaxation induced by 3 µM ACh.


articles

physiological relevance not only of a mammalian TRP protein butalso for a store-operated current in endothelial cells.

These results were obtained by using four experimentalapproaches: (1) measuring currents activated by a store-depletionprotocol; measuring (2) Ca2+ entry activated by a Ca2+

removal/reapplication protocol using fura-2 fluorescence measure-ments; (3) measuring agonist-induced Ca2+ entry in current- andvoltage-clamped cells; and (4) measuring agonist-induced relax-ation of aortic rings. Throughout the four experimental approachesthe inhibitory effect of 1 µM La3+ was observed quite consistently.

The store-operated current in MAECs shares qualitatively someproperties with the well studied CRAC currents in RBL or in Jurkatcells5,6, for example its activation by store-depletion protocols, ahigh Ca2+ permeability and the permeation of monovalent cationsin the absence of Ca2+. This current is, however, not identical withCRAC; for example, inward rectification and Ca2+ selectivity are lesspronounced in MAECs than in RBL or in Jurkat cells. In addition,current densities are extremely small in MAECs (< 1pA pF–1 at–50 mV and 5 mM [Ca2+]e). Future experiments comparing furtherproperties of RBL, Jurkat and other cells, including endothelialcells, might reveal considerable variability in the SOC/CRAC cur-rents of different cell types and provide evidence for additionalsimilarities and differences in the currents of these cells.

Analysis by polymerase chain reaction after reverse transcrip-tion (RT-PCR) analysis showed that MAECs express transcripts ofadditional TRP proteins, including those of TRP3 and TRP6 (ref.22). The lack of SOC currents in TRP4–/– endothelial cells indicatesthat other TRPs do not contribute to functional store-operatedCa2+ channels in the absence of TRP4 or cannot substitute for TRP4in these cells. TRP3 and TRP6 have been implicated in agonist-acti-vated Ca2+ entry24,37–40. As it has been shown previously that TRP3forms agonist-activated Ca2+ permeable ion channels in endothelialcells that are clearly involved in the regulation of the Ca2+

plateau23,24, it might be interesting to find out whether these homo-logues are responsible for the remaining Ca2+ entry observed inTRP4–/– endothelial cells after agonist stimulation.

Finally, mouse vascular endothelial cells provide a system tostudy molecular and functional properties of TRP4 in its native cel-lular environment. Our data provide evidence that TRP4 con-tributes to SOC channel function in MAECs either as an essentialconstituent for channel activation and/or as channel-forming sub-unit. As a subunit, TRP4 might contain the ion conducting poreresponsible for Ca2+ entry. However, the molecular identity of thepore forming region within TRP4 as in other TRP proteins has notbeen revealed so far. TRP4–/–cells may provide a suitable cell systemto address these questions in future studies.

MethodsTargeting vector construction and gene targeting.trp4 DNA was isolated from a murine 129 SvJ genomic library (Genome Systems). A 3.2-kb AflIII frag-

ment containing the splice acceptor site and the following 34 base pairs of the S4/S5 exon and a down-

stream located 4.5-kb intronic EcoRI fragment were obtained from a 13-kb SpeI genomic fragment

which contains the exon encoding transmembrane segments 4 and 5 of trp4 and were cloned 5’ and 3’

of the pgk-promotor driven neomycin resistance gene (neo), respectively, thereby deleting a 278-base-

pair (bp) fragment of this exon and the adjacent splice donor site. For negative selection, the herpes

simplex virus thymidine kinase cassette (tk) from pNTK (a gift from R. Mortensen) was introduced 3’

to the mtrp4 sequence. Embryonic stem (ES) cell culture was essentially done as described41. Briefly, R1

ES cells were electroporated with the linearized targeting vector pKO-27 and plated on irradiated

G418-resistant embryonic feeder cells isolated from cGK II +/– embryos41. Recombinant clones were

selected with G418 (0.5 mg ml–1) and 1-[2’ –deoxy-2’-fluoro-β-D-arabino-furanosyl-]-5-iodouracil

(FIAU) (0.2 µM). Six out of 250 double-resistant colonies showed homologous recombination at the

mtrp4 locus as confirmed by Southern blot hybridization with a 5’ and 3’ probe external to the target-

ing vector, and a neo probe. Germline chimeras were obtained by injection of two correctly targeted ES

cell clones into C57Bl/6 blastocysts and crossed to homozygosity in a C57Bl/6J (outbred) or 129 SvJ

(inbred) background. Mice were kept in essentially specific pathogen-free environment.

Northern blot and immunoblot analysis.Northern blot hybridizations were performed as described33. Poly(A)+ RNA (10 µg) isolated from brain

of TRP+/+ , TRP+/– and TRP–/– mice (Fig. 2c, e) and poly(A)+ RNA (10 µg) isolated from passage four

MAECs (Fig. 2d) were hybridized with the following random labelled cDNA probes: mtrp4 cDNAs

(nucletides 1,588–1,876 and 813–3,043; GenBank accession number U50922), mtrp1 cDNA (1–360;

U40980), htrp3 (163–2,709; U47050), mtrp5 (199–2,897; AJ006204), htrp6 (1–2,796; AJ271066) and a

239-bp PCR fragment from the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA.

Mouse aortic endothelial cells (MAEC) were isolated from adult wild-type mice as described34. Filters

were exposed to X-ray films for two to three weeks (trp probes) or up to two days (GAPDH). For

immunoblot analysis, microsomes from mouse brains or homogenates from MAECs and antibody 236

(ref. 31) directed against amino acids 968–981 of bovine TRP4 were used. Northern and immunoblot

experiments were repeated two to six times using independent preparations and yielded identical

results.

Electrophysiology and Ca2+ measurements.MAECs from TRP4+/+ and TRP4–/– mice, which were obtained from F2 TRP4+/+ or TRP4–/– intercrosses,

were used for all experiments. Electrophysiological methods and Ca2+ measurements have been

described in detail elsewhere20,34,42. In short, whole-cell currents were measured using ruptured patches.

Currents and voltages were monitored in voltage- and current-clamp modes with an EPC-9 (HEKA

Elektronik, Lambrecht, Germany). Currents were monitored during agonist stimulation by repetitive

applications of 650-ms voltage ramps from –100 to +100 mV (sampling interval 0.5 ms, 10-s intervals

between ramps). For measurements of store-operated currents, 50-ms ramps were applied (sampling

frequency 0.05 ms, 2-s interval between ramps). The standard external solution contained (in mM):

150 NaCl, 6 KCl, 1.5 CaCl2, 1 MgCl2, 10 mM HEPES, and 10 glucose, osmolarity was 320 ± 5 mOsm.

The standard pipette solution contained (in mM): 40 KCl, 100 K-aspartate, 1 MgCl2, 0.1 EGTA,

4 Na2ATP, 10 HEPES, pH 7.2 with KOH (290 mOsm). Measurements of store-operated currents were

performed in 1.5, 5 and 20 mM Ca2+ with a pipette solution containing (in mM) 12 BAPTA, 145 cae-

sium glutamate, 4 Na2ATP, 2 MgCl2, 30 µM Ins(1,4,5)P3 (Sigma), 20 µM tBHQ (Sigma) in the pipette

solution and/or the bath. The respective extracellular solution contained (mM): 150 NaCl, 6 CsCl,

1.5 CaCl2, 100 mannitol, 10 HEPES, 10 glucose, pH 7.4 with NaOH. When CaCl2 was increased to 5 or

20 mM, an equimolar concentration of mannitol was omitted. ATP, acetylcholine (from Sigma) was

added to the external solution. For measurements of [Ca2+]i, cells were loaded with Fura-2/AM for

25 min at 37 °C. All experiments were performed at 37 °C. Summarized data are given as mean ± s.e.

Permeation ratios were measured from liquid junction potential corrected reversal potentials as

described26.

Contraction measurement on isolated aortic rings. Five-to-six-month-old mice of either sex were anaesthetized by i.p. injection of pentobarbital sodium

(50 mg per kg body weight) and killed by cervical dislocation. The thoracic aorta was dissected out,

and cut into rings of about 3.0 mm. Mechanical responses were recorded from the aortic ring seg-

ments using a home-made myograph43. Each aortic ring was threaded onto two pieces of a 120-µm

tungsten wire. One wire was anchored in the organ bath chamber (1 ml) and the other one connected

to a mechano-transducer (Grass, FT-03) mounted on a three-dimensional manipulator. Optimal rest-

ing tension (0.8–1 g) was applied, and the muscle chamber was perfused at a flow rate of 2.5 ml min–1

with Krebs/Ringer bicarbonate solution (in mM): NaCl 118.3, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, CaCl2

2.5, NaHCO3 25.0, and glucose 11.1), maintained at 37 °C and aerated with 95% O2/5% CO2. For

measurements using La3+ and in the corresponding control experiments (Fig. 6f–j), the same HEPES

buffered solution was used (150 mM NaCl, 6 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES

and 10 mM glucose) as for Ca2+ measurements. Rings were precontracted with 3 µM prostaglandin F2α

(Sigma). Relaxation was induced by ACh (Oterop, Brussels, Belgium).

RECEIVED 10 APRIL 2000; REVISED 1 SEPTEMBER 2000; ACCEPTED 27 SEPTEMBER 2000;PUBLISHED 10 JANUARY 2001

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ACKNOWLEDGEMENTS

We thank S. Buchholz, G. Ulrich and J. Prenen for excellent technical support; F. Zimmermann for

blastocyst injection and transfer; L. H. Philipson for providing mtrp4 cDNA; and R. Vennekens, M.

Hoth and A. Cavalié for helpful discussion. This work was supported by the Deutsche

Forschungsgemeinschaft (V.F.); the Belgian Federal Government, the Flemish Government and the

Onderzoeksraad KU Leuven (B.N.); the Interuniversity Poles of Attraction Program, the Prime

Ministers Office IUAP, “Levenslijn”, and the Fonds der Chemischen Industrie (V.F.).

Correspondance and requests for materials should be addressed to V.F. or to B.N.

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