Store-depletion activated CaT1 currents in RBL mast cells are inhibited by 2-APB

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1 JBC Ppers in Press. Pulished on My 14, 22 s Mnuscript M2372 Store-depletion ctivted CT1 currents in RBL mst cells re inhiited y 2-APB Evidence for regultory component tht controls ctivtion of oth CT1 nd CRAC chnnels Riner Schindl 1, Heike Khr 1, Ingrid Grz 1, Klus Groschner 2 & Christoph Romnin 1* 1 Institute for Biophysics, University of Linz, A-44 Linz, Austri 2 Institute of Phrmcology nd Toxicology, University of Grz, A-81 Grz, Austri Running title: CT1 currents in RBL mst cells Corresponding uthor: Christoph Romnin Institute for Biophysics University of Linz A-44 Linz, Austri Phone ; Fx ; Emil: christoph.romnin@jku.t Downloded from y guest on Jnury 1, 219 Key words: TRPV6, CT1, 2-APB, I CRAC, RBL-2H3 mst cells, Hek293 cells Copyright 22 y The Americn Society for Biochemistry nd Moleculr Biology, Inc. 1

2 Summry The intestinl C 2+ trnsport protein CT1 encoded y TRPV6 hs een reported (Yue, L., Peng, J.B., Hediger, M.A. & Clphm, D.E. (21) Nture 41, 75-79) to e ll or prt of the C 2+ -relese ctivted C 2+ chnnel (CRAC). The mjor chrcteristic of CRAC is its ctivtion following store depletion. We expressed CT1 in Hek293 cells nd RBL mst cells, nd mesured whole-cell currents y the ptch-clmp technique. In Hek293 cells expression of CT1 consistently yielded constitutively ctive current, the size of which ws strongly dependent on the holding potentil nd the durtion of voltge rmps. In CT1-expressing RBL cells, the current ws either ctivted y store depletion or t higher current density constitutively ctive. CT1 currents could e clerly distinguished from endogenous CRAC y their typicl current-voltge reltionship in divlent free solution. 2-minoethoxydiphenyl orte (2-APB) considered s locker of CRAC, ws tested for its inhiitory effect on oth cell types expressing CT1. Endogenous CRAC s well s storedependent CT1-derived currents of RBL-cells were lrgely locked y 75µM 2- APB, wheres constitutively ctive CT1 currents in oth RBL nd Hek293 cells Downloded from y guest on Jnury 1, 219 were slightly potentited. These results indicte tht despite the difference in permetion properties of CRAC nd CT1 chnnels, the ltter re similrly le to form store-depletion ctivted conductnces in RBL mst cells tht re inhiited y 2-APB. 2

3 Introduction Activtion of the inositol triphosphte (IP 3 ) 1 signling cscde leds to the formtion of IP 3 which inds to IP 3 receptors (IP 3 R) nd therey initites the relese of C 2+ from intrcellulr stores. Depletion of the stores is followed y the influx of C 2+ into the cell through storeoperted C 2+ -permele plsm memrne chnnels (1, 2, 3), mechnism tht hs een termed cpcitive C 2+ entry (4, 5). Ion currents ssocited with CCE hve een extensively studied in non-excitle tissues such s mst cells (6) nd T lymphocytes (7, 8). This current hs een nmed I CRAC to signify its ctivtion y C 2+ relese from intrcellulr stores (9). Moleculr cndidtes for SOCs nd CRAC re found in the trnsient receptor potentil (TRP) protein superfmily (1, 11, 12) consisting of diverse group of C 2+ permele ction chnnels tht er structurl similrities to the clssicl Drosophil TRP proteins. The proteins (13) elonging to the TRPC sufmiliy exhiit similrity to voltge-gted N + nd C 2+ chnnels nd form C 2+ permele ction chnnels upon heterologous overexpression (14, 15). Recently, one protein elonging to the vnilloid sufmily of TRP proteins, the C 2+ trnsport protein CT1 (16, 17) encoded y TRPV6 (12) hs een reported (18) to exhiit the unique iophysicl properties of I CRAC when expressed in mmmlin cells, nd it hs een proposed tht CT1 comprises ll or prt of the CRAC pore. However, Voets et l (19) hve Downloded from y guest on Jnury 1, 219 shown tht cler differences in iophysicl properties such s monovlent permeility exist etween CT1 expressed in HEK293 cells nd I CRAC in RBL mst cells. To investigte CT1 chrcteristics in more detil, we mde direct comprison of CT1, heterologously expressed in Hek293 cells, nd CT1 expressed in rt sophilic leukemi (RBL) cells. The ltter cells were chosen s they re the source of the CT1 clone used in this study nd hve een widely employed s model system to study I CRAC (9). We demonstrte tht the regultory nd phrmcologicl chrcteristics of CT1 depend on size of the current nd on 1 The revitions used re: IP 3, inositol triphosphte; I CRAC, clcium-relese ctivted clcium chnnel current; TRP, trnsient receptor potentil; CT1, clcium trnsporter protein; Hek293, humn 3

4 the cell type where it is expressed nd suggest tht 2-APB sensitive component plys key role in coupling CT1-ctivtion to store depletion in RBL mst cells. Downloded from y guest on Jnury 1, 219 emryonic kidney cells; RBL, rt sophilic leukemi; 2-APB, 2-minoethoxydiphenyl orte; DVF divlent free 4

5 Mterils nd methods Cell culture nd trnsfection Experiments were performed on secreting suline (2H3) of RBL cells mintined in monolyer culture. The cells were grown in sttionry flsks using miniml essentil medium Erle s slts (GIBCO) supplemented with 1% fetl clf serum, 2 mm glutmine, 2 U/ml penicillin nd 2 mg/ml streptomycin, in humidified tmosphere with 5% CO 2 t 37 C. The cells were trnsfected y electroportion with 4µg of ptrcercmv2-rct1 (ccession numer AF16798; kindly provided y M.A. Hediger nd D.E. Clphm, Hrvrd Medicl School, Boston, USA) or 2µg pcdna 3 -rct1 cdna. Hek293 cells were cultured in DMEM supplemented with L-glutmine (2 mm), streptomycin (1 µg/ml), penicillin (1U/ml) nd 1% fetl clf serum t 37 o C in humidity controlled incutor with 5% CO 2. Cells were used for pssges nd were trnsferred every 4 dys. Trnsfection ws performed using SuperFect (Qigen, Hilden, Germny). In rief, cells exhiiting confluence of out 2-4% were trnsfected with 8 µg of ptrcercmv2-rct1 cdna. Electrophysiology Electrophysiologicl experiments were performed t 2-24 C, using the ptch-clmp technique (2) in the whole-cell recording configurtion. Soft glss pipettes immersed in extrcellulr solution exhiited resistnce of 3-5 MΩ. Voltge rmps were pplied every 5s Downloded from y guest on Jnury 1, 219 from holding potentil of mv or +7mV, covering rnge of 9 to 9 mv over 1s or 1-2ms. In RBL cells, ctivtion of I CRAC ws monitored pplying the 1s voltge rmp t holding potentil of mv. The effect of 2-APB ws usully studied y employing the 2 ms rmp pplied from holding potentil of +7 mv for oth RBL nd Hek293 cells using extrcellulr solution. In ll recordings tht were performed in divlent free (DVF) solutions, 1 ms rmp (-9 mv to +9 mv) ws pplied every 2 s from holding potentil of mv ccording to (19). Endogenous inwrd currents (mesured t 74 mv) of mock-trnsfected Hek293 cells were in the rnge of.67 pa/pf independent of the voltge protocol pplied. 5

6 Current signls were detected using n L/M EPC7 mplifier nd for nlysis low-pss filtered t 1 khz. Pipette solution used to pssively deplete intrcellulr C 2+ stores contined 145 mm Cs methne sulphonte, 8 mm NCl, 1, 3 or 1 mm MgCl 2, 1 mm HEPES, 1 mm EGTA, ph 7.2. Active depletion ws otined y the ddition of 2µM IP 3 nd 3.5 mm CCl 2 (clculted to yield 6 nm free C 2+ ). In experiments to study the effect of 2-APB on RBL cells in extrcellulr solution (Fig. 6) 1 µm thpsigrgin ws usully dded to the pipette solution to prevent I CRAC inctivtion. Extrcellulr solution consisted of 145 mm NCl, 5 mm CsCl, 1 mm MgCl 2, 1 mm HEPES, 1 mm glucose, 1mM CCl 2, ph 7.4 Divlent free (DVF) solution contined of 165 mm NCl, 5 mm CsCl, 1 mm HEPES, 1 mm glucose, 1mM EDTA, ph 7.4/CsOH. Sttistics Results re presented s mens +/- S.E.M. clculted for the indicted numer of experiments. Student s two-tiled t-test ws used for sttisticl comprison considering differences sttisticlly significnt t p <.5. Downloded from y guest on Jnury 1, 219 6

7 Results To initilly chrcterize CT1-derived currents we heterologously expressed the chnnel in Hek293 cells. CT1-expressing cells, s identified y their GFP fluorescence, exhiited lrge inwrd currents immeditely fter otining whole-cell configurtion with 1 mm extrcellulr C 2+ (Fig. 1). Similr constitutively ctive inwrd currents (n=3) were oserved when C 2+ ws dded to the 1 mm EGTA contining pipette solution to yield 6 nm free C 2+. The CT1-derived current ws strongly inwrd-rectifying nd reversed t potentil >+3 mv. In dependence of the pplied voltge protocol, the inwrd current stedily declined (Fig. 1A, C) or exhiited iphsic time course with n initl trnsient increse (Fig. 1C, E) followed y decy to stedy-stte level (Fig. 1E). The voltge protocols pplied were vried in the holding potentil which ws set to mv (Fig. 1A,) or +7 mv (Fig. 1C; E), nd y pplying voltge rmps of different durtion (1 ms, Fig.1A, C; 2 ms, Fig. 1E). The decline in inwrd currents s depicted in Fig. 1A nd C reveled type of inhiition tht ws lrgely prevented when oth short voltge rmp nd rther positive holding potentil were pplied s evident in Fig. 1E. Moreover, sustntil fcilittion ws oserved within ~5 s fter strting whole-cell recording (Fig. 1C nd E) which my correspond to relief from inhiition. The inhiitory effect my e prtilly ttriuted to n increse of intrcellulr C 2+ due to CT1-medited C 2+ influx, in ccordnce with the recently reported inhiition of CT1 currents y intrcellulr C 2+ (19, 21). Downloded from y guest on Jnury 1, 219 It hs een suggested (18) tht CT1 mnifests the pore properties of the clcium-relesectivted clcium current (I CRAC, s originlly chrcterized in RBL mst cells. However, distinct pore properties of CT1 nd CRAC hve een reported (19). As the rct1 clone hs een derived from RBL cells, we studied the consequences of its overexpression in this cell type (Fig. 2) nd compred CT1-medited currents in RBL cells with those recorded in Hek293 cells. RBL cells were trnsiently trnsfected employing electroportion, nd CT1- overexpressing cells were identified y their GFP fluorescence. As control, I CRAC ctivtion induced y pssive (EGTA) store depletion ws initilly recorded in mock-trnsfected RBL 7

8 cells. I CRAC showed the typicl chrcteristics (22) with reversl potentil ove +4 mv. In RBL cells which were trnsfected to overexpress CT1, inwrd current chrcteristics (Fig. 2C) were similr to mock-trnsfected RBL cells. When stores were pssively depleted, the dely of current ctivtion nd the time to rech the mximum current were not different etween RBL nd CT1-RBL cells (inset in Fig. 2A nd C) suggesting similr mechnism of current ctivtion. Moreover, chnging the voltge protocol y switching the holding potentil from mv to +7 mv nd shortening the rmp durtion from 1 ms to 2 ms, induced similr increse in inwrd current densities to 165±8 % (n=4) nd 168±21 % (n=5) in oth RBL nd CT1-RBL cells, respectively. Active store depletion (IP 3, 6 nm C 2+ ) clerly reduced the dely of current ctivtion nd the time to rech the mximum current, yielding similr time-courses in oth RBL nd CT1- RBL cells (Fig. 2E) sustntiting store depletion s trigger for current ctivtion. However, current densities otined upon mximl ctivtion were significntly lrger (Fig. 2F) in CT1-RBL cells. Inwrd current densities otined y pssive (EGTA) or ctive (IP nm C 2+ ) store-depletion were not significntly different oth in RBL nd CT1-RBL cells (Fig. 2F). All of the mesurements reported ove to yield store-depletion ctivted CT1 currents were performed 1-2 dys fter electroportion of RBL cells. To look for constitutive CT1 ctivity, dditionl experiments were crried out 8-12 hours following electroportion, s RBL mst Downloded from y guest on Jnury 1, 219 cells suffering from prolonged C 2+ influx through constitutively ctivted CT1 chnnels my hve een eliminted due to complete degrnultion fter 1-2 dys. Constitutive CT1 ctivity ws indeed oserved in ~ 5% of cells 8-12 hours fter electroportion (Fig. 3), the reminder lredy exhiited store-operted CT1 ctivtion. The constitutive CT1 ctivity exhiited similr chrcteristics s tht shown previously in HEK293 cells (compre with Fig. 1A, B). Furthermore, ctivtion of endogenous CRAC expected to occur within 5s to 15s ws not visile when CT1 chnnels were constitutively ctive in RBL cells, possily due to C 2+ -dependent inhiition of CRAC chnnels (1). The intrcellulr presence of 1 mm EGTA is oviously not enough to sufficiently uffer the influx of C 2+ through constitutively ctive 8

9 CT1 chnnels locted long with CRAC chnnels in the plsm memrne, prticulrly during hyperpolrizing potentils. To evlute differences etween store-dependent nd constitutive CT1 ctivity in RBL cells, we determined the current density t s nd 15s in control (mock-trnsfected) nd CT1-trnsfected RBL cells 8-12 h nd >24 h following electroportion (Fig. 3C). Control RBL cells nd trnsfected (> 24h) showed cler inwrd currents ctivted upon store depletion, wheres 5% of RBL cells, 8-12 h fter trnsfection, exhiited constitutively inwrd currents the densities of which were significntly lrger (p <.1) thn those of the store-depletion ctivted currents. Hence, RBL cells in contrst to Hek293 cells pprently provide distinctly different cellulr environment cple of suppression of the constitutive ctivity of CT1 nd t the sme time coupling its ctivtion to store depletion. However, higher expression levels of CT1 suggested from lrger current densities yield constitutively ctive CT1 currents proly due to interference with the regultory mechnism ctivting CT1 in dependence of store depletion. To further sustntite tht the increse in current density oserved in CT1-RBL cells is due to expressed CT1 rther thn n up-regultion of endogenous CRAC, we used the currentvoltge reltionship typicl of CT1-medited currents (19) when extrcellulr, monovlent ctions function s chrge crrier in the presence intrcellulr Mg 2+ (Fig. 4). This pproch ws first estlished with CT1-Hek293 cells. Switching from C 2+ -contining extrcellulr Downloded from y guest on Jnury 1, 219 to divlent free (DVF) solution reveled negtive slope in the current-voltge reltionship etween -85 mv nd -7 mv (Fig. 4A). To minimize or even exclude possile contriution of LTRPC7 (23, 24, 25) to the oserved CT1 current-voltge reltionship in DVF solution, intrcellulr Mg 2+ concentrtion ws incresed to 3 mm (Fig. 4A) or 1 mm (not shown) Mg 2+. Under oth conditions CT1 current-voltge reltionship looked identicl excluding contmintion y LTRPC7 in HEK293 cells nd supporting our pproch to use this chrcteristic s cler test for CT1-medited currents in RBL cells. Therefore, monovlent currents were initilly studied in mock-trnsfected control RBL cells to estlish the current-voltge reltionship of endogenous I CRAC. Intrcellulr Mg 2+ ws gin 9

10 vried etween 3 nd 1 mm Mg 2+ to revel possile contriution of LTRPC7 (Fig. 4 C-F). The current-voltge reltionships of mock-trnsfected RBL cells in DVF solution were significntly different using 3 mm (Fig. 4D) or 1 mm (Fig. 4F) intrcellulr Mg 2+, which suggested sustntil contriution of LTRPC7 t 3 mm Mg 2+ prticulrly t positive potentils. At 1 mm intrcellulr Mg 2+ the remining inwrd current in DVF solution exhiited sustntil inctivtion over time (Fig. 4E) nd pronounced inwrd rectifiction in the current-voltge reltionship (Fig. 4F) oth typicl for I CRAC, however, clerly lcked the CT1-specific negtive slope eyond 7 mv (compre with Fig. 4B). CT1-expressing RBL cells were similrly studied s CT1-HEK293 cells to evlute the contriution of CT1 to inwrd currents in DVF solution (Fig. 5). Constitutively ctive CT1 currents in RBL cells (Fig. 5A) clerly exhiited the negtive slope in the current-voltge reltionship (Fig. 5B) s previously estlished in CT1-HEK293 cells, confirming CT1 chnnel expression. Further, store-operted CT1 chnnels ctivted y pssive store depletion either in the presence of 3 mm (Fig. 5D) or 1 mm (Fig. 5F) intrcellulr Mg 2+ similrly showed the negtive slope in DVF solution indicting tht the store-dependence of CT1 did not interfere with this typicl chrcteristic of CT1. Hence, the negtive slope in the current-voltge reltionship oserved in DVF solution suggest tht CRAC nd CT1 currents re medited y different chnnel proteins though the ctivtion of oth chnnels is Downloded from y guest on Jnury 1, 219 coupled to store depletion in RBL cells. To further evlute whether CT1 ctivity in RBL cells shres other fetures with the endogenous CRAC, we compred its sensitivity to 2-minoethoxydiphenyl orte (2-APB), known locker of I CRAC (26-3), with the CT1-derived current expressed in Hek293 cells (Fig. 6). We initilly confirmed tht 75 µm 2-APB cused lock of endogenous I CRAC in RBL cells (Fig. 6A). In RBL cells overexpressing CT1, lockde y 75 µm 2-APB occurred with similr chrcteristics s oserved with endogenous I CRAC (Fig. 6A). Interestingly, CT1-RBL rther thn RBL cells showed initilly smll, trnsient increse in inwrd current 1

11 upon ppliction of 2-APB efore lockde occurred (Fig. 6A). In mock-trnsfected nd CT1-trnsfected RBL cells (Fig. 6B) the 2-APB sensitive component showed similr inwrd rectifiction nd reversl potentil > +3 mv. In contrst to the pronounced locking effect of 2-APB on depletion-ctivted CT1 nd CRAC chnnels in RBL cells, the CT1-medited current in Hek293 cells ws slightly potentited y out 2% followed y slow decline to control vlues upon ppliction of 75 µm 2-APB (Fig. 6C nd D) consistent with [19]. Moreover, preincution with 75 µm 2-APB completely eliminted store-dependent ctivtion of CRAC (dt not shown) nd of CT1 chnnels in RBL cells (Fig. 6E), ut hd no significnt effect on the constitutively ctive CT1-derived currents in Hek293 cells (Fig. 6F). These results suggest tht the mechnism of store-dependent ctivtion for CRAC nd CT1 currents is similr nd possily involves the sme component sensitive to lockde y 2-APB, which is missing in Hek293 cells. To sustntite the involvement of the 2-APB sensitive component provided y the RBL cell (Fig. 7), monovlent currents of store-operted CT1 re rpidly nd strongly inhiited y 2- APB (Fig. 7A). CT1 lockde y 2-APB is clerly visile (Fig 7B), s the negtive slope in the current-voltge reltionship typicl of CT1 disppered. In contrst, those currents crried y constitutively ctive CT1 chnnels in RBL cells responded y trnsient ~2% stimultion (Fig. 7C, D) similrly s previously oserved with constitutively ctive CT1 in HEK293 cells (compre Fig. 6C, D). Downloded from y guest on Jnury 1,

12 Discussion This report demonstrtes tht the regultory nd phrmcologicl properties of heterologously expressed CT1 re dependent on the cell type used s expression system. We compred the properties of CT1 chnnels generted y expression in Hek293 nd RBL cells. The typicl current-voltge reltionship of monovlent CT1 currents enled us to differentite them from endogenous I CRAC in RBL cells. Bsed on this iophysicl chrcteristic we identified store-operted CT1 currents in RBL cells tht were highly sensitive to lockde y 2-APB. In contrst, lrger current densities of CT1 currents in RBL cells suggesting higher CT1 expression levels generted constitutively ctive currents which were slightly stimulted y 2- APB. In Hek293 cells, CT1 currents were lwys constitutively ctive nd stimulted y 2- APB, independent of their current densities. Yue et l. (18) hve reported tht CT1 mnifests the pore properties nd store dependence of CRAC. However, very recently (19), it hs een demonstrted tht the ion selectivity, inwrd rectifiction, intrcellulr Mg 2+ sensitivity nd the effect of 2-APB re different etween CT1-medited current in Hek293 cells nd I CRAC in RBL cells. Hence, it hs een suggested tht the pore properties of CRAC nd CT1 re distinct, therey ruling out the possiility tht CT1 encompsses the full CRAC pore (18). In the present study, the negtive slope in the current-voltge reltionship of CT1 in DVF solution served s indiction to disinguish it from endogenous CRAC in RBL cells. This CT1 chrcteristic ws oserved in ll Downloded from y guest on Jnury 1, 219 trnsfected RBL cells independent of the current densities otined. The oserved iophysicl difference of CT1 nd CRAC chnnels in RBL cells is supported y previous report compring CT1 chnnels in Hek293 with CRAC chnnels in RBL cells (19). Bsed on this significnt iophysicl feture we conclude tht CT1 chnnels tht re formed in RBL cells ehve different to endogenous CRAC chnnels, lthough the regultory nd phrmcologicl properties of CT1 t lower current densities re identicl to those of CRAC chnnels (see elow). Thus, CRAC chnnels re pprently not homotetrmers of CT1 proteins. However, mutle heteromeriztion with nother yet unidentified component grdully chnging 12

13 chnnel chrcteristics in dependence of CT1 expression level cnnot e excluded leving the possiility tht CT1 is prt of the CRAC chnnel. The current densities otined y CT1 expression nd the cell type where it is expressed ffects regultory nd phrmcologicl properties of CT1. Current densities of constitutively ctive CT1 chnnels in RBL cells were significntly lrger tht those reched upon CT1 ctivtion y store depletion. Furthermore, CT1-current densities in Hek293 cells (-6.5±.8 pa/pf; n=6) comprle in size with those of store-operted CT1 currents in RBL cells (- 5.7±.3 pa/pf; n=17) never showed store-dependent ctivtion. Recently, store-dependent regultion of CT1 hs een reported in CHO cells (18), while constitutive ctivity hs een oserved when CT1 (19) or CT-L (31) hs een expressed in Hek293 cells. This celldependent ctivtion of CT1-derived currents is in line with our findings nd suggests the involvement of n uxiliry regultory component coupling CT1 ctivity to store depletion. The expression level of this CT1-intercting component oviously vries etween different cell types. Complementry, the level of CT1 chnnel expression my interfere with the degree of coupling to store depletion. In line with our oservtion it hs een recently suggested tht the level of TRPC3 chnnel expression ffects TRPC3 regultion y intrcellulr stores (32). Hence, high CT1 expression levels my significntly chnge the stoichiometry of CT1 chnnel interction with the 2-APB sensitive (see elow) component yielding non-intercting CT1 chnnels tht re constitutively ctive. Downloded from y guest on Jnury 1, 219 Here we demonstrte tht the phrmcologicl chrcteristics of CT1 chnnels chnge with ltertion in their regultory properties. Constitutively ctive CT1 currents responded to 75 µm 2-APB y slight increse, wheres store-operted CT1 were sustntilly inhiited. These effects occurred independent of the chrge crrier used oth in C 2+ -contining or DVF solutions. Our oservtions complement recent study (19) reporting 2-APB induced potentition of CT1-derived currents in Hek293 cells in contrst to 2-APB medited lockde of CRAC. The finding of the contrry effect of 2-APB on CT1 currents suggests the existence of two distinct trgets (3) in RBL cells mediting stimultion or inhiition. It is 13

14 tempting to speculte tht the stimultory effect is ccomplished y direct interction with CT1, wheres the inhiitory effect involves CT1 intercting component, which my provide the key function of current ctivtion y store depletion s primrily proposed for the IP 3 receptor (11). Recent reports (26-3) imply tht the trget for 2-APB lock is exposed to the extrcellulr medium suggesting trnsmemrne protein. The missing 2-APB sensitive component my e found mong the TRPC fmily possily within the more C 2+ -selective nd store-operted TRPC4 nd TRPC5 proteins (1, 11). In ccordnce, distinct ction of inhiitors such s 2-APB or Gd 3+ on htrpc3-medited ction entry hs een recently reported to result from different modes of ctivtion reflecting differences in sic chnnel structure (33). Additionlly, chnnel properties such s locker sensitivity, nd storedependent ctivtion my well e determined y uxiliry suunits, which do not directly contriute to the permetion pthwy. The ckground expression of such suunits is expected to vry etween expression systems, resulting in different properties of the chnnels generted y expression of CT1. Importntly, our results strongly suggest the existence of regultory protein, which on the one hnd suppresses the constitutive ctivity of CT1, nd on the other hnd enles regultion of the chnnels y the filling stte of intrcellulr stores. In ddition, the CT1 ssocited regultory component confers sensitivity of the chnnel to lock y 2-APB. Recently (34) it hs een suggested tht the trget for 2-APB is component of the coupling mchinery linking store depletion to current ctivtion. We suggest tht such Downloded from y guest on Jnury 1, 219 regultory component is present in RBL ut not in Hek293 cells nd my e shred mong CT1 nd endogenous CRAC chnnels. Model of CT1 current properties in RBL mst cells CT1 expression genertes highly C 2+ selective chnnels of different regultory nd phrmcologicl properties, depending on the type of expression system nd the current density. Endogenous CRAC chnnels in RBL cells re ctivted y store depletion nd locked y 2-APB (Fig. 8A). CT1 currents derived from moderte expression of CT1 chnnels resemle I CRAC in terms of ctivtion y store depletion nd inhiition y 2-APB. 14

15 However, sed on the different iophysicl properties, CRAC nd CT1 chnnels re not identicl (Fig. 8B). Lrger current densities otined y higher CT1 expression levels in RBL cells generte constitutively ctive CT1 chnnels tht re resistnt to the inhiitory effect of 2-APB. These CT1 chnnels re now present in excess to the 2-ABP sensitive component, thus lcking the interction required to couple chnnel ctivtion to store depletion (Fig. 8C). In future studies it will e importnt to identify the 2-APB sensitive component proly y the help of CT1. Downloded from y guest on Jnury 1,

16 Acknowledgements We would like to thnk S. Buchegger nd B. Kend for their excellent technicl ssistence. This work ws supported y the Ntionl nque funds (NB7538, NB9343) nd the Austrin Science Foundtion (P15387 to C.R.; P1495 nd SFB Biomemrnes F715 to K.G.). Downloded from y guest on Jnury 1,

17 References 1. Lewis, R.S. (1999) Adv. Second. Messenger Phosphoprotein Res. 33, Elliot, A.C. (21) Cell Clcium 3, Peterson, O.H., nd Fedirko, N.V. (21) Curr. Biol. 11, R Putney, J.W. (1986) Cell Clcium 7(1), Putney, J.W., nd McKy, R.R. (1999) BioEssys 21, Hoth, M., nd Penner, R. (1993) J. Physiol. 465, Zweifch, A., nd Lewis, R.S. (1993) Proc. Ntl. Acd. Sci. 9(13), Lepple-Wienhues, A., nd Chln, M.D. (1996) Biophys J. 71(2), Prekh, A.B., nd Penner, R. (1997) Physiol. Rev. 77, Clphm, D.E., Runnels, L.W., nd Strüing, C. (21) Nture Reviews Neuroscience 2, Montell, C. in Science s STKE : Montell, C., Birnumer, L., Flockerzi, V., Bindels, R.J., Bruford, E.A., Cterin, M.J., Clphm, D.E., Hrmeck, C., Heller, S., Julius, D., Kojim, I., Mori, Y., Penner, R., Prwitt, D., Schrenerg, A.M., Schultz, G., Schimizu, N., nd Zhu, M.X. (22) Mol. Cell 9, Hrdie, R.C. nd Minke, B. (1993) Trends Neurosci.1, Vc, L., Sinkins, W.G., Hu, Y., Kunze, D.L., nd Schilling, W.P. (1994) Am. J. Physiol. 267, C Xu, X.Z., Li, H.S., Guggino, W.B., nd Montell, C. (1997) Cell 89, Peng, J.-B., Chen X.-Z., Berger. U.V., Vssilev P.M., Tsukguchi, H., Brown, E.M., nd Hediger, M.A. (1999) J. Biol. Chem. 274, Hoenderop, J.G.J., Muller, D., Suzuki, M., vn Os, C.H., nd Bindels, R.J. (2) Current Opinion Nephrol. Hypert. 9(4), Yue, L., Peng, J.B., Hediger, M.A., nd Clphm, D.E. (21) Nture 41, Voets, T., Prenen, J., Fleig, A., Vennekens, R., Wtne, H., Hoenderop, J.G., Bindels, R.J., Droogmns, G., Penner, R., nd Nilius, B. (21) J. Biol. Chem.276, Hmil, O.P., Mrty, A., Neher, E., Skmnn, B., nd Sigworth, F.J. (1981) Pflügers Archiv 391, Niemeyer, B.A., Bergs, C., Wissench, U., Flockerzie, nd Trost, C. (21) Proc. Ntl. Acd. Sci. 98, Reinsprecht, M., Rohn, M.H., Spdinger, R.J., Pecht, I.., Schindler, H. nd Romnin, C. (1995) Mol. Phrmcol. 47, Runnels, L.W., Yue, L., nd Clphm, D.E. (21) Science 291, Downloded from y guest on Jnury 1,

18 24. Ndler, M.J., Hermosur, M.C., Ine, K., Perrud, A.L., Zhu, Q., Stokes, A.J., Kuroski T., Kinet J.P., Penner. R., Schrenerg, A.M., nd Fleig, A. (21) Nture 411, Hermosur, M.C., Monteilh-Zoller M.K., Schrenerg, A.M., Penner, R. (22) J. Physiol. 539, M, H.T., Ptterson, R.L., vn Rossum, D. B., Birnumer, L., Mikoshi, K., nd Gill, D.L. (2) Science 287, Kukkonen, J.P., Lund, P.-E., nd Akermn, K.E.O.(21) Cell Clcium 3, Bkwoski, D., Glitsch, M.D., nd Prekh, A.B. (21) J.Physiol. 532, Brun, F.J., Brod, L.M., Armstrong, D.L., nd Putney, J.W., Jr. (21) J. Biol.Chem. 276, Prkriy, M., nd Lewis, R.S. (21) J. Physiol. 536, Wissench, U., Niemeyer, B., Fixemer, T., Schneidewind, A., Trost, C., Cvlie, A., Reuss, K., Meese, E., Bonkhoff, H., nd Flockerzi, V. (21) J. Biol. Chem. 276, Vzquez, V., Lievremont, J.-P., Bird, G.St. J., nd Putney Jr., J.W. (21) Proc. Ntl. Acd. Sci. 98, Trek, M., Bird, G.St.J., McKy, R.R., nd Putney Jr., J.W.(22) M M, H.-T., Venktchlm, K., Prys, J.B., nd Gill. D.L.(22) J. Biol. Chem 277(9), Downloded from y guest on Jnury 1,

19 Figure legends Fig. 1. Expression of CT1 in Hek293 cells yields constitutively ctive C 2+ inwrd current. Time-course of whole-cell inwrd currents determined t 74 mv during voltge rmp from 9 mv to +9 mv employing the voltge protocol s specified in (A), (C) nd (E). Voltge rmps re pplied every 5s. Corresponding current trces from representtive experiments re shown in (B), (D) nd (F), respectively, mrked with nd denoting their time-point of recording. All experiments were performed >24 hours following trnsfection. Fig. 2. Expression of CT1 in RBL cells increses current density of store depletionctivted C 2+ inwrd current. Time-course of whole-cell inwrd currents in (A, E) mock- nd (C, E) CT1-trnsfected RBL cells re visulized y ppling voltge rmps ( 9 mv to +9 mv over 1s) every 5s from holding potentil of mv. Current ctivtion ws induced y pssive (A-D) or ctive (E) store depletion. Corresponding current trces recorded in representtive experiments t the time-points indicted in A nd C re shown in (B) nd (D), respectively. The insets in A nd C depict the dely to current ctivtion nd the time to rech mximum pek current. (F) CT1-expressing RBL cells exhiited significnt (p<.1) lrger inwrd current densities thn Downloded from y guest on Jnury 1, 219 mock-trnsfected RBL cells, independent of the mode of store-depletion. All experiments were performed >24 hours following electroportion. Fig. 3. Both constitutively ctive nd store-dependent CT1 chnnels re oserved in RBL mst cells. Time-course of constitutively ctive CT1 currents in RBL cells (A) recorded t 74mV y pplying voltge rmp (-9 to +9mV over 1s) from holding potentil of mv. CT1- trnsfected RBL cells were studied 8-12 h fter electroportion. 5% of trnsfected cells 19

20 exhiited constitutively ctive inwrd currents. Corresponding current trces from representtive experiment re shown in (B) for the time-points (, ) indicted in A. The current densities t s nd 15s fter otining whole-cell configurtions (C) re compred etween mock-trnsfected, constitutively ctive CT1 (8-12 hours fter electroportion) nd store-operted CT1 RBL cells (> 24 hours fter electroportion). Current densities derived from constitutively ctive CT1 chnnels (t s) re significntly (p <.1) lrger thn those otined upon mximum ctivtion (t ~15s) of store-operted CT1 chnnels in RBL cells. Fig. 4. CT1 -derived currents in Hek293 cells cn e distinguished from I CRAC in RBL cells y their current-voltge reltionships in divlent free solution. Time-courses of whole-cell inwrd currents of CT1-HEK293 (A) nd RBL (C, E) cells monitored y pplying repetitive voltge rmps (-9 to +9mV over 1ms) from holding potentil of mv. Exchnge of extrcellulr solution y divlent free (DVF) solution is followed y rpid increse in current density s mesured t 74mV. Intrcellulr solutions contin 3 mm Mg 2+ (A-D) or 1mM Mg 2+ (E, F) to evlute the contriution of LTRPC7. Time-courses re reltive to perfusion strt with DVF solution. Current-voltge reltionships of corresponding, representtive experiments (B, D, F) re shown for divlent-contining extrcellulr () nd DVF () solution. All experiments were performed >24 hours following trnsfection. Downloded from y guest on Jnury 1, 219 Fig. 5. Both constitutively ctive nd store-operted CT1 chnnels depict the typicl current-voltge reltionship in DVF solution. Time-courses of whole-cell inwrd currents of constitutively ctive CT1 (A), store-operted CT1 t 3mM (C) nd 1 mm (E) intrcellulr Mg 2+ in RBL cells otined in divlentcontining nd DVF solution. Time-courses re shown reltive to perfusion strt with DVF solution. Experiments in (A) nd (C, E) were performed 8-12 hours nd >24 hours, respectively, following electroportion. Corresponding current trces from representtive 2

21 experiments re shown in (B), (D) nd (F) recorded t the time-points (, ) s indicted in A, C nd E, respectively. Voltge protocols pplied to record CT1-currents in RBL cells re identicl to those used in Fig 4. Fig. 6. Store-operted CT1 chnnels in RBL cells re inhiited y 2-APB in contrst to constitutively ctive CT1 chnnels in Hek293 cells. Time-courses of whole-cell inwrd currents in CT1-RBL (A, E) nd CT1-Hek293 (C, F) cells displying the effect of extrcellulr ppliction of 75 µm 2-APB. CT1 nd CRAC currents re evoked y 2ms voltge rmp ( 9 to +9mV) every 5 s from holding potentil of +7 mv. The time-scle in A nd C is reltive to the strt of 2-APB perfusion set to 5s nd 15s, respectively. All experiments were performed >24 hours following trnsfection. Corresponding current trces from representtive experiments re shown in (B) nd (D) t the time-points (, ) indicted in A nd C respectively. Pre-incution of CT1- RBL (E, +2-APB) nd CT1-HEK (F, +2-APB) cells with 75 µm 2-APB ws performed for 1-2 min. Fig 7. 2-APB exerts n opposite effect on store-operted nd constitutively ctive CT1 chnnels in RBL cells. Time-courses of store-operted (A) nd constitutively ctive (C) CT1 currents from RBL Downloded from y guest on Jnury 1, 219 cells in DVF solution re mesured y voltge rmp ( 9 mv to +9 mv over 1ms) pplied every 2s from holding potentil of mv. Time scle in A nd C is reltive to perfusion strt with 75µM 2-APB. In (C) inwrd current densities immeditely efore ddition of 2-APB re set to 1%. Experiments in (A) nd (C) were performed >24 hours nd 8-12 hours following electroportion. Current-voltge reltionships from representtive experiments re shown in (B) nd (D) t the time points (, ) indicted in A nd C. Fig. 8. Model of CT1 current properties in RBL mst cells 21

22 Figure 1 A HP = mv rmp 1 ms (n = 6) B Potentil [mv] C 5 HP = + 7 mv -5 rmp 1 ms (n =3) D Potentil [mv] Downloded from y guest on Jnury 1, 219 E 2 F HP = + 7mV rmp 2ms -6 (n = 7) Potentil [mv] 22

23 A C Figure (n = 14) 15 (n = 17) Time[s] RBL Dely Time to Pek store-operted CT1-RBL -6 (n = 3) Dely Time to Pek (n = 3) B D Potentil [mv] Potentil [mv] Downloded from y guest on Jnury 1, 219 E µM IP 3 + 6nM C 2+ RBL (n = 4) CT1-RBL (n = 4) F mximum current [pa/pf] (n = 17) (n = 4) (n = 14) (n = 4) pssive ctive pssive ctive RBL CT1-RBL 23

24 Figure 3 A 2 constitutively ctive CT1-RBL (8-12 Std. fter electroportion) -2-4 (n = 5) C (n = 14) B (n = 5) Potentil [mv] (n = 17) Downloded from y guest on Jnury 1, 219 s 15s s 15s s 15s RBL CT1-RBL CT1-RBL (8-12h) (>24h) 24

25 Figure 4 A constitutively ctive CT1-HEK DVF 3 mm [Mg 2+ ] i (n = 4) B Potentil [mv] C E RBL 3mM [Mg 2+ ] i DVF (n = 3) D F Potentil [mv] RBL 1 2 DVF 1mM [Mg 2+ ] -1 i -2-2 (n = 4) Potentil [mv] Downloded from y guest on Jnury 1,

26 Figure 5 A constitutively ctive CT1-RBL 3mM [Mg 2+ ] i DVF (n = 5) B Potentil [mv] C E store-operted CT1-RBL 5 DVF -5 3mM [Mg 2+ ] i (n = 4) store-operted CT1-RBL DVF 1 mm Mg (n = 4) D F Potentil [mv] Potentil [mv] Downloded from y guest on Jnury 1,

27 Figure 6 A store-operted CT1-RBL RBL (n = 3) -8-1 CT1-RBL (n = 7) APB B 2-APB sensitive current 4 RBL ( - ) -4-8 CT1-RBL ( - ) Potentil [mv] C Current [%] E constitutively ctive CT1-HEK store-operted CT1-RBL 2-APB (n = 3) + 2-APB (n=5) - 2-APB (n =3) D F Potentil [mv] constitutively ctive CT1-HEK - 2-APB (n = 5) + 2-APB (n = 5) Downloded from y guest on Jnury 1,

28 Figure 7 A store-operted CT1-RBL DVF 2-APB (n = 4) B Potentil [mv] C Current [%] constitutively ctive CT1-RBL DVF 2-APB (n = 4) D Potentil [mv] Downloded from y guest on Jnury 1,

29 Figure 8 A CRAC chnnel locked y 2-APB B Store-operted CT1 chnnel locked y 2-APB C 2+ store C 2+ store C Constitutively ctive CT1 chnnel stimulted y 2-APB CRAC chnnel CT1 chnnel Component coupling ctivtion to store-depletion mediting lockde y 2-APB C 2+ store store-operted C 2+ influx constitutively ctive C 2+ influx 29 Downloded from y guest on Jnury 1, 219

30 Store-depletion ctivted CT1 currents in RBL mst cells re inhiited y 2-APB: Evidence for regultory component tht controls ctivtion of oth CT1 nd CRAC chnnels Riner Schindl, Heike Khr, Ingrid Grz, Klus Groschner nd Christoph Romnin J. Biol. Chem. pulished online My 14, 22 Access the most updted version of this rticle t doi: 1.174/jc.M2372 Alerts: When this rticle is cited When correction for this rticle is posted Click here to choose from ll of JBC's e-mil lerts Downloded from y guest on Jnury 1, 219