Calcium influx factor directly activates store-operated cation channels in vascular smooth muscle cells.

Recently, we described a novel 3-pS Ca(2+)-conducting channel that is activated by BAPTA and thapsigargin-induced passive depletion of intracellular Ca(2+) stores and likely to be a native store-operated channel in vascular smooth muscle cells (SMC). Neither Ca(2+) nor inositol 1,4,5-trisphosphate or other second messengers tested activated this channel in membrane patches excised from resting SMC. Here we report that these 3-pS channels are activated in inside-out membrane patches from SMC immediately upon application of Ca(2+) influx factor (CIF) extracted from mutant yeast, which has been previously shown to activate Ca(2+) influx in Xenopus oocytes and Ca(2+) release-activated Ca(2+) current in Jurkat cells. In bioassay experiments depletion of Ca(2+) stores in permeabilized human platelets resulted in the release of endogenous factor, which activated 3-pS channels in isolated inside-out membrane patches excised from SMC and exposed to permeabilized platelets. The same 3-pS channels in excised membrane patches were also activated by acid extracts of CIF derived from human platelets with depleted Ca(2+) stores, which also stimulated Ca(2+) influx upon injection into Xenopus oocytes. Specific high pressure liquid chromatography fractions of platelet extracts were found to have CIF activity when injected into oocytes and activate 3-pS channels in excised membrane patches. These data show for the first time that CIF produced by mammalian cells and yeast with depleted Ca(2+) stores directly activates native 3-pS cation channels, which in intact SMC are activated by Ca(2+) store depletion.

Depletion of intracellular Ca 2ϩ stores activates store-operated (capacitative) Ca 2ϩ influx in a variety of nonexcitable cells (1)(2)(3)(4)(5). However, the nature of the store-operated channels (SOC) 1 in different cells as well as the mechanism of their activation remain obscure. Two major types of SOC have been described so far: native Ca 2ϩ release-activated Ca 2ϩ selective (CRAC) channels found in some nonexcitable cells (6,7) and certain members of the TRP channel family (8 -10).
Recently, we found a novel Ca 2ϩ -conducting channel that is likely to be a native store-operated channel responsible for capacitative Ca 2ϩ influx and contraction in vascular smooth muscle cells (SMC). 2 Poor cation selectivity of this 3-pS channel distinguishes it from the CRAC channel (6,7), and although it resembles some channels from the TRP family, none of them described so far has such low single channel conductance (8 -10). These 3-pS nonselective cation channels rarely opened in resting SMC but were activated upon depletion of intracellular Ca 2ϩ stores induced by either chelation of intracellular Ca 2ϩ with BAPTA or by thapsigargin (TG)-induced inhibition of sarco/endoplasmic reticulum Ca 2ϩ -ATPase (SERCA). Neither excision of membrane patches from resting (untreated) SMC nor a variety of second messengers (including Ca 2ϩ , InsP 3 , InsP 4 , GTP␥S, cAMP, cGMP, ATP, and ADP) activated these channels in inside-out membrane patches.
These native 3-pS channels provide a powerful new tool to address the mechanism of activation of these channels in SMC and specifically the link to depletion of intracellular Ca 2ϩ stores. In previous work, partially purified extracts of calcium influx factor (CIF) from pmr1 yeast, which are genetically deficient in SERCA (and therefore depleted in organellar Ca 2ϩ ), were shown to evoke Ca 2ϩ influx when injected into Xenopus oocytes and to activate I CRAC when introduced via a patch pipette into Jurkat cells (12). Here we tested if the 3-pS channels from SMC could be activated directly by this putative CIF (12)(13)(14)(15) or if conformational coupling with InsP 3 receptors in the stores is required for their activation (16 -18). We also addressed the question of whether or not the channel is already present in the plasma membrane of resting SMC or if the channel is delivered and incorporated into the membrane only upon depletion of the stores (19,20).
We show that the 3-pS channels are immediately activated in inside-out membrane patches by the putative CIF extracted from either SERCA-deficient pmr1 yeast or from human platelets with depleted Ca 2ϩ stores. This is the first evidence of direct activation of native store-operated channels in inside-out membrane patches by a putative CIF that was chemically extracted or obtained in bioassay from different cell types following depletion of their Ca 2ϩ stores.

EXPERIMENTAL PROCEDURES
SMC Preparation-SMC were isolated from mouse thoracic aorta as described before 2 using collagenase (4 mg/ml), elastase (2 mg/ml), and trypsin inhibitor (2 mg/ml), (1-h treatment at 37°C), placed on coverslips in 1% Dulbecco modified Eagle medium (Life Technologies, Inc.) supplied with 1% fetal bovine serum (Sigma), and kept at 37°C in 5% CO 2 for 2-5 days. Under these conditions, SMC attached to the coverslips and spread slightly attaining a spindle-like shape but did not divide. These SMC stained positively for ␣-actin.
Electrophysiology-Single channel currents were recorded in insideout membrane patches from resting (unstimulated) SMC as recently described (21) with a low noise Axopatch 200B amplifier (Axon Instruments). To improve the signal-to-noise ratio, pipettes were coated with Sylgard (Dow Corning Corp.) and polished to a resistance of 10 -20 M⍀ (when filled with high sodium pipette solution). pCLAMP 6 software (Axon Instruments) was used for data acquisition and analysis. Data were filtered at 1 kHz and stored for later analysis. Representative traces of single channel currents were later additionally filtered at 100 -200 Hz for better visual resolution of a 3-pS single channel on the figures. Liquid junction potential was corrected. Experiments were conducted at 20 -22°C.
The open channel probability (NP o ) was analyzed and plotted over time to illustrate the time-course of channel activity. The amplitude of single channel currents was analyzed using all-point histograms or amplitude histograms obtained from the event list, because both methods gave the same single channel current amplitude in our experiments as was described before. 2 Standard bath solution contained: 140 mM NaCl, 2.8 mM KCl, 2 mM MgCl 2 , 5.5 mM glucose, 10 mM HEPES (pH 7.4). Pipette solution contained: 140 mM NaCl, 10 mM tetraethylammonium chloride, 0.2 mM EGTA (pH 7.4). In some experiments Ca 2ϩ (1 or 10 mM) was added to the pipette or bath solutions (instead of EGTA). The pipette solutions contained 100 M niflumic acid and 100 nM iberiotoxin to suppress Ca 2ϩ -dependent Cl Ϫ and K ϩ currents, respectively. In control experiments neither of these inhibitors affected 3-pS channels or TG-induced Ca 2ϩ influx in SMC. 2 CIF Preparations-Acid extracts from wild type and pmr1 mutant yeast were prepared as described previously (12). Human platelets were obtained from the regional Red Cross following 5-7 days of storage. Unstimulated platelets were kept at room temperature. The Ca 2ϩ stores in platelets were depleted by either TG (2 M, 20 min), overnight hypothermic treatment (4°C), or by a combination of both stimuli. Acid extracts from platelets were prepared as described previously (12). A starting platelet count of about 10 11 cells yielded crude extract, which corresponded to ϳ5 ϫ 10 11 platelets/ml. CIF extracts were diluted 1:30 and applied at a final concentration corresponding to about 1.7 ϫ 10 10 platelets/ml. Anion exchange HPLC purification of the crude extracts was performed using a Keystone Scientific Partisil 10 SAX column eluted with a linear salt and pH gradient from 5 to 750 mM (NH 4 )H 2 PO 4 and from pH 2.8 to 3.7, respectively.
Xenopus Oocyte Assay-CIF activities were assayed by microinjection of extracts into fura-2 loaded albino Xenopus laevis oocytes as described (12). Changes in intracellular Ca 2ϩ were measured on an Olympus IX70 inverted microscope through a 10x UplanAPO objective, NA ϭ 0.17, equipped with a rapid excitation filter changer alternating between 340 and 380 nm (Ludl Electronics) and a CCD camera (Sensys, Photometrics). Changes in fluorescence were analyzed in a 600 ϫ 400 m area of the oocyte close to the injection site. Data are expressed as the 340/380 nm ratio.
Bioassay of CIF from Permeabilized Platelets-Human platelets were concentrated from platelet-rich plasma and filtered through Sepharose-2B gel into regular HEPES buffer: 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 3.3 mM NaH 2 PO 4 , 5.5 mM glucose, 3.8 mM HEPES (pH 7.4), 0.1 mM aspirin, 6 milliunits/ml apyrase. Filtered platelets were transferred into an intracellular solution containing: 110 mM KCl, 10 mM NaCl, 1 mM KH 2 PO 4 , 5 mM KH 2 CO 3 , 20 mM HEPES (pH 7.1), 2 mM Mg-ATP, ATP-regenerating system (creatine phosphate 5 mM creatine phosphokinase 15 units/ml) and permeabilized with 50 g/ml saponin for 10 min, washed, and resuspended in saponin-free intracellular solution. Within the next 30 min the suspension of permeabilized platelets was added to the chamber (at a concentration of ϳ1.75 ϫ 10 9 platelets/ml) containing an inside-out membrane patch excised from SMC. Before application to the membrane patches, each preparation of permeabilized platelets was tested for TG (1 M)-induced emptying of their stores which was monitored as described earlier (22).
Platelet Lysate-Approximately 10 11 platelets (filtered as described above) were exposed to ϩ4°C for 12 h, then concentrated into 3 ml of standard bath solution used for patch-clamp (see above), and sonicated in a Branson sonifier at 40 watts, 4 times for 5 s (at ϩ4°C). Lysate was centrifuged at ϩ4°C for 25 min at 20,000 ϫ g. The supernatant was collected and used during the day. It was applied to inside-out membrane patches at a concentration corresponding to ϳ2.5 ϫ 10 9 cells/ml.
Drugs-All the drugs were from Sigma. Statistics-The data are presented as mean Ϯ S.E. with n showing the number of experiments in the text. Statistical significance was assessed using the t test and ANOVA (analysis of variance). Values of p Ͻ 0.05 were considered to be significant.

RESULTS
To assess the possible mechanism of activation of the 3-pS nonselective cation channels, the inside-out membrane patches with little or no single channel activity were excised from resting SMC. Application of CIF extracts from genetically modified (SERCA-deficient) pmr1 yeast (12) (at a final concentration corresponding to ϳ4.5 ϫ 10 8 cells/ml) to the cytoplasmic side of membrane patch immediately activated 3-pS channels in 16 of 18 experiments (Fig. 1). Although the single channel currents were very small (about 0.3 pA at 100 mV), they could be clearly resolved (Fig. 1a). The histograms of the current amplitude (Fig. 1b) revealed the presence of several (two to five) channels with the same current amplitude in each membrane patch. Fig. 1c shows an example of CIF-activated single channel currents in an inside-out membrane patch recorded at different membrane potentials with all-point histograms showing single channel current amplitudes. CIF-activated channels had the same electrophysiological characteristics as those previously described for the native 3-pS channel activated by BAPTA and TG in intact SMC. 2 The I/V relationship of the CIF-activated channel (Fig. 1d) was linear with a slope conductance of 3.3 Ϯ 0.4 pS (n ϭ 7) in the absence of extracellular Ca 2ϩ , and 3.2 Ϯ 0.2 pS (n ϭ 6) and 3.1 Ϯ 0.4 pS (n ϭ 3) in the presence of 1 and 10 mM of Ca 2ϩ , respectively. The dependence on membrane potential of the NP o of CIF-activated channels, which did not change at negative, but significantly increased at high positive membrane potentials (Fig. 1d), was identical to that of native store operative 3-pS channels. 2 Thus, CIF extracts activated 3-pS channels with the properties indistinguishable from the channels activated by TG and/or BAPTA in intact SMC. In contrast, similar extracts from wild-type yeast, which do not produce CIF (12), did not activate 3-pS channels in inside-out membrane patches (in four of four experiments).
We also sought a relatively abundant mammalian source for CIF preparations. In recent publications we described capacitative Ca 2ϩ influx in human platelets and showed its SERCAdependent regulation by nitric oxide (22). Because of these results and the availability of relatively large quantities of platelets, we initiated a series of experiments to determine if a CIF similar to that derived from yeast (12) could also be produced by human platelets and if it also activates 3-pS channels in excised membrane patches from SMC.
The first series of experiments was designed to bioassay CIF produced by human platelets during depletion of their Ca 2ϩ stores. The cytoplasmic side of an inside-out membrane patch from SMC was exposed to suspension of permeabilized platelets (Fig. 2a), and the activity of the 3-pS channel was monitored before and during TG-induced depletion of the platelets' Ca 2ϩ stores. Fig. 2b shows that the addition of permeabilized platelets alone (1.75 ϫ 10 9 platelets/ml) did not activate channels in the patch (NP o was 0.010 Ϯ 0.006 before and 0.011 Ϯ 0.004 after addition of platelets). However, the subsequent addition of TG to permeabilized platelets activated 3-pS channels in the isolated patch (in five of six experiments), and at the peak of activity NP o reached 0.23 Ϯ 0.06 (at Ϫ100 mV, n ϭ 5). The time course and peak activity of the channels in this bioassay system were similar to the time course of TG-induced channel activation in cell-attached patches in intact SMC. 2 The channel could be blocked by a high concentration of La 3ϩ (2 mM) applied from the cytoplasmic side (n ϭ 5, Fig. 2b). The addition of TG to inside-out patches in the absence of platelets did not produce activation of 3-pS channels (n ϭ 12). Thus, it appears that upon depletion of Ca 2ϩ stores, a factor is produced and released from the platelets through their permeabilized plasma membrane that is capable of activating 3-pS channels in isolated membrane patches.
In another series of experiments, to avoid the use of TG, platelets were activated by exposure to low temperature, and the lysates prepared from the platelets were applied to insideout membrane patches from SMC. Application of the lysate at concentration equivalent to ϳ2.5 ϫ 10 9 platelets/ml to insideout membrane patches immediately activated 3-pS channels in 8 of 12 experiments (Fig. 2c). When diluted 10 times (equivalent to 2.5 ϫ 10 8 cells/ml) the same lysate was without an effect in five of five experiments.
Next, acid extracts were prepared from platelets and partially purified as described previously (12). To test if extracts from platelets whose stores were depleted either by TG or exposure to cold (23) have similar CIF activity as those obtained from SERCA-deficient yeast, platelet extracts were injected into Xenopus oocytes, and Ca 2ϩ influx was assessed. As shown in Fig. 3a, extracts from activated platelets caused a time-dependent increase in intracellular Ca 2ϩ that spread across the oocyte from the point of injection similar to that described for CIF derived from yeast and Jurkat cells (12). Fig.  3b shows that the Ca 2ϩ rise strictly depended on the presence of extracellular Ca 2ϩ and was maximal in response to extracts from platelets that had been activated by both TG and exposure to cold. Extracts prepared similarly but from nonactivated platelets showed no significant Ca 2ϩ rise in the same assay (Fig. 3b).
To determine if CIF extracts from platelets could also activate 3-pS channels, they were applied (at concentration equivalent to 1.7 ϫ 10 10 platelets/ml) to inside-out membrane patches from resting SMC. Fig. 3c shows that partially purified extract prepared from resting platelets (no. 5) did not activate any channels in the patch (n ϭ 9). However, the subsequent addition of extracts from TG-treated platelets (no. 1) immediately activated 3-pS channels in five of seven membrane patches (Fig. 3c). Importantly, when CIF extract was applied at a concentration equivalent to 1.7 ϫ 10 10 platelets/ml (nearly 10 times higher than used in permeabilized platelet bioassay), NP o of 3-pS channels reached 0.96 Ϯ 0.19 (at -100 mV, n ϭ 5), which was about four times higher than NP o under similar conditions in the bioassay experiments (Fig. 2c). The activity of the channels in the presence of CIF extracts from platelets was so high that even at negative membrane potentials more than one single channel current level was observed (Fig. 3d).
We then determined if further purification steps of the acid extract from platelets would provide evidence for a single molecular species capable of inducing Ca 2ϩ influx in oocytes and also activating single 3-pS channels in membrane patches isolated from SMC. The partially purified acid extracts from activated platelets utilized in Fig. 3 were subjected to anion exchange HPLC. The absorbance at 262 nm of the column effluent is shown in Fig. 4a. Initially, pooled fractions were tested for activity in the oocyte assay, and eventually two half-minute fractions (no. 49 and 50 shown in the inset in Fig.  4a) were found to activate Ca 2ϩ influx in oocytes (Fig. 4b). Fraction no. 51 was found to have no activity in the oocyte assay (Fig. 4b). When these same fractions were applied to FIG. 3. CIF extracts purified from activated platelets activate Ca 2؉ influx in Xenopus oocytes and single channel currents in inside-out membrane patches from SMC. a, pseudo-colored ratiometric images of an albino Xenopus oocyte preloaded with fura-2 following injection at t ϭ 0 of 14 nl of crude acid extract prepared from cold-and TG-treated human platelets. Starting concentration of crude extract corresponded to ϳ5 ϫ 10 11 platelets/ml. Increases in the 340/380 nm ratio relative to those at the time of injection are shown. External Ca 2ϩ was 5 mM. The asterisk in the first frame denotes the point of injection. Data are representative of 10 experiments with independent preparations. b, changes in intracellular Ca 2ϩ (shown as fura-2 340/380 nm fluorescence ratio) in Xenopus oocytes following injection of extracts prepared from platelets: trace 1, treated with 2 M TG for 20 min; trace 2, exposed to cold overnight; traces 3 and 4, exposed to cold and then treated with TG; trace 5, left untreated and kept at room temperature. All the traces except trace 4 were recorded in the presence of 5 mM Ca 2ϩ in the bath. c, the plot of NP o in inside-out membrane patch (at Ϫ100 mV) before and after application of the same acid extracts (at final concentrations corresponding to ϳ1.7 ϫ 10 10 cells/ml) from nontreated resting platelets (no. 5) and from platelets treated with cold and TG (2 M for 20 min) (no. 1) as those described in b. All-point current histogram at the peak of channel activity is shown on the right.
inside-out membrane patches isolated from SMC, fraction no. 51 caused no activation of 3-pS channels (Fig. 4c). However, the subsequent addition of fraction no. 50 immediately activated 3-pS channels, and their activity was so high that up to three single channel current levels could be seen even at negative membrane potentials (Fig. 4, c and d, in five of six experiments). DISCUSSION This study demonstrates for the first time that CIF from mammalian and yeast cells with depleted Ca 2ϩ stores directly activates native 3-pS cation channels, which in intact SMC are activated by Ca 2ϩ store depletion. The results presented here allow us to address several important issues related to the mechanism of store-operated Ca 2ϩ influx in vascular SMC and possibly in other cell types.
Strong evidence was obtained that finalized the proof that the 3-pS channel in vascular SMC is a native store-operated channel. Indeed, this Ca 2ϩ -conducting channel is activated in intact SMC following depletion of their stores (achieved by intracellular Ca 2ϩ chelation with BAPTA or TG-induced inhibition of SERCA-dependent Ca 2ϩ uptake). 2 Demonstration of the direct activation of 3-pS channels by CIF produced upon depletion of intracellular Ca 2ϩ stores (but not other known second messengers) establishes a store-dependent mechanism for 3-pS channel activation in SMC.
Our experiments also directly demonstrate that a native SOC is constitutively present in plasma membrane of SMC. For its activation it does not need to be delivered to or incorporated into the plasma membranes following depletion of the stores, as was previously proposed (19,20). Indeed, 3-pS channels were inactive in silent membrane patches isolated from resting SMC, but up to five channels were immediately activated when CIF was applied to the inside of the membrane patches. Interestingly, after activation in intact SMC, 3-pS channels remain active even when excised from SMC, implying that activation of the channels results in a stable modification rather than in an easily reversible interaction that is common to a variety of second messengers. Excision from the cell may also eliminate the mechanisms responsible for channel deactivation. Simi-larly persistent activity has been recently reported for a storeoperated current (I SOC ) in macro patches excised from Xenopus oocytes (19).
Several attractive models for store-dependent regulation of plasma membrane channels have been proposed and supported by strong experimental evidence in nonexcitable cells (for recent reviews see Refs. 4, 5, and 24), although all models still face unresolved questions and the mechanism of SOC regulation remains obscure.
Our study for the first time provided solid evidence for a CIF-mediated mechanism of store-operated Ca 2ϩ entry at the level of single store-operated channels activated by CIF in isolated membrane patches. CIF with identical biological activities was found to be produced upon store depletion in both yeast and human platelets. Importantly, CIF activates single SOC in isolated membrane patches independently of whether it is released by permeabilized platelets during store depletion or chemically extracted from platelets after store depletion. The results with permeabilized platelets dismiss the concern about the possible induction of nonphysiological activity as a result of chemical extraction. They also clearly show, at least for the native SOC from SMC, that the direct physical coupling of this SOC to intracellular Ca 2ϩ stores during their depletion is not required for channel activation. The question that remains open is whether the mechanism of SOC regulation is the same for different store-operated channels and different cell types.
Strong arguments against a CIF-based model of SOC activation have been previously raised by some experimental data that were apparently inconsistent with a freely diffusible and long lived messenger traveling from Ca 2ϩ stores to the plasma membranes (19,25,26). Other recent data have provided compelling evidence for the importance of secretory processes and vesicle docking and/or membrane fusion in the activation of store-operated Ca 2ϩ influx (19,27). In addition, direct interaction between the InsP 3 receptor and the TRP3 channel has been described (11, 16 -18) and calls into question the role of a diffusible CIF in activation of store-operated channels (24). Although multiple mechanisms of activation of Ca 2ϩ influx in different cell types are possible, it remains attractive to con- FIG. 4. Specific HPLC fraction of CIF extract purified from activated platelets activates Ca 2؉ influx in Xenopus oocytes and single channel currents in inside-out membrane patches from SMC. a, OD 262 absorbance of the effluent of a Partisil 10 anion exchange column loaded with 200 l of coldand TG-treated human platelet extract. Chromatography was performed as described under "Experimental Procedures." 30-s fractions were collected. An expanded view of the region containing fractions 48 -51 is shown in the inset. b, increases in fluorescence ratio in Xenopus oocytes preloaded with fura-2 AM following injection of 14 nl of fractions no. 50 and 51 (described in a) in the presence of 5 mM extracellular Ca 2ϩ . The result of injection of fraction no. 50 in the absence of extracellular Ca 2ϩ is also shown (#50/ 0Ca). c, the plot of NP o in an inside-out membrane patch (at Ϫ100 mV) before and after application of HPLC-purified platelet CIF fractions no. 51 and 50. The panel on the right represents an all-points current histogram at the peak of channel activity.
sider new unifying models that could account for much of the presently available experimental data. For instance, it is possible that in intact cells vesicular transport and/or docking, rather than simple diffusion, is required to deliver CIF from depleted Ca 2ϩ stores to the vicinity of the plasma membrane. Physiological process could be more complicated than the free diffusion observed upon injection of exogenous CIF into the cell. Another possibility could be that a portion of CIF-producing sarcoplamic/endoplasmic reticulum needs to be docked near the plasma membrane for sufficient levels of CIF to escape sequestration or cytoplasmic degradation to reach and activate SOC. Also, cytoplasmic CIF and not Ca 2ϩ store depletion per se might be necessary for altering the association of the InsP 3 receptor with TRP channels. In any case, our data show that a putative CIF is sufficient for the activation of native SOC in isolated membrane patches. It is our strong hope that the utilization of excised membrane patches containing single SOC will facilitate further investigations of the molecular identity of CIF and aid in the elucidation of the processes responsible for activation of store-operated Ca 2ϩ influx.