Distinct Contributions of Orai1 and TRPC1 to Agonist-Induced [Ca2+]i Signals Determine Specificity of Ca2+-Dependent Gene Expression

Regulation of critical cellular functions, including Ca2+-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca2+ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca2+]i due to intracellular Ca2+ release and Ca2+ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca2+]i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca2+]i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca2+ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca2+ store depletion in these cells, it is not clear how the cells decode individual Ca2+ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca2+]i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca2+ entry generates [Ca2+]i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca2+ entry generates sustained [Ca2+]i elevation at high [CCh] and contributes to frequency of [Ca2+]i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca2+ dependent gene expression pathways, consistent with the different patterns of [Ca2+]i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca2+]i generated by Orai1, independent of global [Ca2+]i changes or TRPC1-mediated Ca2+ entry. In contrast, Ca2+ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca2+ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca2+-dependent gene expression pathways.


Introduction
Stimulation of cells with physiologically relevant agonists that target G protein-or tyrosine kinase-coupled receptors leads to increases in cytosolic [Ca 2+ ] ([Ca 2+ ] i ) as a result of inositol 1,4,5triphosphate (IP 3 )-induced Ca 2+ release from intracellular Ca 2+ stores via the IP 3 receptors (IP 3 Rs) and Ca 2+ influx via plasma membrane Ca 2+ channels. The temporal and spatial pattern of [Ca 2+ ] i signals generated in response to agonist stimulation are utilized by the cells to regulate various critical functions, such as gene expression, ion channel activation and fluid secretion [1,2]. High levels of agonist typically induce sustained elevations in baseline [Ca 2+ ] i , whereas lower [agonist] elicit oscillatory [Ca 2+ ] i responses [1,3,4]. Two types of oscillations are seen; baseline oscillations that are usually seen at very low [agonist] or oscillations over a sustained elevation in baseline [Ca 2+ ] i at relatively higher [agonist]. Such oscillatory responses have been proposed to represent the physiological mode of signaling in many cell types and have been observed in almost all cell types, including cell lines as well as primary cell preparations from various tissues [3,5,6,7,8]. These oscillations primarily reflect repetitive cycles of Ca 2+ release from the ER stores via IP 3 Rs, inhibition of Ca 2+ release, and reuptake into the store due to SERCA pump activity. In several cell types, sustained [Ca 2+ ] i oscillations require extracellular Ca 2+ influx to achieve refilling of the ER store after every release event, thus priming it for the next release cycle. Even in cells where the oscillations are sustained for longer periods in the absence of external Ca 2+ , intracellular Ca 2+ stores are eventually depleted and there is a run-down of [Ca 2+ ] i oscillations.
Store-operated calcium entry (SOCE) is activated in response to depletion of Ca 2+ within the ER as a result of IP 3 -induced Ca 2+ release following agonist stimulation of cells [1,9]. SOCE has been shown to the primary determinant of agonist-induced [Ca 2+ ] i oscillations in a number of cells. Removal of extracellular Ca 2+ , or inhibition of Ca 2+ influx with La 3+ , induced cessation of [Ca 2+ ] i oscillations [5,10,11]. A major component of SOCE is STIM1, an ER Ca 2+ binding protein that serves as a sensor for ER-[Ca 2+ ] and regulates plasma membrane calcium channels mediating SOCE. [12,13,14]. Orai1, the pore-forming subunit of the highly Ca 2+selective Ca 2+ release-activated Ca 2+ (CRAC) channel has now been established as an essential component of SOCE [15,16,17].
Orai1 determines critical cellular functions including T-lymphocyte activation and mast cell degranulation. Suppression of Orai1 or STIM1 expression or function leads to elimination of SOCE and CRAC channel function [12,13,15,18,19]. Transient receptor potential 1 (TRPC1) is also activated in response to stimulation of cells by agonists or thapsigargin (Tg), and is a major contributor to Ca 2+ influx in some cell types [1,2,20,21,22,23,24]. We have previously shown that TRPC1 forms a dynamic complex with STIM1 and Orai1 in response to store depletion [25,26,27]. Further, data from several laboratories have demonstrated that while TRPC1 is gated by STIM1, its function depends on Orai1 [26,27,28,29]. Our recent finding provide evidence that Orai1mediated Ca 2+ entry triggers recruitment of TRPC1 into the plasma membrane where it is activated by STIM1 [27]. However, once activated, the two channels appear to have distinct functional contributions. In TRPC12/2 mice, decrease in Ca 2+ entry in salivary acinar cells is associated with loss of salivary fluid secretion as well as Ca 2+ -dependent K + channel activation [20]. These findings suggest that TRPC1 generates [Ca 2+ ] i signals that are specifically required for the activation of K Ca channels in acinar cells, which cannot be achieved or are not compensated for by the residual Orai1 channel in these cells. A similar decrease in Ca 2+activated Clchannel activity has been shown in pancreatic acini from TRPC12/2 mice [8].
CRAC channel activity has been associated with [Ca 2+ ] i oscillations in RBL cells and T lymphocytes [30,31,32,33]. Importantly, CRAC channel-mediated [Ca 2+ ] i oscillations have been shown to underlie regulation of Ca 2+ -dependent gene expression via activation of the transcription factor, nuclear factor activated T cells (NFAT). NFAT is translocated in an ''all-ornone'' manner following its activation which involves its complete dephosphorylation by calcineurin, a Ca 2+ -CaM dependent phosphatase [5]. By contrast, the nuclear factor kappa-light-chain enhancer of activated B cells (NFkB) pathway is activated through DAG-and Ca 2+ -dependent degradation of the inhibitor of NFkB (IkB), and is suggested to exhibit a strong dependence on peak amplitude, rather than duration, of a [Ca 2+ ] i signal. In B cells, a high level of [Ca 2+ ] i , exceeding that achieved by CRAC channel activity alone, is required for activation of NFkB. [34].
Our previous study suggested that Orai1-mediated Ca 2+ influx is more relevant for NFAT-activation than TRPC1, but is less relevant for K Ca activation. However, both TRPC1 and Orai1 are required for NFkB activation [27]. Since both Orai1-STIM1 and TRPC1-STIM1 channels contribute to agonist-or Tg-stimulated [Ca 2+ ] i increases in human submandibular gland (HSG) cells, but only Orai1-mediated Ca 2+ entry regulates the activation of NFAT, it is unclear how the cells decode individual Ca 2+ signals generated by the two channels for the regulation of specific cellular functions. We hypothesized that the two channels induce distinct [Ca 2+ ] i signals which determine their functional specificity in the regulation of Ca 2+ -dependent gene expression.

Orai1 and TRPC1 Channels Contribute Distinct [Ca 2+ ] i Signals in a Single Cell
Following CCh stimulation of HSG cells, a fairly sustained [Ca 2+ ] i elevation was seen, with an initial rapid increase followed by a sustained elevation above baseline that slowly declined over time ( Figure 1A shows the average response in a cell population). In the absence of external Ca 2+ , only a transient increase in [Ca 2+ ] i was seen ( Figure 1B), suggesting that the sustained [Ca 2+ ] i increase following agonist stimulation is dependent on extracellular Ca 2+ entry. Knockdown of STIM1 expression by siSTIM1 eliminated the sustained [Ca 2+ ] i elevation, producing a pattern similar to that in the absence of external Ca 2+ ( Figure 1C). Complete elimination of sustained [Ca 2+ ] i elevation was also seen with expression of Orai1E106Q (a dominant negative Orai1 mutant, [35,36]) or siOrai1 (.90% decrease; Figure 1, D, E and H). On the other hand, expression of shTRPC1 or STIM1-KK/ EE [29] induced .60% decrease in sustained [Ca 2+ ] i elevation (responses at 250 s were 0.04660.009 and 0.06260.008, respectively, which are both significantly higher than that in Orai1E106Q-or siOrai1-expressing cells (0.01660.007 and 0.01660.003 respectively), and lower than in control cells (0.19360.014) (p,0.001; Figure 1, F to H). Based on our previous studies, we can conclude that cells expressing siOrai1 or Orai1E106Q lack both TRPC1 and Orai1 functions [26,27]. While these findings demonstrate that Ca 2+ entry via Orai1 and TRPC1 determine sustained the [Ca 2+ ] i elevation seen in CChstimulated cells, the individual contributions of the two channels cannot be resolved using such measurements.
To determine the characteristics of [Ca 2+ ] i signals generated by Orai1 and TRPC1, we carried out detailed analysis of the [Ca 2+ ] i changes induced by CCh in individual HSG cells. Stimulation with 1 mM CCh induced a rapid initial increase [Ca 2+ ] i which was followed by oscillatory increases over a sustained elevation of baseline [Ca 2+ ] i (Figure 2A). The initial response primarily represents Ca 2+ release from the ER, while the subsequent oscillatory responses and sustained baseline elevation are determined by the influx of Ca 2+ (c.f. trace in absence of external Ca 2+ , Figure 2B). Knockdown of STIM1 using siRNA (siSTIM1) reduced both the oscillations and sustained elevation of baseline [Ca 2+ ] i ( Figure 2C The relative proportions of cells affected by these maneuvers are shown in Figure 2H. In the control group, 66% of cells showed oscillatory [Ca 2+ ] i responses over a sustained elevation in baseline [Ca 2+ ] i while none of the cells displayed sustained baseline oscillations or transient responses (cells that did not respond or those that showed a sustained elevation without oscillations were not included). Interestingly in cells expressing STIM1-KK/EE, the proportion of cells with [Ca 2+ ] i oscillations over an elevated baseline decreased to 10% (from 66% in control cells) and those with baseline oscillations increased to 63%, whereas only 6% of cells showed transient responses. Similar changes were seen in cells expressing shTRPC1. In the case of Orai1E106Q-and siOrai1-expressing cells, the proportion of cells with transient response was increased to 40% or 67%, respectively, as would be expected if the SOCE process was inhibited (about 14% or 9% of cells displayed normal response and 15% or 8% showed baseline oscillations, these likely represent either non-transfected cells or those expressing low levels of Orai1 mutant or siOrai1 respectively).
In aggregate, the data in Figure 2   dependent on the STIM1-activated Orai1 channel ( Figure S1A). When TRPC1 and STIM1 were co-expressed in HEK293 cells, the pattern of baseline [Ca 2+ ] i oscillations was converted to oscillations over a sustained elevation ( Figure S1B). In contrast, expression of STIM1-KK/EE in HEK293 cells did not significantly alter CCh-induced [Ca 2+ ] i oscillations ( Figure S1C; c.f. control cells in Figure S1A). Furthermore, as described earlier for HSG cells, these CCh-induced oscillations in HEK293 cells were dependent on Ca 2+ entry and could not be sustained with the absence of Ca 2+ in the extracellular milieu ( Figure S1D). Thus, when both Orai1 and TRPC1 are activated simultaneously, the typical response achieved is oscillations over a sustained elevation of baseline [Ca 2+ ] i . While both channels contribute to SOCE in HSG cells, the Orai1 channel predominantly mediates SOCE in HEK293 cells. Together, the data discussed above suggest that inclusion of functional TRPC1 channels leads to modulation of the [Ca 2+ ] i signals generated by Orai1 channels.

NFAT is Regulated by Orai1-mediated Ca 2+ Influx
We then investigated whether the cellular mechanisms regulating activation of NFAT can decode the different [Ca 2+ ] i signals generated by Ca 2+ entry via Orai1 and TRPC1 channels. Translocation of GFP-NFAT into the nucleus, with a corresponding decrease of protein in the cytosol, was detected within 2 min of  Figure 3A). NFAT activation was exclusively driven by Ca 2+ entering the cell since application of 1 mM La 3+ extracellularly blocked nuclear translocation of GFP-NFAT following stimulation with 1 mM CCh ( Figure 3B). At this concentration, La 3+ blocks both Ca 2+ entry via plasma membrane Ca 2+ channels as well as Ca 2+ extrusion via the plasma membrane Ca 2+ -ATPase pump thereby retaining the Ca 2+ released from the ER in the cytoplasm. NFAT translocation into the nucleus was also abrogated in cells where Orai1 function was suppressed (compare A to C and D in Figure 3). However, in cells where only TRPC1 expression was suppressed, the residual Orai1 activity was sufficient to support activation of NFAT ( Figure 3E and F). We had reported similar specificity for Orai1 in NFAT-dependent gene expression following Tg stimulation of HSG cells [27]. Together the data in Figures 1, 2   NFAT activation was also assessed in single cells stimulated with 100 mM CCh. Nuclear translocation of NFAT was not faster than that in cells stimulated with 1 mM CCh, although the number of cells responding to the stimulus was significantly higher ( Figure 5A c.f. Figure 3A, .90% cells vs. ,70% cells displayed NFAT translocation). Further, as seen in cells stimulated with 1 mM CCh, NFAT translocation was completely prevented by expression of Orai1E106Q and siOrai1 but was not affected by STIM1-KK/EE and shTRPC1 ( Figure 5, B Figure 6A). Initial oscillations due to intracellular Ca 2+ release (cells stimulated in Ca 2+ -free medium) were slightly more prolonged (up to 250 s; Figure 6C; similar results were obtained in cells expressing Orai1E106Q in Figure 6D) probably due to the low levels of Ca 2+ store depletion. In order to determine the Ca 2+ influx component, [Ca 2+ ] i was monitored for a period of 10 min and the value at 350 s was used to determine the amplitude. The number of oscillations generated between 300-600 s were also counted (note that oscillations subside by this time in a Ca 2+ -free medium). Importantly at this low level of stimulation, there was a detectable contribution of TRPC1 to the frequency of [Ca 2+ ] i oscillations as cells expressing STIM1-KK/EE showed a rundown of oscillations with .75% decrease in the number of oscillations (Figure 6, B, E and F). Based on these findings, we examined the [Ca 2+ ] i oscillations in cells stimulated at 1 mM CCh between 300-600 s (representative traces are shown in insets of Figure 6A and B). As shown in Figure 6F, there was ,50% decrease in the frequency of oscillations due to suppression of TRPC1 activity in cells stimulated at 1 mM CCh, c.f. ,62% decrease with 300 nM CCh. Thus, Ca 2+ influx via TRPC1 also contributes to [Ca 2+ ] i oscillations. We suggest that this is likely due to more efficient refilling of the Ca 2+ stores in cells when both Orai1 and TRPC1 channels are active.
A major finding of this study was the dissociation between [Ca 2+ ] i changes and NFAT activation seen in cells stimulated with 300 nM CCh. Despite detection of [Ca 2+ ] i oscillations in 30% of the cells, only 4.8% of the cells exhibited nuclear translocation of NFAT (compare with .90% and ,70% of cells stimulated with 100 or 1 mM CCh, respectively). Since the number of oscillations at this very low [CCh] is about 40-50% less and amplitude of the [Ca 2+ ] i signal is ,75% lower than that at 1 mM CCh ( Figure 6E and F, c.f. Figure 1H), we hypothesized that a lower number of Orai1 channels are activated and therefore, the local increase in [Ca 2+ ] i is lower than the threshold required for NFAT activation. To test this, extracellular [Ca 2+ ] was raised from 1 to 10 mM to increase the driving force for Ca 2+ entry via Orai1, which should increase the local [Ca 2+ ] i near the channel. Consistent with our prediction, this maneuver resulted in a significant increase in the number of cells exhibiting nuclear translocation of NFAT to 38.8% ( Figure 6G and H). Similarly, NFAT translocation was seen in very few cells expressing STIM1-KK/EE unless extracellular [Ca 2+ ] was increased to 10 mM (5.8% at 1 mM to 53.6% at 10 mM Ca 2+ ; Figure 6G and H). Furthermore, a sustained elevation of [Ca 2+ ] i with minimal oscillations was seen when extracellular [Ca 2+ ] was increased to 10 mM ( Figure 6H). In aggregate, these findings further establish that only local [Ca 2+ ] i near Orai1 channels is involved in NFAT activation.
A recent study showed that there is a threshold for local [Ca 2+ ] i generated by Orai1-mediated Ca 2+ influx which is critical for dephosphorylation of NFAT [37]. These investigators showed that while stimulation with 120 nM leukotriene C 4 (LTC 4 ) was insufficient to induce activation of NFAT1, a second pulse of 120 nM LTC 4 (total = 240 nM) within 10 min of the first was sufficient to activate NFAT1. We have observed similar results when two pulses of 300 nM CCh within 3 min of each other were added to the cells. The number of cells showing nuclear translocation of NFAT increased from about 4.35% (n = 184) to 11.7% (n = 171). Furthermore, this was not altered by suppression of TRPC1 function in STIM1-KK/EE-expressing cells, where 14.4% of cells (n = 118) showed NFAT  activation following two pulses of 300 nM CCh (c.f. 5.8% (n = 86) for a single pulse). We suggest that the first pulse of CCh was not sufficient to completely dephosphorylate NFAT, while the second pulse of agonist achieved the extra [Ca 2+ ] i increase needed to fully dephosphorylate NFAT, a requirement for its nuclear translocation. Collectively, the data presented in Figure 6 demonstrate that although TRPC1 contributes to oscillatory [Ca 2+ ] i responses at very low [agonist], nuclear translocation of NFAT is solely dependent on local [Ca 2+ ] i signals generated by Orai1. Further, the lack of NFAT translocation at low levels of stimuli when compared to the relatively higher levels, suggests that local [Ca 2+ ] i required to drive this process is likely to depend on the number of Orai1 channels activated at any given [agonist] and that NFAT regulation is mediated by an ''all-or-none'' mechanism in which complete dephosphorylation of the transcription factor is required for its nuclear translocation. What is more important is that TRPC1-mediated Ca 2+

Specific Regulation of NFAT-and NFkB-driven Luciferase Activities by Orai1 and TRPC1 Channels
To further establish whether the functional specificity of TRPC1 and Orai1 seen in short-term responses (i.e. nuclear translocation of NFAT) is also retained for long-term effects at the level of gene expression in the nucleus, we measured NFAT-or NFkB-driven luciferase activities. Such long-term effects would indicate that a [Ca 2+ ] i signal ''memory'' is retained even after the initial [Ca 2+ ] i elevation has declined. Both NFAT-and NFkBdriven luciferase (NFAT-luc and NFkB-luc, respectively) activities were clearly detected following 100 mM CCh treatment of cells (substantial variability was seen with lower [CCh], i.e. 10 and 1 mM, possibly due to the lower percentage of cells responding to CCh at these concentrations). Consistent with the findings shown in Figures 3 and 5, loss of Orai1 function, but not that of TRPC1, significantly abrogated CCh-stimulated increase in NFAT-dependent promoter activity (Figure 7). Thus, both early and late events in NFAT signaling are exclusively dependent on Orai1. In contrast, loss of either Orai1 or TRPC1 function significantly and severely reduced CCh-stimulated NFkB-driven promoter activity (Figure 7). We had earlier shown that Orai1-mediated Ca 2+ entry is required for the assembly of functional TRPC1-STIM1 channels in the plasma membrane [27]. Therefore abrogating Orai1 function eliminates TRPC1 channel activity and accounts for the decrease in NFkB-luc in shTRPC1-or siOrai1-treated cells. Further, the similar levels of NFkB-luc activity measured in the two groups of cells suggest that TRPC1 is the primary determinant in the regulation of this transcription factor. In aggregate, our findings provide strong evidence for the functional specificity of Orai1 and TRPC1 channels in the regulation of Ca 2+ -dependent gene expression.

Discussion
In some cell types, including salivary gland cells, more than one channel contributes to agonist stimulated [Ca 2+ ] i signals. It is not fully understood how cells decode [Ca 2+ ] i signals originating from multiple sources for the regulation of specific Ca 2+ -dependent functions. Variations in the pattern of individual [Ca 2+ ] i signals generated by the two channel types is most likely the primary determinant of the functional specificity of the channels in regulation of cell function. Here we have studied the contributions of endogenous TRPC1 and Orai1 to agonist-stimulated [Ca 2+ ] i signals in a single HSG cell. We show that Ca 2+ entry via each channel generates a specific pattern of [Ca 2+ ] i elevation, with Orai1 controlling the generation of [Ca 2+ ] i oscillations and TRPC1 mediating sustained [Ca 2+ ] i elevation at higher [agonist] and contributing to the frequency of baseline [Ca 2+ ] i oscillations. Even more significant is the finding that the channels display functional specificity in the activation of Ca 2+ -dependent transcription factors and gene expression. Consistent with the oscillatory [Ca 2+ ] i signals generated by Orai1, NFAT translocation and NFAT-dependent gene expression were exclusively dependent on Orai1-mediated Ca 2+ entry, without any contribution of TRPC1. Our data suggest that NFAT is strictly regulated by the [Ca 2+ ] i achieved locally near the Orai1 channel, likely due to localization of calmodulin-calcineurin-NFAT within the Orai1associated microdomain, such that the Ca 2+ entering via Orai1 can be locally sensed by the calcium sensor. Moreover, since Ca 2+ entering into this microdomain via Orai1 rapidly rises to concentrations that exceed a threshold level required for activation, NFAT activation did not reflect global [Ca 2+ ] i changes achieved at the various stimulus intensities. We also show that NFAT activation follows an ''all-or-none'' mode of activation which is strictly dependent on Orai1; if an insufficient number of Orai1 channels is activated, NFAT dephosphorylation is not completed and nuclear translocation does not occur. However, we cannot rule out the possibility that at even higher level of stimulus (e.g. a more potent agonist or involving different receptor pathways) or if more channels were expressed, this pattern could vary and sustained [Ca 2+ ] i elevations could be induced by Orai1 (e.g. in lymphocytes or in HEK293 cells overexpressing Orai1+S-TIM1). In contrast to the regulation of NFAT, we show that NFkB is primarily regulated by TRPC1. Furthermore, we previously reported that Ca 2+ entry via TRPC1, but not Orai1, is required for sustained activation of K Ca in HSG cells as well as acinar cells isolated from mouse salivary glands [20]. However, it remains to be fully understood whether local [Ca 2+ ] i achieved near the TRPC1 channel or global [Ca 2+ ] i changes mediated by TRPC1 are involved in the activation of NFkB and K Ca channel. In aggregate, these findings provide conclusive evidence that Orai1 and TRPC1 generate functionally specific local and global [Ca 2+ ] i signals.
An important determinant in the generation of local [Ca 2+ ] i signals is the clustering of the channels within the signaling microdomain [38,39,40,41]. Furthermore, localization of other Ca 2+ signaling components, such as Ca 2+ pumps in the ER and plasma membrane or mitochondria, could also affect the amplitude as well as temporal and spatial aspects of [Ca 2+ ] i signals [5,7,31]. More recently, stimulation with different types of agonist has been shown to recruit different STIM proteins to activate Orai1 channel and generate different [Ca 2+ ] i signals that are decoded to induce NFAT-driven gene expression in RBL cells [32]. In this context, a recent report describes that distinct modes of Ca 2+ signaling are triggered by Ca v 1 and Ca v 2 channels within the same neurons, which are differentially used for regulating gene   [42]. Both Ca v 1 and Ca v 2 channels are activated following plasma membrane depolarization and employed the same calmodulin kinase (CaMK)-dependent pathway to activate CREB-dependent gene expression. Nonetheless, Ca 2+ entry via both channels contributed to different pools of Ca 2+ within a single neuron with Ca v 1 contributing to the local [Ca 2+ ] i but Ca v 2 to the global [Ca 2+ ] i increases. Interestingly, Ca v 1 was generally clustered close to the puncta of bCaMKII, the predominant CaMKII isoform in neurons, while Ca v 2 clusters were located supramicrons away. Another important point was that Ca 2+ entry via Ca v 2 channels was preferentially buffered by the ER and mitochondria. Hence, Ca v 2-mediated Ca 2+ signaling was dampened to a greater degree than Ca v 1 and requires a greater depolarizing stimulus for channel activation [42].

[Ca 2+ ] i Measurements
Fura-2 fluorescence was measured in single HSG cells as described previously [25,27]. Cells were loaded with 2 mM Fura-2AM (Invitrogen) for 45 min at 37uC, fluorescence was recorded using a Polychrome V spectrofluorimeter (TILL Photonics, Victor, NY) and MetaFluor imaging software (Molecular Devices, Sunnyvale, CA). Each fluorescence trace (340/380 nm ratio) represents an average from at least 50 cells. For the bar graphs, data presented show change in Fura-2 ratio due to influx where the fluorescence value at 250 s or 350 s was subtracted from the baseline (F t 2F 0 ).

Measurement of NFAT Translocation into the Nucleus
Translocation of NFAT in transfected HSG cells was observed using an Olympus IX81 motorized inverted microscope (Olympus, Center Valley, PA) and a TIRF-optimized Olympus Plan APO 606 (1.45 NA) oil immersion objective. Excitation was achieved [25] using the 488 nm laser for excitation of GFP, and emission detected using a Lambda 10-3 filter wheel (Sutter Instruments, Novato, CA) containing the 525-band pass (BP50m) filter. Images were collected using a Rolera EM-C 2 camera (Q-Imaging, Surrey, BC) and the MetaMorph imaging software (Molecular Devices). MetaMorph was also used to measure the fluorescence intensity in the nucleus and cytoplasm before and after stimulation with CCh. Briefly, regions of interest were selected to obtain the values for their fluorescence intensities during a time course experiment. These values were then plotted using the Origin 8 software (OriginLab, Northampton, MA). Due to the low responsiveness of HSG cells to stimulation with 300 nM CCh, a 206 fluorescence objective was used to screen larger numbers of cells.

Measurement of NFAT and NFkB Luciferase Activities
HSG cells were seeded at 15610 3 per well in a 96-well plate one day prior transfection. The shTRPC1 (0.25 mg/well) and siOrai1 (200 nM/well) constructs were transiently transfected into cells using Lipofectamine 2000 or RNAiMAX (Invitrogen), respectively, following manufacturer's protocol. After 24 h, the firefly luciferase reporter constructs for NFAT (pGL4.30[luc2P/ NFAT-RE/Hygro]) or NFkB (pGL4.32[luc2P/NF-kB-RE/ Hygro]) were transfected with the renilla luciferase reporter construct (pGL4.74[hRluc/TK]; to monitor transfection efficiency) into HSG cells using Lipofectamine 2000 for another 24 h. All three luciferase constructs were obtained from Promega (Madison, WI). Cells were then left untreated or treated with CCh at various concentrations for 6 h at 37uC. For NFAT luciferase activity, cells were stimulated with CCh alone. For NFkB luciferase activity, cells were stimulated with CCh in the presence of PMA (10 ng/ ml). Luciferase activities were determined using Dual-Glo Luciferase Assay, as per manufacturer's instructions (Promega). Luminescence intensity was monitored in using the FLUOStar OMEGA microplate reader (BMG Labtech, Cary, NC). At least 3 separate experiments were performed using samples in triplicates. The firefly luciferase values were normalized to renilla luciferase values. All data were presented as fold-change relative to the vector control.

Statistics
Data analysis was performed using Origin 7.0 (OriginLab). Statistical comparisons were made using Student's t-test. Exper-imental values are expressed as mean6SEM. Differences in the mean values were considered to be significant at p,0.001.