Suppression of TRPC3 Leads to Disappearance of Store-operated Channels and Formation of a New Type of Store-independent Channels in A431 Cells*

In most non-excitable cells, calcium (Ca2+) release from the inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores is coupled to Ca2+ influx through the plasma membrane Ca2+ channels whose molecular composition is poorly understood. Several members of mammalian TRP-related protein family have been implicated to both receptor- and store-operated Ca2+ influx. Here we investigated the role of the native transient receptor potential 3 (TRPC3) homologue in mediating the store- and receptor-operated calcium entry in A431 cells. We show that suppression of TRPC3 protein levels by small interfering RNA (siRNA) leads to a significant reduction in store-operated calcium influx without affecting the receptor-operated calcium influx. With single-channel analysis, we further demonstrate that reduction of TRPC3 levels results in suppression of specific subtype of store-operated calcium channels and activation of store-independent channels. Our data suggest that TRPC3 is required for the formation of functional store-operated channels in A431 cells.

In non-excitable cells, activation of phospholipase C (PLC) 2 mediates calcium (Ca 2ϩ ) release from the inositol 1,4,5trisphosphate (InsP 3 )-sensitive intracellular Ca 2ϩ stores and Ca 2ϩ influx through the plasma membrane Ca 2ϩ channels. Two distinct pathways for calcium influx have been identified: the receptor-operated pathway which is directly mediated by downstream signaling cascades of PCL, and the store-operated pathway which is activated by depletion of intracellular calcium stores (1)(2)(3). Several types of store-operated calcium currents have been characterized in various tissues. Depending on the cell type, these currents display variability in biophysical characteristics and modes of regulation (4 -6, 7, 8), suggesting that different proteins may be involved in forming store-operated channels (SOC) in the plasma membrane and/or regulating SOC activity. Several lines of evidence have suggested that STIM1 (9,10) and Orai1 (11)(12)(13) play essential roles in activation of calcium release-activated Ca 2ϩ currents (I CRAC ) (4 -6). Recent studies have shown that STIM1 acts as a sensor of the intracellular Ca 2ϩ stores (9,14) while Orai1 may be directly involved in forming I CRAC channels in the plasma membrane (15)(16)(17). Although it is well established that I CRAC is activated by store depletion, I CRAC may only represent one subtype of store-operated channels since the SOC currents with properties distinct from I CRAC have been characterized (7,8). Despite extensive calcium imaging and electrophysiological studies, the molecular composition of SOC types other than I CRAC remains poorly understood.
The members of mammalian TRP-related family of ion channels have been implicated to both receptor-and store-operated Ca 2ϩ influx (reviewed in Ref. 18). TRPC3 has been shown to form non-selective cation channels that can be activated in a PLC-dependent manner (19). Although most studies suggest that overexpressed TRPC3 forms receptor-operated channels (20 -24), several reports provided the evidence for regulation of TRPC3 by depletion of the intracellular calcium stores (25)(26)(27). In different cell types, activation of overexpressed TRPC3 channels have been shown to depend on InsP 3 , DAG, Ca 2ϩ , G-proteins, and store depletion. For example in DT40 cells, transiently expressed TRPC3 forms the InsP 3 R-dependent and InsP 3 R-independent channels (28,29). These controversial observations may at least in part be reconciled by the fact that TRPC3 forms heterooligomers with the other members of TRPC subfamily (30). Interestingly, recent knockdown studies suggested that native TRPC3 is essential for store-operated calcium entry in HEK293 cells (31,32).
Our previous studies have shown that A431 carcinoma cell line expresses highly selective I CRAC currents and moderately selective I SOC currents. We found that I SOC currents are at least partly mediated by I min channels, whose activity is controlled by downstream products of PLC (33)(34)(35)(36)(37)(38) and could be triggered by passive depletion of intracellular Ca 2ϩ stores with Tg and/or BAPTA-AM (39). In the present study, we aimed to determine the role of TRPC3 in mediating the receptor-and store-operated calcium influx in A431 cells. Using calcium imaging and whole cell recordings, we found that suppression of TRPC3 expression with siRNA results in significant reduction in storeoperated calcium entry. Because most of the functional studies linking TRP family to receptor-operated and store-operated Ca 2ϩ influx utilized Ca 2ϩ imaging or whole cell current recordings, we further extended our analysis with single channel recordings to obtain more detailed information on functional properties of individual types of channels supporting store-operated Ca 2ϩ influx. We found that suppression of TRPC3 in A431 cells results in selective inhibition of specific subtype of store-operated calcium channels and activation of store-independent channels. Together, these data indicate that TRPC3 is essential for formation of functional SOCs in A431 cells.

EXPERIMENTAL PROCEDURES
Cell Culture-Human carcinoma A431 cells (Cell Culture Collection, Institute of Cytology, St. Petersburg, Russia; ATCC CRL-1555) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 80 g/ml gentamicin, and 2 mM glutamine. The cells were maintained in 37°C incubator (5% CO 2 ). For patch clamp and Ca 2ϩ -imaging experiments, the cells were plated on glass coverslips and maintained in culture for 1-3 days before use.
The synthesized double-stranded oligonucleotides were annealed and ligated into pUB/Bsd/H1 RNAi vector containing a cassette for blasticidin selection (40). The oligonucleotide inserts were confirmed by sequencing.
Transfections-Cells were transfected by Effectin Transfection Reagent (Qiagen) with the appropriate siRNA vectors, and placed into selection medium containing 10 g/ml blasticidin. After 2 weeks of selection, the individual clones were picked and expanded. The protein expression in individual clones was tested by immunoblotting. The clone with highest TRPC3 suppression level was generated with siRNA type III. It was designated siTRPC3 and used for experiments. For control of anti-TRPC3 antibodies specificity, cells were transient transfected with pcDNA 3.1 vector containing Myc-tagged human TRPC3 cDNA (generously provided by Dr. C. Montell, The Johns Hopkins University School of Medicine).
Western Blotting-A431 and siTRPC3 cells were grown on 5-cm dishes under the conditions described above. Cells were lysed in radioimmune precipitation assay buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% Nonidet P-40, 2 mM EDTA, 0,2 mM phenylmethylsulfonyl fluoride, inhibitor mixture). The protein concentration was measured by BCA kit (Pierce). Total protein extract (50 -150 g) was applied on 8% SDS-PAGE (10 cm ϫ 12 cm gels). The proteins were trans-ferred onto Immobilon P membrane (Millipore Inc.) and treated with first polyclonal anti-TRPC1 or anti-TRPC3 antibodies (1:200) and anti-TRPC4 or anti-TRPC6 antibodies (1:500) (Alomone Labs), then with secondary anti-rabbit antibody (1:5000) (Sigma) and developed with SuperSignal Chemiluminescent Substrate (Pierce) at a suitable time so as not to saturate the film. Anti-␣-tubulin monoclonal antibody (1:5000) (Sigma) was used to test for equal protein loading. For the control of specificity of anti-TRPC3 antibody the control A431 cells and cells transient transfected with Myc-tagged TRPC3 were lysed and subjected to SDS-PAGE. The proteins were then transferred onto Immobilon P membrane and immunoblotted with anti-Myc antibodies (1:1000) (Santa Cruz Biotechnology) and anti-TRPC3 antibodies. Western blots were repeated at least three times using different cell lysates.
Ca 2ϩ Imaging-Control A431 and A431-siTRPC3 cells were loaded with 5 M Fura-2AM in the presence of 0.025% Pluronic for 40 min at room temperature. Loaded cells were illuminated by alternating 340 and 380-nm excitation light at 2 Hz. Emission fluorescence intensity was measured at 510 nm with the InCyt Basic I/P dual wavelength fluorescence imaging system (Intracellular Imaging Inc., Cincinnati, OH). The change in cytosolic Ca 2ϩ concentration was expressed as the ratio of emission fluorescence intensity at 340 and 380 nm excitation wavelengths (340/380 ratio).
Electrophysiology-All electrophysiological experiments were performed with Axopatch 200B patch clamp amplifier (Axon Instruments). pClamp6 software (Axon Instruments) was used for data acquisition and off-line data analysis. All experiments were carried out at room temperature (22-24°C).
The whole cell recordings were performed with 3-5 MW sylgard-coated, fire-polished glass pipettes. The pipette solution contained (in mM) 145 NMDG aspartate, 10 Cs-EGTA, 10 Cs-HEPES pH 7.3, 1.5 MgCl 2 , and either 4.5 CaCl 2 (pCa 7.0) or 0 CaCl 2 (pCa Ͼ 9). Extracellular solution contained (in mM) 140 NMDG aspartate, 10 BaCl 2 , 10 Cs-HEPES, pH 7.3. The currents were sampled at 5 kHz and filtered digitally at 500 Hz. In all experiments, the holding potential was 0 mV. Periodically (once every 5 s) the holding potential was shifted to Ϫ100 mV (for 30 ms) and a 170-ms voltage ramp to ϩ70 mV was applied. The traces recorded before current activation were used as templates for leak subtraction. Whole cell currents were normalized to the cell capacitance. The mean value of cell capacitance was 25 Ϯ 4 pF (n ϭ 45).
The single channel recordings were performed with 8 -15 M⍀ glass pipettes. The pipette solution contained (in mM): 105 BaCl 2 , 10 Tris-HCl (pH 7.3). In cellattached experiments, the bath solution contained 140 mM KCl and 2 mM CaCl 2 to nullify the resting membrane potential. In store depletion experiments, 1 mM TPEN, or 1 M thapsigargin were added to the bath solution containing (in mM): 140 KCl, 5 NaCl, 10 K-Hepes, and 2 EGTA (pH 7.4). In inside-out experiments, the intracellular solution contained (in mM): 140 potassium glutamate, 5 NaCl, 1 MgCl 2 , 10 K-HEPES pH 7.4, 2 K-EGTA, and 1.13 CaCl 2 (pCa 7.0), with or without InsP 3 . The drugs were applied by bath perfusion. The time required for a complete exchange of solution around the patch pipette was less than 1 s. The single channel recordings were digitized at 5 kHz and filtered at 100 Hz for analysis and presentation. The amplitudes of single-channel currents were determined from the current traces and all-point amplitude histograms. The channel open probabilities (NP o ) were determined by using the following equation: NP o ϭ (I)/i, where (I) is a mean channel current; and i is the unitary current amplitude. The (I) was estimated from the time integrals of the currents above the baseline, and i was determined from the current traces and all-point amplitude histograms. The data were collected from the current traces after channel activity reached steady state.

Suppression of TRPC3 Expression Reduces Store-operated
Calcium Influx in A431 Cells-Our biochemical analysis has shown that A431 cells express several isoforms of TRPC protein family, including TRPC3 (Fig. 1A). This data are in agreement with the unpublished observations of Dr. Schilling. 3 Additional evidence of TRPC3 expression in A431 was obtained using transient transfection of Myc-tagged human TRPC3 cDNA in these cells (Fig. 1B). To assess the role of TRPC3 in mediating agonist-or store-induced currents in A431 cells, we knocked down TRPC3 expression using three different siRNA constructs and tested the protein levels in individual clones by immunoblotting. A clone that showed the highest reduction in TRPC3 expression (siTRPC3) was expanded and used for electrophysiological and imaging analyses (Fig. 2). To determine the role of TRPC3 in mediating agonist-evoked Ca 2ϩ entry, we compared the UTP-induced Ca 2ϩ entry in control A431 cells and the cells expressing TRPC3 siRNA (siTRPC3). Application of 100 M UTP to Fura-2-loaded cells maintained in Ca 2ϩ -free media, resulted in transient elevation of cytosolic Ca 2ϩ levels due to calcium release from the intracellular InsP 3 -sensitive Ca 2ϩ stores (Fig. 3A). Subsequent re-addition of 2 mM extracel-3 W. Schilling, personal communication.  In every individual experiment, a calcium response form ϳ100 cells was recorded. A, control and siTRPC3 cells display similar calcium responses mediated by the extracellular application of UTP. The Ca 2ϩ release evoked by application of UTP in Ca 2ϩ -free medium is followed by a Ca 2ϩ elevation upon re-addition of Ca 2ϩ to the extracellular medium. B, suppression of TRPC3 expression reduces Ca 2ϩ influx mediated by depletion of intracellular calcium stores with Tg. C, suppression of TRPC3 levels reduces the Ca 2ϩ influx mediated by depletion of intracellular calcium stores with TPEN. Store-depletion was induced by TPEN added to the Ca 2ϩ -free bath medium. Subsequent addition of 2 mM extracellular Ca 2ϩ resulted in Ca 2ϩ entry, which was significantly decreased in siTRPC3 cells when compared with A431 cells. D, gadolinium (Gd 3ϩ , 10 M) inhibits Ca 2ϩ influx mediated by depletion of intracellular store in both control A431 and siTRPC3 cells. AUGUST 10, 2007 • VOLUME 282 • NUMBER 32 lular Ca 2ϩ induced the second peak of intracellular Ca 2ϩ with practically equal transients in A431 and siTRPC3 cells.

The Role of TRPC3 in Forming SOC in A431 Cells
To test whether TRPC3 forms or regulates store-operated channels in A431 cells, we compared Ca 2ϩ entry mediated by passive store depletion in control and siTRPC3 cells. In contrast to the agonist-induced responses, thapsigargin (Tg) or TPENinduced Ca 2ϩ entry was reduced by respectively 40 and 17% in siTRPC3 cells (Fig. 3, B and C). Analysis of the areas underneath the transient Ca 2ϩ peaks showed that control and siTRPC3 cells released similar amounts of calcium from the intracellular stores in response to Tg treatment. These results suggest that TRPC3 suppression does not affect calcium storage or calcium release from the intracellular stores. To further assess the role of TRPC3 in mediating calcium influx in response to store depletion, we analyzed the effect of gadolinium (Gd 3ϩ , 10 M). At micromolar concentrations, Gd 3ϩ blocks the calcium entry triggered by store depletion (41)(42)(43). We found that in the presence of Gd 3ϩ , the Tg-induced calcium peaks were reduced by ϳ10and ϳ2-fold in control and siTRPC3 cells, respectively (Fig. 3, B and D). Together, these results suggest that suppression of TRPC3 in A431 cells, results in significant reduction of calcium influx mediated by depletion of the intracellular calcium stores.
We next employed the whole cell recordings to investigate the effects of suppression of TRPC3 on cation currents induced by agonists and store depletion. In these experiments, we used 10 mM Ba 2ϩ as a charge carrier. Under these conditions, extracellular application of UTP induced the cation currents that reached the maximum at ϳ4 min after addition of the agonist (Fig. 4A). Consistent with the calcium imaging experiments, we found that both control and siTRPC3 cells displayed similar UTP-mediated responses (Fig. 4, A  and B). Passive store depletion with Tg also induced the cation currents that displayed kinetics of on-set similar to the agonist-induced currents (Fig. 4C). In a few cells (2 out of 15 in control group and 3 out of 13 in siTRPC3 group), we only observed the highly selective I CRAClike inward calcium currents that were not affected by suppression of TPRC3 (Fig. 4D). We therefore excluded these experiments from the final analysis. In the remaining experiments, the cation currents induced by Tg were reduced on average to 38% in siTRPC3 cells when compared with control (Fig. 4,  C and E). Thus, our calcium imaging and whole-cell experiments consistently show that reduction in TRPC3 levels results in selective inhibition of calcium influx mediated by depletion of the intracellular calcium stores. We next used the single channel analysis to explore the effects of TRPC3 suppression on various types of calcium-and cation-selective channels expressed in A431 cells.
TRPC3 Does Not Regulate I min and I max Cation Channels-We have previously shown that A431 cells express highly selective I min calcium channels that could be activated by extracellular UTP, intracellular InsP 3 and depletion of intracellular Ca 2ϩ stores (35,36,39, see also Fig. 5F). We also found that I min channels can be directly activated by N-terminal ligand-binding domain of InsP 3 R (33). To test whether suppression of TRPC3 affects I min channels in A431 cells, we performed single channel recordings and tested the effects of extracellular application of UTP or TPEN to the cell-attached patches, and intracellular application of InsP 3 to the inside-out patches (Fig. 5). We found that similarly to control A431 cells, siTRPC3 cells express I min channels that can be activated by extracellular agonists (UTP), store depletion and InsP 3 (Fig. 5). The biophysical characteristics of single I min channels monitored in A431 cells with reduced levels of TRPC3 were identical to I min channels that we previously described in control A431 cells: the single channel conductance measured in the presence of 105 mM extracellular Ba 2ϩ was 1.2 pS (Fig. 5D); the extrapolated reversal potential was higher than ϩ30 mV, indicating high selectiv- ity for Ca 2ϩ and Ba 2ϩ over K ϩ (Fig. 5D); and the channel mean open time was ϳ8.8 ms (Fig. 5E). The occurrence of I min channels in siTRPC3 cells stimulated with UTP or store-depletion agents was not significantly different form the control A431 cells. However, these channels could rarely be evoked in siTRPC3 cells stimulated with InsP 3 (Fig. 5F).
The reversal potential of single I min channels observed in A431 cells (Fig. 5D) is not consistent with the reversal potential of receptor-or store-operated currents that we detected in whole cell mode (Fig. 4, B and E). Are there additional less or non-selective receptor-and store-operated channels in A431 cells? How does suppression of TRPC3 affect these channels? Analysis of 87 single channel experiments revealed that in addition to I min channels, extracellular application of UTP resulted in activation of less-selective channels that displayed a larger unitary conductance (Fig. 6A). These channels could also be activated by passive store depletion with TPEN (Fig. 6B) or Tg (data not shown). The unitary current-voltage relationship of these channels was nonlinear (Fig. 6G) with the slope conductance of 18 pS at the negative holding potentials. The extrapolated reversal potential was ϩ15 mV (Fig. 6G). The open-time distribution of these channels could be fitted by sum of two exponential functions with the time constants o1 2.5 ms and o2 23.1 ms (Fig. 6C). The channels with similar gating and regulation properties were described by us in HEK293 cells and we have named them I max (44). Similarly to the I min channels, I max in A431 cells could be activated by application of InsP 3 to inside-out patches (Fig. 6H). We found that in siTRPC3 cells, I max could also be evoked by UTP (Fig. 6D) or depletion of intracellular stores with TPEN (Fig. 6E). In addition, in both control and siTRPC3 cells, I max channels had similar conductance and gating characteristics (Fig. 6, G and F), suggesting that TRPC3 is not involved in formation or regulation of I max channels.
Non-selective Channels in A431 and siTRPC3 Cells-Further single-channel analysis indicated that in addition to I min and I max channels, A431 cells express non-selective cation channels. These channels can be activated by UTP (10 out of 87 experiments, Fig. 7A) or store-depletion (13 out of 74 experiments, Fig. 6B) but not by InsP 3 (n ϭ 119, Fig. 7D) or 10 M OAG (n ϭ 12, data not shown), indicating receptor-independent mode of their activation. We previously characterized the channels with similar characteristics in HEK293 cells, and named them I NS (44). In A431 cells, I NS channels displayed the following basic properties: the reversal potential near 0 mV; the single-channel conductance of 4.5 pS (Fig. 7J); the lack of voltage dependence of channel open probability (Fig. 7E); and the mean open time ϳ3.4 ms (Fig. 7C). In contrast, no I NS channel activity was observed in siTRPC3 cells treated with Tg or TPEN (n ϭ 58, Fig.  7I). Selective suppression of I NS in siTRPC3 cells suggests that TRPC3 is an essential subunit or regulator of I NS channels. Because the I CRAC , I min and I max currents were similar in control A431 and siTRPC3 cells, suppression I NS channel activity in siTRPC3 cells is the most likely explanation for reduction of Tg-induced Ca 2ϩ influx that we observed in our Ca 2ϩ imaging experiments. On the other hand, Ca 2ϩ -imaging data suggests that suppression of TRPC3 did not affect the agonist-induced Ca 2ϩ entry (Fig. 3A). The later observation indicates that receptor-operated calcium influx is compensated in siTRPC3 cells. Indeed, in 15% of the single-channel experiments, the agonist application to siTRPC3 cells induced the activity of a new type of Ca 2ϩ -permeable channels (n ϭ 62, Fig. 7F). In 10 experiments (16%, n ϭ 62) the moderate constitutive activity of these channels was observed and was further enhanced after application of UTP (data not shown). In contrast to I NS , the open probability of these channels was highly voltage-dependent with increased activity observed at the membrane potentials above Ϫ50 mV (Fig. 7, E and G). Kinetic characteristics of these channels were also different from the I NS . Analysis of open-time histograms revealed the time constants of 6.6 ms (Fig. 7H), which is 2-fold higher than the time constant of I NS . Similar to I NS in control A431 cells, the channels observed in siTRPC3 displayed the reversal potential near 0 mV and the unitary conductance of ϳ4.5 pS (Fig. 7J). Thus, we called these channels I NS-2 , whereas the non-selective channels (I NS ) in A431 cells were renamed as I NS-1 Further analysis has shown that activity I NS-2 could not be evoked by store depletion with Tg or TPEN (n ϭ 58). Neither constitutive nor UTP-induced I NS-2 channel activity could be blocked by Gd 3ϩ (10 M) (n ϭ 22, data not shown). Together these data indicate that in contrast to I NS-1, I NS-2 are store-independent cation channels. In addition, activ- ity of I NS-2 in siTRPC3 cells could not be evoked by application of 2.5 M InsP 3 to the inside-out patches (n ϭ 51, Fig. 7I) or 10 M OAG to the cell-attached or inside-out patches (n ϭ 10 and data not shown). Thus, I NS-2 and I NS-1 channels display distinct biophysical properties and modes of activation. Our singlechannel analysis indicates that in A431 and siTRPC3 cells, agonist-induced Ca 2ϩ influx is mediated by diverse set of ion channels. Considering that the single-channel conductance, selectivity and the observation frequency of I NS-1 and I NS-2 are very similar, we conclude that suppression of I NS-1 is compensated by up-regulation of I NS-2 channels that contribute to the agonist-induced Ca 2ϩ influx in siTRPC3 cells. Thus, suppression of TRPC3 in A431 cells has not altered the agonist-evoked Ca 2ϩ entry, but affected its regulation which can be elucidated only by single-channel analysis. The different situation was observed with store-dependent Ca 2ϩ influx. Because I NS-2 channels are store-independent, and do not mediate the store depletion-evoked Ca 2ϩ influx, the decrease of Tg-induced Ca 2ϩ entry in siTRPC3 cells was observed in both Ca 2ϩ imaging and whole cell experiments.

DISCUSSION
By using a combination of Ca 2ϩ imaging and electrophysiological recordings we found that in A431 cells, the native TRPC3 is essential for activity of Ca 2ϩ permeable channels that support store-operated Ca 2ϩ influx. Despite the increasing evidence that TRPC proteins mediate store-operated currents, the role of TRPCs in forming or regulating SOC channels remains controversial. Although some overexpression studies have suggested that recombinant TRPC3 forms receptor-operated channels (20 -24), several knockdown studies indicate that TRPC3 is required for SOCs activity. For example, the TRPC3 antisense decreased the storedependent calcium influx in HSY and PS1 cells (45,46). In addition, TRPC3 antisense and siRNA reduced receptor-and store-operated Ca 2ϩ influx in HEK293 cells (31,32). In our previous studies we demonstrated that HEK293 cells express four types of agonist-and store-activated Ca 2ϩ channels that display distinct permeability and activation profiles: I CRAC , I min , I max , and I NS (44). We also characterized the highly selective I CRAC and moderately selective I SOC currents in A431 cells, and shown that in these cells, I SOC are partly supported by I min channels (33). In the present study, we investigated the role or native TRPC3 in mediating receptor-and store-operated currents in A431 cells. We generated a stable cell line in which the expression levels of native TRPC3 were significantly reduced (siTRPC3, Fig. 2). We found that in siTRPC3 cells, the UTP-induced Ca 2ϩ influx was normal (Figs. 3A and 4A) suggesting that in these cells, TRPC3 is not directly involved in regulation of receptor-operated cation channels. However, it is also possible that the effect of reduction of TRPC3 on receptor-operated calcium influx was compensated by other TRP homologues. In contrast to the agonist-induced currents, the calcium currents triggered by passive store-depletion were significantly reduced in siTRPC3 cells (Figs. 3 and 4). Thus, in A431 cells, TRPC3 is essential for Ca 2ϩ entry mediated by depletion of intracellular stores. The Tg-induced currents observed in control A431 cells and siTRPC3 cells, displayed different sensitivity to Gd 3ϩ (Fig. 3D) suggesting that reduction of TRPC3 levels affects the composition of functional cation channels in the plasma membrane. In siTRPC3 cells, more than 45% of Ca 2ϩ influx was store-independent and Gd 3ϩ -insensitive indicating that Ca 2ϩ influx in these cells is supported by constitutive activity of store-independent Ca 2ϩ channels. Thus, we investigated the single cation channels expressed in A431 and siTRPC3 cells.
Our detailed single-channel analysis has shown that A431 cells express several types of cation channels. Although we could not analyze the I CRAC channels because their unitary conductance is too small (6), we found that similarly to the HEK293 cells, A431 cells express three distinct types of channels: I min , I max , and I NS . The I min channels displayed a 1.2 pS unitary conductance for divalent cations (35,36), a 10 pS conductance for monovalent cations (33) and high selectivity for divalent cations (36). In A431 cells, I min could be activated by extracellular UTP, intracellular InsP 3 , depletion of intracellular Ca 2ϩ stores with Tg, or BAPTA-AM (35-39, see also Fig.  5F). The I max channels displayed a 18 pS unitary conductance for divalent cations and lower selectivity than I min (Fig. 6G). Similar to the HEK293 cells (44), activity of I max channels in A431 cells could be evoked by extracellular UTP or intracellular InsP 3 (Fig. 6, A and H). Together, these data indicate that in A431 cells, I min and I max channels function in both receptor-and store-operated modes. In addition to I min and I max , we also identified non-selective 4.5 pS I NS-1 channels (Fig. 7H). The I NS-1 channels could be activated by store-depletion but not by intracellular InsP 3 (Fig. 7I). Thus, I NS-1 function exclusively as store-operated channels. We found that siTRPC3 cells also express I min and I max channels that display the biophysical characteristics and modes of regulation similar to control A431 cells. In addition, the observation frequency of I min and I max channels was not affected by suppression of TRPC3. These findings rule out the possibility that TRPC3 directly acts as a subunit for I min and I max channels.
In contrast to control A431 cells, we could not detect active I NS-1 channels in the plasma membrane of siTRPC3 cells. Neither passive store depletion, nor agonist application could activate I NS-1 in siTRPC3 cells indicating that TRPC3 is essential for forming functional I NS-1 channels. However, in 15% cell-attached experiments we registered a new type of nonselective, Gd 3ϩ -insensitive I NS-2 channels. I NS-2 dis- played a unitary conductance of 4.5 pS conductance for divalent cations (Fig. 7H) and high voltage-dependence with increased activity at membrane potentials above Ϫ50 mV (Fig. 7, E and F). I NS-2 could be activated by UTP but not by InsP 3 or depletion of intracellular calcium stores (Fig. 7I). The application of high resolution single-channel patch clamp analysis in our study revealed that reduction of TRPC3 expression level caused complex changes in functional properties of store-and receptoroperated Ca 2ϩ influx channels in A431 cells. These changes could not be clearly identified using low-resolution methods such as Ca 2ϩ -imaging techniques (Fig. 3) or whole-cell Ca 2ϩ current recordings (Fig. 4). In the future, similar single-channel approach can be taken to dissect functional roles of other proteins proposed to serve as subunits of store-operated Ca 2ϩ channels in cells.