Gαi-mediated TRPC4 activation by polycystin-1 contributes to endothelial function via STAT1 activation

Hypertension and aneurysm are frequently associated with autosomal dominant polycystic kidney disease (ADPKD) caused by polycystin-1 (PC1) mutations, which is closely related to endothelial dysfunction. PC1 is an atypical G-protein-coupled receptor that activates G-proteins by self-cleavage; currently, however, the molecular and cellular mechanisms of the associated intracellular signaling and ion channel activation remain poorly elucidated. Here, we report an activation mechanism of a calcium-permeable canonical transient receptor potential 4 (TRPC4) channel by PC1 and its endothelial function. We found that the inhibitory Gαi3 protein selectively bound to the G-protein-binding domain on the C-terminus of PC1. The dissociation of Gαi3 upon cleavage of PC1 increased TRPC4 activity. Calcium influx through TRPC4 activated the transcription factor STAT1 to regulate cell proliferation and death. The down-regulation of PC1/TRPC4/STAT1 disrupted migration of endothelial cell monolayers, leading to an increase in endothelial permeability. These findings contribute to greater understanding of the high risk of aneurysm in patients with ADPKD.

SCIeNtIfIC RepoRts | (2018) 8:3480 | DOI: 10.1038/s41598-018-21873-1 classical TRP (TRPC) is a receptor-operated channel (via G protein-coupling), which is primarily activated in response to PLC activation 21 or inhibitory Gα (Gα i ) interactions 22 . By inducing dissociation of heterotrimeric Gα i/o /βγ proteins by PC1, we hypothesized that PC1 could activate the TRPC4 channel via Gα i/o . Vascular endothelial cell Ca 2+ entry through TRPC4 leads to vascular smooth muscle relaxation in an endothelial acetylcholine-dependent manner, as well as endothelial hyperpermeability via disruption of cell junction complexes or cytoskeletal reorganization 23 . Cerebral aneurysms are more common in ADPKD patients with loss of function or missense mutations in the PC1-encoding PKD1 gene [24][25][26] . Accordingly, the functional interaction of TRPC4 with PC1 is essential for Ca 2+ regulation in the endothelium, but their pathophysiological mechanisms remain unknown.
In the present study, we overexpressed recombinant TRPC4 and PC1 in HEK cells and analyzed ion channel activity using electrophysiological techniques and Ca 2+ -dependent signaling using molecular biological methods. Furthermore, we confirmed the functional interaction of TRPC4 with PC1 in HUVECs. Taken together, these findings are consistent with Gα i -dependent TRPC4 activation by the PC1 protein. The Gα i dissociation by GPS cleavage of PC1 activates TRPC4, which in turn elevates Ca 2+ levels available for triggering the activation of STAT1 in endothelial cells.

Results
Identification of polycystin-1 (PC1). Polycystin-1 (PC1) is a large plasma membrane glycoprotein that undergoes several proteolytic cleavages, including autocatalytic cleavage at the G protein-coupled receptor proteolytic site (GPS) (Fig. 1). The cleaved PC1 consists of an N-terminal fragment (NTF) associated with a C-terminal fragment (CTF). At least three other cleavages liberate portions of the cytoplasmic C-terminal tail (CTT) of PC1 (Fig. 1A,B). To determine PC1 expression patterns, we used variously tagged (e.g., GFP, Flag, and HA) constructs at each terminus of PC1 and performed Western blot analysis with several detection antibodies using lysates of HEK293 cells expressing human PKD1 (Fig. 1B). Using an antibody against the PC1 N-terminus (7E12), two bands at ≥460 kDa, full-length (FL) at 520 kDa and NTF at 440 kDa, were observed in a PC1 construct flag-tagged at the N terminus (PC1-Flag (FL-Flag)) and a PC1 construct GFP-tagged at the C terminus (PC1-GFP (FL-GFP)) ( Fig. 1C, Supplementary Fig. 9). With an antibody against the PC1 C-terminus (A-20), FL and CTF (130 kDa) were detected in PC1-Flag, and FL-GFP and CTF-GFP (157 kDa), a cleavage product of hPC1-GFP, were detected in PC1-GFP (Fig. 1C, Supplementary Fig. 9). With an antibody against GFP (GFP), FL-GFP and CTF-GFP were detected in PC1-GFP. With an antibody against Flag (Flag), CTF was detected in CTF-Flag.

Characterization of PC1 as a G protein-coupled receptor (GPCR). A fundamental feature of PC1 is
post-translational modification via cleavage at the juxtamembrane GPCR proteolysis site (GPS) motif that is part of the larger GAIN domain 27 . The PC1 C-terminal cytosolic domain also has a G protein activation region, which is defined as a sequence of ≤25 amino acids with the consensus motif BB…..BBxB or BB…..BBxxB (B = R, K, or H) ( Fig. 2A). Signaling pathways of PC1 may be mediated by activating or binding to heterotrimeric G proteins 13 . Therefore, PC1 may act as an atypical GPCR, which belongs to the GPCR family with GAIN domain.
To identify binding between PC1 and specific Gα protein subunits, we heterologously expressed CTF of PC1 (PC1(CTF)) and Gα subunits in HEK293 cells and analyzed them via co-immunoprecipitation (Co-IP) and FRET imaging (Fig. 2). The CTF of PC1 has a C-terminal cytoplasmic tail (CTT), including a G protein activation region and a coiled-coil domain. The CTF of PC1 was used because the N-terminal extracellular region does not participate in cleavage signaling. Protein was immunoprecipitated with anti-A-20 and then probed with anti-Gα antibodies. The IP band was observed only for the co-expression of PC1(CTF) with Gα i3 , indicating that PC1 specifically interacts with Gα i3 (Fig. 2B, Supplementary Fig. 10). The FRET efficiency between PC1(CTF) and Gα i was also measured. When we co-transfected CFP-tagged PC1(CTF) and YFP-tagged Gα i , the highest FRET efficiency was recorded between PC1(CTF) and Gα i3 (Fig. 2C). Next, the interaction between full-length PC1 (PC1(FL)) and Gα i3 was investigated ( Supplementary Fig. 1A). The binding was weaker than for PC1(CTF) (Supplementary Fig. 1C). A GPS site deletion mutant (ΔGPS) was also used to evaluate the interaction with Gα i3 (Supplementary Fig. 1B). Other G proteins, e.g., Gα s , Gα q , and Gα 12 , did not bind with PC1(CTF) (Supplementary Fig. 2A). These results suggest that PC1 is able to bind to specific G protein subunits and likely acts as a GPCR in cells and tissues.
The activation of the TRPC4β channel by PC1 cleavage. Since PC1 is considered a GPCR, albeit an atypical one, and TRPC4β can be activated by GPCR, we investigated whether PC1 affects TRPC4β channel currents. The TRPC4β channels expressed in HEK293 cells had lower basal activity. The measurements of TRPC4β activity could be manipulated by altering the extracellular ion composition. To efficiently measure TRPC4β activity, we used 140 mmol/L Cs + -rich solution on the basis of the high permeability of Cs + ions in TRPC4. The amplitude of TRPC4 currents was 4 ± 1 pA/pF (n = 7) in 140 mmol/L Cs + -rich solution. When TRPC4β was co-expressed with PC1(FL), TRPC4β current was increased by PC1(FL) (41 ± 14 pA/pF, n = 7) under Cs + -rich conditions (Fig. 3A,B). The co-expression of TRPC4β with PC1(CTF) did not increase TRPC4β currents (7 ± 3 pA/pF, n = 3) compared with controls (Fig. 3B). To activate TRPC4β, we used intracellular GTPγS, which activates different types of G proteins, through a patch pipette. Intracellular GTPγS administration fully activated TRPC4β. GTPγS-induced Cs + currents in TRPC4β by PC1(FL) or PC1(CTF) showed no differences compared with controls (Fig. 3C,D). We investigated whether Gα i3 that was dissociated from CTT by cleavage of PC1 affected TRPC4β activity. We observed the interaction of TRPC4β with Gα i3 protein in both the presence and absence of PC1(FL) using Co-IP. Expression of PC1(FL) significantly increased their physical interaction (Fig. 3E, Supplementary Fig. 11). TRPC4β current was inhibited by a dominant negative form of Gα i3 protein (Gα i3 G202T) in HEK cells transfected with PC1(FL) (Fig. 3F). To determine whether PC1 constitutively activates the TRPC4 channel, we performed Ca 2+ measurements and analyzed the relative increase in calcium. PC1(FL) SCIeNtIfIC RepoRts | (2018) 8:3480 | DOI:10.1038/s41598-018-21873-1 significantly increased TRPC4β-mediated Ca 2+ influx when the external Ca 2+ was increased from 0 mmol/L to 2 mmol/L (Fig. 3G,H). The pretreatment of 100 ng/ml PTX partially attenuated the relative increase of Ca 2+ in HEK cells co-expressed with PC1 and TRPC4β (Fig. 3I). Finally, we investigated whether PC1 increases TRPC4β currents by regulating channel expression. PC1 did not increase the surface expression of TRPC4β channel ( Supplementary Fig. 3). These results suggest that the activation of TRPC4β by PC1 is mediated by Gα i3 , leading to increased intracellular Ca 2+ levels. PC1 is constitutively cleaved at His-Leu↓Thr3049 (between Leu3048 and Thr3049) within the GPS domain 28,29 . The most well-characterized mechanism is the cis-autoproteolysis, a self-catalyzed protein rearrangement that results in cleavage at HX↓(T/S/C) 30 , where X indicates all amino acids. Mutants of non-cleavable PC1 were generated by site-specific mutagenesis to confirm cleavage at the GPS domain and to investigate the role of consensus HLT sequences for cleavage (Fig. 4A). Such a mechanism of cis-autoproteolysis requires Thr, Ser, and Cys, which contain nucleophilic side chains (-OH or -SH group) to support cleavage. Substitution of Thr by Ser or Cys did not disrupt the cleavage. In contrast, substitution of Thr by Val, Gly, or Arg and deletion of the GPS domain (ΔGPS) blocked the cleavage. To confirm the correlation between cleavage and pathologic mutations affecting the sequence at or near GPS, two germline mutants (L2993P and Q3016R) were examined. Each of the mutants almost completely inhibited the cleavage (Fig. 4B, Supplementary Fig. 12). Next, we investigated whether the activation of TRPC4β depended on PC1 cleavage at the GPS site. Non-cleavable mutants (L2993P, Q3016R and T3049G/R/V) did not activate TRPC4β channels. In contrast, TRPC4β currents were increased in cells transfected with T3049C or T3049S, where cleavage occurs at the GPS (Fig. 4C). These results suggest that cleavage at the HL↓T of PC1 is required for the activation of TRPC4 channels. STAT1 phosphorylation by PC1-mediated activity of TRPC4β. PC1 has been implicated in a variety of intracellular signaling events, including JAK-STAT signaling. Overexpression of full-length PC1 can activate signal transducer and activator of transcription 1 (STAT1), STAT3 and STAT6 9,31,32 , which mediate signaling involved in proliferation, differentiation and death. STATs are phosphorylated and activated by protein tyrosine kinases, including growth factor receptors such as EGFR, and non-receptor tyrosine kinases (e.g., Src and JAK) 33 . To confirm whether STAT1 activation by PC1 was dependently regulated by Src kinase activity, we transiently transfected HEK 293 cells with dominant-negative Src (Src DN ) mutants. The basal level of phosphorylated STAT1 was not detected in HEK cells or changed by Src DN expression. STAT1 was activated by PC1 and wild-type Src (Src WT ), and co-expression of PC1 and Src further increased STAT1 activation levels. Activation of STAT1 by PC1 was not affected by Src DN (Fig. 5A,D, Supplementary Fig. 13). These results suggest that PC1 leads to Src-independent activation of STAT1 through tyrosine phosphorylation.
We have also investigated whether activation of the TRPC4β channel by PC1 affects STAT1 phosphorylation. STAT1 was not activated by the TRPC4β channel itself. Activation of STAT1 was increased by PC1-mediated activation of TRPC4β when TRPC4β was co-expressed with PC-1 (Fig. 5B,E, Supplementary Fig. 13). Since it has been reported that CTT of PC1 is released by γ-secretase-mediated cleavage 34 , we investigated whether the phosphorylation levels of STAT1 through PC1-mediated TRPC4β activity was inhibited by the γ-secretase inhibitor DAPT ( Supplementary Fig. 4). Transfection mixtures of PC1 and/or TRPC4β were added drop-wise to cell culture media containing 80 μmol/L DAPT and incubated for 24 hours. STAT1 activity of PC1/TRPC4β was inhibited by DAPT, indicating that STAT1 phosphorylation is associated with CTT cleaved from PC1. To investigate the potential role of Ca 2+ entry through the TRPC4β channel in increased STAT1 phosphorylation, we observed the effects of removing extracellular calcium ions using Ca 2+ -free media. The increased level of STAT1 was significantly attenuated in Ca 2+ -free medium compared with normal conditions (Fig. 5C,F, Supplementary  Fig. 13). These results suggest that the CTT of PC1 activates TRPC4β via Gα i3 protein and that calcium influx through TRPC4β is required for the activation of STAT1.
Effects of the PC1/TRPC4β/STAT1 pathway on endothelial migration. The TRPC family plays a role in normal and pathophysiological vascular functions 35,36 . TRPCs are involved in vascular tone (e.g., TRPC4, TRPV1, and TRPV4), regulation of vascular permeability (e.g., TRPC1, TRPC4, TRPC6, and TRPV1), hypoxia-induced vascular remodeling (e.g., TRPC4), angiogenesis (e.g., TRPC4 and TRPC6), endothelial cell proliferation, and apoptosis 37 . TRPC4 plays significant roles in normal and pathophysiological vascular function. PC1 also has an important role in vascular function. PC1 knock-out mice died in mid-gestation with a variety of phenotypes, including a vasculopathy characterized by profound edema 38,39 . To investigate the relationship between the PC1/TRPC4β pathway and endothelial function, we first investigated the expression of TRPC4 and PC1 in HUVECs using Western blot analysis. Expression of PC1 was detected with anti-7E12 or anti-A-20 antibodies in endothelial cells (Fig. 6A, Supplementary Fig. 14). TRPC4β was predominantly expressed in endothelial cells compared with TRPC4α isoforms (Fig. 6B, Supplementary Fig. 14). In addition, Gα i3 was expressed in the given cell lines ( Supplementary Fig. 5). These results show that all signaling proteins related to the PC1-Gα i3 -TRPC4β pathway are expressed in HUVECs.
First, we examined the silencing effect of TRPC4β and PC1 on HUVEC migration. In wound-healing assays, endothelial cells treated with vehicle were able to close ~100% of the wound. In contrast, endothelial cells treated with 20 μmol/L ML204, a TRPC4 inhibitor, were only able to close ~40% of the wound (Fig. 6E). The migration rate of HUVECs transfected with PKD1 siRNA or TRPC4 siRNA was also significantly decreased (Fig. 6C,D). To identify whether STAT1 affects endothelial migration, STAT1 siRNA was transfected into HUVECs. The migration of STAT1-knocked-down cells was inhibited, as predicted (Fig. 6F). To test siRNA efficiency, PC1, TRPC4 and STAT1 transcript levels were evaluated by Western blot. The tested siRNAs effectively reduced the levels of protein expression (Supplementary Fig. 6). These results suggest that PC1/TRPC4β/STAT1 pathway plays an important role in endothelial migration.
To evaluate the functional activity of TRPC4, we measured the channel currents and the intracellular Ca 2+ levels by knock-down of TRPC4. In HUVECs transfected with siControl, basal TRPC4 activity showed constitutively active current, which was induced by CsCl. Englerin A (EA), a TRPC4 agonist, slightly increased the inward current of TRPC4. Both basal and EA-evoked currents showed typical double-rectifying current-voltage relationships known to be features of the TRPC4 channel (Fig. 6G). By knock-down of TRPC4, the TRPC4-like current was completely reduced. EA-induced Ca 2+ influx was also reduced by knock-down of TRPC4 (Fig. 6G,  Supplementary Fig. 7A).
Effects of the PC1/TRPC4β/STAT1 pathway on adherens junctions. To determine whether PC1 or TRPC4β affects vascular permeability, we investigated the distribution of an endothelial-specific cadherin, VE-cadherin, and leakage of Evans blue dye in HUVECs. We evaluated the endothelial permeability using Evans blue dye. Upon siTRPC4 or ML204 treatment with or without siPKD1, the permeability of the endothelial cell monolayers to Evans blue dye was significantly increased (Fig. 7A,B). Next, the junction protein VE-cadherin was visualized via immunofluorescence staining. Treatment with siPKD1, ML204 or siSTAT1 decreased junction-localized VE-cadherin levels (Fig. 7C). Subsequently, the expression of VE-cadherin was assessed by Western blot analysis. There was no significant difference in the protein level of VE-cadherin ( Supplementary  Fig. 8). These data show that PC1 knock-down, ML204 or siSTAT1 treatment on endothelial cells induced VE-cadherin internalization. These results suggest that PC1-mediated TRPC4β activation plays an important role in the stability of endothelial junctions and permeability.

Discussion
In the present study, we found that PC1-mediated TRPC4β activity plays a key role in endothelial migration and permeability. We have shown that TRPC4β can be activated by PC1, although its mechanism is not clear. Several lines of evidence suggest that PC1 functions as a G protein-coupled receptor. First, the C-terminal tail of PC1 contains a G protein-binding region. This region is conserved across many species. In our hands, PC1 coimmunoprecipitated with Gα i3 , indicating that PC1 specifically interacts with the Gα i3 protein. FRET efficiency between PC1 and Gα i3 also increased compared with other Gα i isoforms. Second, PC1 is cleaved at the GPS, which is in an immediate location of the first transmembrane domain, and this cleavage results in G protein-mediated signaling cascades. The GPS motif was first identified as a neuronal GPCR, CIRL/latrophilin 40 , and has recently been recognized as a part of the larger GPCR autoproteolysis-inducing (GAIN) domain, which is also found in PC1. Interestingly, the cleaved PC1 N-terminal fragment remains non-covalently attached to the membrane-bound C-terminal fragment 29 . Such a heterodimeric PC1 is required to transduce the signal through G proteins and plays an important role in biological functions. In contrast, it has been reported that deletion of the NTF results in constitutive activation of several aGPCRs 41 , suggesting that NTF association might normally prevent constitutive activation. Interestingly, all of the disease-associated missense mutations located in the GAIN domain and the adjacent REJ module of PC1 analyzed to date impair or disrupt cleavage. Defective GPS cleavage of PC1 has been found in a subset of ADPKD patients with aneurysmal rupture 42 . We generated non-cleavable mutants of PC1 and identified the loss of functional properties of PC1 in activating the TRPC4β channel through the Gα protein.
Third, PC1 is a large integral membrane protein with 11 transmembrane segments that structurally resembles a receptor or adhesion molecule. Thus, these multiple lines of evidence demonstrate that PC1 represents structural or functional characteristics of GPCR.
In a previous report, we showed that Gα i proteins played an essential and novel role in the activation of TRPC4 22 . In our hands, PC1 activated the TRPC4β channel through the Gα protein. First, PC1(FL) but not PC1(CTF) activated TRPC4β. PC1(CTF) alone did not activate TRPC4β because NTF is required for G protein-mediated signaling through the C-terminal tail of PC1. Second, non-cleavable mutants of PC1 did not activate TRPC4β current when cleavage of PC1 N-terminus and C-terminus was blocked by missense mutations at the GPS domain. Third, when intracellular Ca 2+ levels were measured using Fura-2, PC1(FL) increased Ca 2+ influx through the TRPC4β channel. Fourth, intracellular 0.2 mmol/L GTPγS-induced TRPC4β activation was  not significantly different in the presence or absence of PC1. Fifth, activation of TRPC4β by PC1(FL) was inhibited by expression of a dominant-negative Gα i3 variant. Therefore, these results demonstrate unambiguously that TRPC4β is activated by an atypical Gα i3 coupled-receptor, PC1.
Next, we identified intracellular signaling cascades through a rise in cytosolic Ca 2+ due to PC1/Gα i3 /TRPC4β. Many studies have reported that disturbances in the balance between cell proliferation and apoptosis cause ADPKD. Abnormal proliferation in tubular epithelial cells plays a crucial role in cyst development and/or growth in PKD 43 . The kidneys from patients with ADPKD demonstrate high levels of apoptosis, as well as cellular proliferation 44 . Intracranial aneurysm is also believed to develop as a result of disruption of the balance between cell proliferation and apoptosis 45 . Indeed, proliferation and apoptosis must be tightly regulated. Among many regulatory factors of proliferation and apoptosis, we observed activation of STAT1 by PC1/Gα i3 /TRPC4β. STAT1 is predominantly phosphorylated by activation of PC1-mediated TRPC4β. The Ca 2+ influx can also lead to phosphorylation of STAT1 on tyrosine residues. PC1, which is localized to the primary cilium, functions as flow-sensitive mechanosensor 46 . Cleavage of the PC1 protein is a response to various stimuli, such as extracellular fluid flow 47 . The products of these cleavages perform important physiological functions. Nevertheless, it is still unclear how and where PC1 is cleaved. According to previously reported data, there are cleavage-inducible factors, such as mechanical stimuli 47 , polycystin-2 (PC2) 48 , and γ-secretase 34 . Thus, perfusion flow in calcium imaging and patch clamp experiments may generate mechanosensory stimulation. In our hands, the GPS domain was used to induce autoproteolysis of PC1 into NTF and CTF, whereas the CTT of PC1 was released by γ-secretase-mediated cleavage. The increased levels of STAT1 phosphorylation via PC1-mediated TRPC4β activity were inhibited by treatment with the γ-secretase inhibitor DAPT (Supplementary Fig. 4). Thus, CTT of PC1 activated TRPC4β via the Gα i3 protein, and extracellular calcium through TRPC4β was required for the activation of STAT1.
Endothelial dysfunction is a hallmark of aneurysm. Aneurysm is one of the most common manifestations in ADPKD. The dysregulation of TRPC4 results in vascular endothelial dysfunction. A role of TRPC4 in vascular endothelial function was previously reported that acetylcholine-induced vasorelaxation occurred due to acetylcholine-dependent NO production in aortic endothelial cells, which was largely reduced in TRPC4 knock-out mice 49 . Reduced calcium entry in TRPC4-deficient lung endothelial cells was also associated with reduced thrombin-induced formation of actin stress fibers, reduced endothelial cell retraction, and reduced microvascular endothelial permeability evoked by a PAR-1 agonist peptide in isolated lungs 50 . A missense SNP (TRPC4-I957V) in the TRPC4 human gene was further found to be associated with a reduced risk of myocardial infarction. The proposed mechanism underlying protection involved improved endothelial function 51 . In addition, a TRPC4-mediated Ca 2+ signaling pathway evoked by EGF was specifically identified in sub-confluent, proliferating clusters of human microvascular endothelial cells. The abundance of TRPC4 in the plasma membrane and its contribution to Ca 2+ entry depend on the proliferation state, and its activity is regulated by cellcell contact formation in a β-catenin-dependent manner 23 . In our hands, inhibition of PC1-mediated TRPC4β activity induced endothelial dysfunction, as demonstrated by the reduction of endothelial migration (Fig. 6C-E) and cell-cell junctions (Fig. 7C) by down-regulation of PC1 or TRPC4. Although histamine and thrombin could increase the permeability of HUVECs by a robust Ca 2+ release and Ca 2+ entry ( Supplementary Fig. 7) via receptor-operated and store-operated channel (SOC), sustained Ca 2+ influx through TRPC4, which is constitutively activated by PC1, may play a role in refilling the ER Ca 2+ stores, which require further investigation. Furthermore, Ca 2+ -dependent STAT1 participates in other signaling pathways associated with endothelial function in strengthening tight junctions. Thus, knock-down of PKD1, TRPC4 or STAT1 increases endothelial permeability. Based on these findings, we propose the activation of PC1-mediated TRPC4β in endothelial cells as a tentative molecular target for cerebral aneurysms associated with ADPKD.
In conclusion, the main findings of this study are as follows ( Western blotting, Co-IP, and Surface biotinylation. Cells were plated in 6-well dishes. Lysates were prepared in lysis buffer (0.5% Triton X-100, 150 mmol/L NaCl, 50 mmol/L HEPES, 2 mmol/L MgCl 2 , 2 mmol/L EDTA, pH 7.4) via passage 10-15 times through a 26-gauge needle after sonication. After lysates were centrifuged at 13,000 × g for 10 minutes at 4 °C, the protein concentration in the supernatants was determined. The extracted proteins in sample buffer were loaded onto 5, 8, or 10% Tris-glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. The proteins were transferred onto a PVDF membrane. For details concerning the antibodies, please see the supplementary materials.
In the Co-IP experiments for detection of PC1-Gα subtypes, 500 μL of cell lysates (500-1000 μg) were incubated with 1 μg of anti-PKD1 (A-20) or anti-Gα antibodies and 30 μL of protein G-agarose beads at 4 °C overnight with gentle rotation. After the beads were washed three times with wash buffer (0.1% Triton X-100), the precipitates were then eluted with 30 μL of 2× Laemmli buffer and subjected to Western blot analysis.
For surface biotinylation, cells were first washed with PBS and incubated in 0.5 mg/mL sulfo-NHS-LC-biotin (Pierce, USA) in PBS for 30 minutes on ice. The biotin was quenched by 100 mmol/L glycine in PBS. The cells were then processed as described above for cell extraction. Forty microliters of 1:1 slurry of immobilized avidin beads (Pierce, USA) was added to 300 μL of cell lysates (500 μg protein). After incubation for 1 hour at room temperature, the beads were washed three times with 0.5% Triton X-100 in PBS, and proteins were extracted in sample buffer. The collected proteins were analyzed by Western blot.
Electrophysiology. The transfected cells were trypsinized and transferred to a recording chamber, which was equipped for the application of a number of solutions. Whole-cell currents were recorded using an Axopatch 200B amplifier (Axon Instruments, USA) and Digidata 1440 A Interface (Axon Instruments), and analyzed using a personal computer equipped with pClamp 10.2 software (Axon Instruments) and Origin software (Microcal origin v.8.0, USA). Patch pipettes were made from borosilicate glass and had resistances of 2-4 MΩ when filled with standard intracellular solutions. For whole cell experiments, we used an external bath medium (normal Tyrode solution) of the following composition (in mmol/L): 135 NaCl, 5 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 glucose, and 10 N- [2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES) with the pH adjusted to 7.4 using NaOH. Cs + -rich external solution was made by replacing NaCl and KCl with equimolar CsCl. The standard pipette solution contained the following (in mmol/L): 140 CsCl, 10 HEPES, 0.2 Tris-GTP, 0.5 EGTA, and 3 Mg-ATP with the pH adjusted to 7.3 using CsOH. Intracellular 50 nmol/L, 200 nmol/L, or 5 μmol/L free Ca 2+ pipette solutions were chosen on the basis of previous studies of TRPC5. Voltage ramp pulses were applied at −60 mV with a holding potential from +100 to −100 mV for 500 ms. A salt-agar bridge was used to connect the ground Ag-AgCl wire to the bath solution for the experiments that used reducing agents. All current traces are the selected values at −60 or +80 mV of the ramp pulses. The inward current amplitudes at −60 mV are summarized for all bar graphs. The current recording was performed as previously described 52 . Fluorescence Resonance Energy Transfer (FRET) measurements. Three FRET images (cube settings for CFP, YFP, and Raw FRET) were obtained from a pE-1 Main Unit to 3 FRET cubes (excitation, dichroic mirror, filter) via a fixed collimator. The excitation LED and the filter were sequentially rotated, and the rotation period for each filter cube was ~0.5 s. All of the images were obtained within 1.5 seconds. Each image was captured on a cooled 10 MHz (14 bit) CCD camera (ANDOR technology, USA) with 100 ms of exposure time with 2 × 2 binning (645 × 519 pixels). Using IX70, an Olympus microscope equipped with a 60× oil objective, the three-cube FRET efficiency was analyzed using MetaMorph 7.6 software (Molecular Devices, USA). For details of FRET ratio and FRET efficiency computation, please see the supplementary materials. Intracellular Ca 2+ measurements with Fura-2. The ratiometric measurement of [Ca 2+ ] i was performed using Fura-2-AM (molecular probe, USA). The cells were seeded in 24-well dishes and loaded with 5 μmol/L of Fura-2-AM for 30 minutes at 37 °C. The Fura-2 fluorescence was measured at 510 nm emission with 340/380 nm dual excitation using a DG-4 illuminator. The experiments were performed in a normal solution containing 145 mmol/L NaCl, 3.6 mmol/L KCl, 10 mmol/L HEPES, 1.3 mmol/L CaCl 2 , 1 mmol/L MgCl 2 , and 5 mmol/L glucose with pH adjusted to 7.4 using NaOH.
Wound-healing assay. HUVECs transfected with PKD1, TRPC4 or STAT1 siRNA were seeded in 6 well culture plates and incubated with ML204, a selective TRPC4 channel inhibitor. Cells were grown as high-density monolayers, scratched with a 200 μL pipette tip, and allowed to migrate for the indicated times after three washes to remove detached cells. Migration was recorded using a Nikon ECLIPSE TS100 microscope equipped with a Lumenera's INFINITY1-3 digital camera. The area covered by the monolayer was measured using ImageJ (National Institutes of Health, USA). All migration assays are representative of at least three independent experiments.
In vitro endothelial permeability assay. To measure permeability changes of endothelial cells in vitro, HUVECs were cultured to confluence on cell culture inserts (3.0 μm, BD Biosciences) placed in a 12-well plate. 5 × 10 5 HUVECs were seeded on each cell culture insert. In control samples, 500 μL of Evans blue dye (0.67 mg/ mL) was added to the upper compartment to demonstrate that the endothelial layers were impermeable to this dye. Experimental wells were treated with siPKD1, siTRPC4 or ML204 for 48 hours, and 500 μL dye was added to the upper compartments. The liquid was collected from the lower wells after 10 minutes and measured by spectrophotometric absorbance at 570 nm.
The cells were fixed with 4% paraformaldehyde for 15 minutes. After washing in PBS, cells were incubated with an anti-VE-cadherin antibody at 4 °C overnight. VE-cadherin was localized using Alexa Fluor 488 (Invitrogen, USA). Nuclei were stained with Hoechst 33342 (Invitrogen, USA). The cells were examined using a fluorescence microscope, and images were analyzed using Metamorph software (Molecular Devices, USA).

Statistics.
The results are presented as the means ± s.e.m. They were compared using Student's t-tests between two groups, or ANOVA followed by post-hoc tests among three or more groups. p < 0.05 was considered statistically significant. The number of cells for electrical recordings is provided as an n value in the bar graphs.
Data availability. The data generated during the current study are available from the corresponding author upon reasonable request.