Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited diseases. ADPKD is characterized by the progressive expansion, in both kidneys, of multiple fluid-filled cysts, which gradually replace normal renal tissue and ultimately result in end-stage renal failure [1]. In ADPKD-causative genes, the polycystic kidney disease 1 (PKD1) and PKD2 genes are involved in the activation of cation-permeable currents by regulation of G-protein signaling, as well as the control of epithelial cell population growth, migration, differentiation and apoptosis [1,2]. PKD1 (also known as polycystin-1 [PC-1]) is a glycoprotein that consists of approximately 4,302 amino acids, PKD1 functions as an atypical G protein coupled receptor (GPCR), binds heterotrimeric G_αi/o proteins and regulates calcium flux through PKD2 (also known as PC-2 or transient receptor potential polycystin 2 [TRPP2]) by releasing G_βγ subunits [1]. Thus, PKD1 and PKD2 are related to disturbed intracellular Ca²⁺ homeostasis and cyclic adenosine monophosphate (cAMP) accumulation, leading to abnormal cell proliferation and the growth of multiple cysts [3,4].

Transient receptor potential (TRP) channels make up a family of seven cationic channels, which are divided into 7 subfamilies based on amino acid similarity. TRP channels can form functional homo- or hetero-tetrameric channels with intra-subgroups or even with inter-subfamilies [5]. The classical TRP (TRPC) is a receptor-operated channel (via G protein-coupling), which is primarily activated in response to phospholipase C (PLC) activation [6] or inhibitory G_α (G_αi) interactions [7,8]. We identified the mechanism by which PKD1 activates the calcium-permeable TRPC4 channel and its role in regulating endothelial function. Our results show that the inhibitory G_αi3 protein selectively binds to the G-protein-binding domain located on the C-terminus of PKD1. Upon PKD1 cleavage, G_αi3 dissociates, leading to enhanced TRPC4 activity. The subsequent calcium influx through TRPC4 activates the transcription factor STAT1 (signal transducer and activator of transcription 1), which controls cell proliferation and apoptosis. Downregulation of the PKD1/TRPC4/STAT1 pathway impairs endothelial cell monolayer migration, resulting in increased endothelial permeability [9].

In the absence of PKD1, PKD2 primarily localizes to the endoplasmic reticulum (ER) membrane, where it mediates calcium (Ca²⁺) leak, with its function modulated by other ER membrane proteins. Endogenously, PKD2 is found on the plasma membrane and the membrane of primary cilia, while heterologously expressed PKD2 predominantly targets the ER membrane [10,11]. TRPC1 also functions as a homotetramer calcium release channel in the ER, while at the plasma membrane, it forms agonist-activated heterotetramers with TRPC4 or TRPC5 [12]. Originally, PKD1 and PKD2 were proposed to function as sensors of fluid flow, triggering calcium flux through PKD2 due to their localization on the plasma membrane of primary cilia [13,14]. A key limitation of the ciliary hypothesis is that it overlooks the role of polycystin-dependent calcium signaling originating from the ER or the plasma membrane. Furthermore, recent evidence suggests that cilia are largely impermeable to calcium, which challenges the validity of the ciliary hypothesis [15].

Although the modulation of Ca²⁺ by polycystins and TRPC, particularly between PKD1 and the TRPC4 channel, has been documented [9], the interaction between the TRPC subfamily and PKD2 has remained unclear. TRPC1 protein has been shown to interact with proteins beyond the classical TRP channel monomers, particularly with PKD2 [16-18], forming heteromeric channels, despite PKD and TRPC belonging to different groups based on amino acid homology and CryoEM structures. Both proteins are suggested to function as Ca²⁺ release channels in the ER. Moreover, TRPC1 and PKD2 require their respective subfamily members to transport from the ER to the plasma membrane. However, with the structural elucidation of each ion channel [7,19], the existence of these heteromers has become questionable. In this study, we explored how TRPC interacts with and modulates the PKD2 channel. First, we aimed to confirm whether TRPC1 physically interacts with PKD2. Following this, we expanded our experiments to investigate the effects of PKD2 on TRPC4 and TRPC5. Our results demonstrate that TRPC5 modulates PKD2 activity, while TRPC1 does not. Additionally, PKD2 regulates TRPC5 by influencing channel activity at the plasma membrane and ER calcium levels, but it does not affect TRPC1 in the same manner.

Cell culture, transient transfection, and chemicals

Human embryonic kidney (HEK)-293 cells (American Type Culture Collection) were maintained in Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin according to the supplier’s recommendations. Prior to transient transfection, cells were seeded in 6- or 12-well plates. The following day, 0.5–2 μg/well TRPC and PKD1 (PC-1, polycystin-1) or PKD2 (PC-2, polycystin-2) cDNA was transfected into cells using the transfection reagent FuGENE 6 (Roche Molecular Biochemicals) for electrophysiological experiments, according to the manufacturer’s protocol. For molecular biology experiments, Lipofectamine 2000 (Invitrogen) was used as the transfection reagent. All experiments were performed 20–30 h after transfection. Englerin A was purchased from Sigma Aldrich.

Plasmids

Human PKD1(FL(full-length)) in pGFP-N1 and human PKD1(FL)-Flag in pCI-neo plasmids were kindly provided by Eric Honoré and Gregory Germino, respectively. HA-human PKD1(CTF)-Flag in pCI and EGFP-human PKD1(FL) in pCI were kindly provided by Feng Qian (Johns Hopkins University). Human PKD1(FL) was subcloned into the pECFP-N1 and pEYFP-N1 vectors. Mouse PKD2 in pGFP-N1 were kindly provided by Yao Xiaoqiang (Hong Kong University). Human PKD2-myc and Pet-15b mouse PKD2 were kindly provided by Yiqiang Cai (Yale University). Human PKD2 and mPKD2 were subcloned into the pECFP-N1 and pEYFP-N1 vectors. Mouse PKD2 was subcloned into the pEGFP-N1 vector. PKD2 R740X, mutagenesis was conducted using QuickChange site-directed mutagenesis kit (Stratagene).

Mouse TRPC4 and TRPC5 were kindly provided by Dr. M. Schaefer and cDNA human TRPC4 and TRPC5-GFP was kindly provided by Dr. Shuji Kaneko. Human TRPC4 and TRPC5 were subcloned into the pECFP and pEYFP vectors. Mouse TRPC4 and TRPC5 were subcloned into the pECFP and pEYFP vectors. Mouse TRPC4-Flag and TRPC5-Flag were also subcloned into pcDNA3. cDNA of human TRPC1 isoform long tagged with CFP at N-terminus was kindly donated by Dr. Luis Vaca.

Human G_αi1 Q204L, G_αi2 Q205L, G_αi3 Q204L, G_αs WT, G_αs Q277L, G_αo Q205L, human G_αq Q209L, and human M₃ receptor, were kindly provided by Dr. Yong-Sung Juhn. Src Y527F and GSK3β were kindly provided by Juhong Jeon.

Western blotting, co-immunoprecipitation (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 MgCl2, 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 min 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 antibodies part. In the Co-IP experiments for detection of TRPC-PKD2, 500 μl of cell lysates (500–1,000 μg) were incubated with 1 μg of anti-PKD2 (H-280) or anti-GFP antibody 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 phosphate buffered saline (PBS) and incubated in 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) in PBS for 30 min 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) was added to 300 μl of cell lysates (500 μg protein). After incubation for 1 h 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) 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). 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 CaCl2, 1 MgCl2, 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. 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 recorded at −60 or +80 mV during the ramp pulses. The bar graphs summarize the inward current amplitudes at −60 mV. The current recordings were performed according to previously established protocols.

Fluorescence resonance energy transfer (FRET) measurements

Three FRET images (cube set tings 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 sec. All of the images were obtained within 1.5 sec. Each image was captured on a cooled 10 MHz (14 bit) CCD camera (ANDOR technology) 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).

Antibodies

The following commercial antibodies were used: anti-PC1 (PKD1, sc-130554 or sc-10371; Santa Cruz Biotechnology); anti-GFP (A11122; Life Technologies); anti-Flag (F3165; Sigma Aldrich); anti-STAT1 (#9172; Cell Signaling); anti-p-STAT1 (#9167; Cell Signaling); STAT3 (Cell Signaling, #9132); pSTAT3(Y705) (Cell Signaling, #4133); PKD2(H-280); Anti-STAT6 (Cell Signaling, #9362); Anti-p-STAT6(Tyr641) (Cell Signaling, #9361); anti-TRPC (73-119; Neuromab); anti-β-tubulin (T-4026; Sigma Aldrich); Anti-JNK (Santa Cruz Biotechnology, sc-571); Anti-p-JNK(Thr183/Tyr185) (Cell Signaling, #9251s); Anti-c-Jun (Cell Signaling, #9165); Anti-p-c-Jun(Ser63) (Cell Signaling, #9261); Anti-p38 MAPK (Cell Signaling, #9212); Anti-p-p38 MAPK(Thr180/Tyr182) (Cell Signaling, #9216); Anti-c-Src (Santa Cruz Biotechnology, sc-8056); Anti-p-Src(Tyr416) (Cell Signaling, #2101); Anti-NFATc1 (Santa Cruz Biotechnology, sc-7294); Anti-NFATc2 (Santa Cruz Biotechnology, sc-7296); Anti-NFATc3 (Santa Cruz Biotechnology, sc-8321); Anti-NFATc4 (Santa Cruz Biotechnology, sc-13036); Anti-Akt (Cell Signaling, #9272); Anti-Akt(Ser473) (Cell Signaling, #4058); Anti-GSK3α/β (Santa Cruz Biotechnology, sc-7291); Anti-p-GSK3β(Ser9) (Cell Signaling, #9336); Anti-ERK1/2 (Cell Signaling, #9102); Anti-p-ERK(Thr202/Tyr204) (Santa Cruz Biotechnology, sc-7383); Anti-PDK1 (Cell Signaling, #3062); Anti-p-PDK1(Ser241) (Cell Signaling, #3061).

Statistics

The results are presented as the means ± S.E.M. They were compared using Student’s t-tests between two groups. p < 0.05 was considered statistically significant.

The trafficking of PKD from the ER to the plasma membrane

PKD2 primarily resides in the ER and reaches the plasma membrane with special conditions. Specifically, it has been shown that the amount of PKD2 in the plasma membrane is dynamically regulated by interacting proteins (PKD1 [20,21], Polycystin-2 interactor, Golgi- and ER-associated protein-14 (PIGEA-14) [22]), posttranslational modifications (serine phosphorylation by casein kinase 2 (CK2: S812) [23] and glycogen synthase kinase 3 (GSK3: S76) [24]), interaction with other channel subunits in the plasma membrane (PKD1 [20,21], TRPC1 [16-18]) (Fig. 1A). We investigated whether specific factors, such as PKD1 or GSK3, promote the translocation of PKD2 to the plasma membrane. As shown in Fig. 1, both PKD1 (Fig. 1B) and GSK3β (Fig. 1C) significantly increased PKD2 surface expression. Furthermore, we generated a PKD2 mutant by deleting its ER retention signal. The C-terminal region of PKD2 contains several important functional domains, including the ER retention signal, Ca²⁺ binding domains, EF-hand, and a coiled-coil domain. This region also mediates interaction with the C-terminus of PKD1. The hPKD2 R742X mutation, which affects the C-terminal region, is known to cause ADPKD [14]. The resulting PKD2 R740X mutant efficiently localized to the plasma membrane (Fig. 1D). Our result confirmed more surface expression R740X mutant without ER retention deletion. From these results, we reconfirmed certain factors regulating the surface expression of PKD2.

Figure 1. The effects of PKD1 and GSK3 β on PKD2 expression. (A) Schematic illustration of factors affecting the trafficking of PKD2 between the endoplasmic reticulum (ER) and the plasma membrane (PM). (B, C) Surface expression of PKD2-GFP in HEK cells coexpressing PKD1 (B) or GSK3β (C). The surface expression of PKD2 was increased by PKD1 or GSK3β, as determined by co-expression and surface biotinylation. (D) Deletion of the PKD2 ER retention signal (PKD2 R740X) increased the surface expression of PKD2. Surface and total PKD2 were detected by immunoblotting with an anti-GFP antibody. PKD, polycystic kidney disease; IB, immunoblot.

PKD1 is a large plasma membrane glycoprotein that undergoes several proteolytic cleavages, including autocatalytic cleavage at the G-protein coupled receptor proteolytic site (GPS). The cleaved PKD1 consists of an N-terminal fragment (NTF) associated with a C-terminal fragment (CTF). As previously reported, PKD1 cleaved to NTF and CTF in our hands (Fig. 2. See also [9,25]). PKD2, together with PKD1, forms a PKD1/PKD2 complex. This complex moves to the plasma membrane, where it creates a functional channel that responds to mechanical stimuli. In this study, we investigated whether PKD1 directly interacts with PKD2 (Fig. 2). Using an antibody against the PC1 N-terminus (7E12) or an antibody against the PC1 C-terminus (A-20), PKD1 was immunoprecipitated. With anti-PKD2 antibody (H-280), Western blot was performed for PKD2. When PKD2 was immunoprecipitated, anti-PKD1 antibody, 7E12 or A-20, was used for Western blot of PKD1. CTF of PKD1 and FL PKD co-immunoprecipitated with PKD2 (Fig. 2). From these results, we reconfirmed FL and CTF of PKD1 interact with PKD2 to make PKD1/PKD2 complex. The regulators for PKD1 cleavage are Ca²⁺, FBS, mechanical stimulus, γ-secretase as well as PKD2 [2,4,26]. When ER Ca²⁺ pump was blocked with thapsigargin (TG) to induce ER stress, there was no cleavage of PKD1 into NTF and CTF (Supplementary Fig. 1). Such cleavage disruption might lead to the mislocation of PKD1 and PKD2. If PKD2 fails to reach the plasma membrane, its accumulation in the ER increases, consequently regulating Ca²⁺ release from the ER. Cyclopiazonic acid (CPA) also inhibited the cleavage of PKD1 into NTF and CTF (Supplementary Fig. 1). In addition, external Ca²⁺ itself increased the surface expression of PKD2 only in the presence of FBS (Supplementary Fig. 2). These results suggest that ER calcium and extracellular calcium play roles of PKD2 trafficking.

Figure 2. The interaction of hPKD1 CTF with hPKD2-myc. PKD1(FL or CTF) flag-tagged at the C-terminus was coexpressed with PKD2. PKD1/PKD2 complex was immunoprecipitated using an antibody against the PKD1 N-terminus (7E12) (A upper panel) or an antibody against the PKD1 C-terminus (A-20) (B upper panel) and detected by anti-PKD2 antibody (H-280). PKD1/PKD2 complex was also immunoprecipitated using anti-PKD2 antibody (H-280) and detected an antibody against the PKD1 N-terminus (7E12) (A lower panel) or an antibody against the PKD1 C-terminus (A-20) (B lower panel). FL and NTF forms of PKD1 were observed with FL-Flag. The cleaved CTF forms and non-cleaved FL were detected by an anti-PKD1 (A-20) antibody. PKD1 FL or CTF co-immunoprecipitated with PKD2 but not PKD1 NTF. PKD, polycystic kidney disease; CTF, C-terminal fragment; FL, full-length; NTF, N-terminal fragment; IP, immunoprecipitation; IB, immunoblot.

In our previous study, we showed that PKD1 activated TRPC4 by G_α protein signaling pathway [9]. Since PKD1 is an adhesion type GPCR, we tested whether various G_α proteins affect the expression of PKD2 at the plasma membrane. Constitutively active G_αs proteins increased the surface expression of PKD2 (Supplementary Fig. 3). As PKD1-G_α protein pathway activated TRPC4, PKD1-G_αs protein pathway increased the surface expression of PKD2. On the other hand, constitutively active Src (Src Y527F) did not increase the surface expression of PKD2 (Supplementary Fig. 3).

The effect of TRPC on the expression of PKD2 at the plasma membrane

TRPC1 is well known for its expression on the ER membrane and its direct interaction with PKD2 [16-18]. TRPC1 and PKD2 assembles together with ratio of 2:2 [17,18]. Numerous studies have suggested that TRPC1 directly binds to PKD2 and enhances its expression at the plasma membrane [16-18]. We investigated the effects of TRPC1, TRPC4, and TRPC5 on PKD2 expression. Contrary to expectations, neither TRPC1 nor TRPC4 had any impact on the surface expression of PKD2. Interestingly, TRPC5 significantly increased the surface expression of PKD2 at the plasma membrane (Fig. 3). TRPC5, but not TRPC1 or TRPC4, promoted PKD2 localization predominantly to the plasma membrane. However, when TRPC1 was coexpressed with TRPC5 to form a TRPC1/5 heteromer, TRPC1 reversed the increase in PKD2 surface expression induced by TRPC5 (Supplementary Fig. 4). These results suggest that the increased surface expression of PKD2 by TRPC5 might be due to 1) direct interaction of TRPC5 with PKD2, 2) some factor related to TRPC5 with TRPC1 reversing the effects or 3) competition of TRPC1 and TRPC5 against PKD2.

Figure 3. The effects of TRPC on the surface expression of PKD2. (A) The surface expression of PKD2 in HEK cells coexpressing TRPC. Surface expression of PKD2-myc was increased by TRPC5 as determined by co-expression and surface biotinylation. Immunoblots of surface and total were detected by anti-PKD2 antibody (H-280). (B) Bar graphs showing mean levels of surface PKD2 relative to total PKD2 protein levels (black bar, n = 5), and mean levels of surface TRPC relative to total TRPC protein levels (white bar, n = 5). Statistical significance is denoted by an asterisk (*p < 0.05). (C) The surface expression of TRPC in HEK cells coexpressing PKD2. Surface expression of TRPC5 was increased by PKD2 as determined by co-expression and surface biotinylation. Immunoblots of surface and total were detected by anti-GFP antibody. (D) The schematic illustration of factors affecting the trafficking of PKD2 between the endoplasmic reticulum and the plasma membrane (PM). TRPC5 increases the surface expression of PKD2. TRPC, classical transient receptor potential; PKD, polycystic kidney disease; IB, immunoblot.

Thus, we investigated how TRPC5 increases the surface expression of PKD2 at the plasma membrane. To investigate the direct interaction of TRPC with PKD2, we performed Co-IP and FRET experiments. First, we performed Co-IP experiments of PKD2 with TRPC channels. Different from the previous reports [16-18], TRPC1 did not directly bind and co-immunoprecipitate with PKD2 (Fig. 4A). TRPC5 and TRPC4 did not Co-IP with PKD2, either. Thus, TRPC5 did not directly bind and Co-IP with PKD2 (Fig. 4). Next, we performed FRET experiments. The FRET efficiency was also much lower compared with that between PKD2 itself or TRPC5 itself (Fig. 4). These results suggest that the increased surface expression of PKD2 by TRPC5 is not due to a direct interaction between TRPC5 and PKD2. TRPC5 forms a TRPC1/5 heteromer with TRPC1 and regulates Ca²⁺ permeability, even though both TRPC1 and PKD2 homotetramers are typically localized in the ER (see also [5,8,10,12]). Based on the results regarding the regulation of PKD2 surface expression—such as ER Ca²⁺, extracellular Ca²⁺, cAMP, and TRPC1—it is possible that Ca²⁺ influx through TRPC5 channels contributes to the increased surface expression of PKD2.

Figure 4. The interactions between TRPC and PKD2. (A) HEK cells were transfected with TRPC1α, TRPC4β-Flag, or TRPC5-Flag along with PKD2-Myc and were used to test co-immunoprecipitation (Co-IP) of TRPC and PKD2. Anti-TRPC1 or anti-Flag antibody was used for immunoprecipitation (IP), and anti-PKD2 antibody was used for detecting PKD2-Myc. TRPC did not Co-IP with PKD2. (B, C) Anti-PKD2 antibody (H-280) was used for IP. TRPC4 or TRPC5 was detected with anti-Flag antibody (B), and TRPC1 was detected with anti-TRPC1 antibody (C). TRPC and PKD2 were not co-immunoprecipitated reciprocally. (D) FRET between TRPC and PKD2. FRET-detectable interactions occur between EYFP-PKD2 and ECFP-PKD2. Representative FRET images of ECFP-PKD2 co-expressed with EYFP-PKD2, EYFP-TRPC4, and TRPC4-EYFP are shown in the upper panel. In the lower panel, FRET images of ECFP-PKD2 co-expressed with EYFP-TRPC5 and a bar graph of FRET efficiency between PKD2 and TRPC are shown. TRPC, classical transient receptor potential; PKD, polycystic kidney disease; FRET, fluorescence resonance energy transfer; IB, immunoblot.

The effects of PKD1 and PKD2 on TRPC5 channels

Although Ca²⁺ modulation by polycystins has been reported between PKD1 and TRPC4 channel or TRPC1 and PKD2, whether the function of TRPC subfamily is regulated by PKD2 has remained elusive. Here, we recorded the activity of TRPC4/C5 heterologously co-expressed with PKD1 or PKD2 in HEK293 cells. First, PKD1 activated TRPC4 (40 ± 14 pA/pF) channel by modulating G-protein signaling without change in TRPC4 translocation as previously reported [9]. PKD1 acts as a GPCR and activates G_αi increased both basal G_αi proteins induced TRPC4 currents [9]. PKD1 also activated TRPC5 channel from 54 ± 8 pA/pF to 114 ± 16 (n = 7) by modulating G-protein signaling without change in TRPC4/C5 translocation (Fig. 5A, B). Intracellular 0.2 mM GTPγS-induced TRPCC5 activation, however, was not significantly different in the presence or absence of PKD1 (Fig. 5C, D). Whereas FL PKD1 increased TRPC5 current, CTF of PKD1 did not affect TRPC5 activity (80 ± 12 pA/pF, n = 7) due to loss of N-terminus containing GPS (Fig. 5C, D). FL PKD1 also increased the La³⁺-induced TRPC5 currents from 27 ± 8 to 58 ± 12 pA/pF (n = 7), whereas CTF did not (Fig. 5E, F). PKD1 increased both the inward and outward TRPC5 currents. These results suggested that PKD1 increased TRPC5 current via G_αi proteins as in TRPC4 currents. In addition, although CTF is required for binding with PKD2, NTF is also needed for increasing TRPC5 currents. Interestingly, PKD1 increased both basal and La³⁺ induced TRPC5 current in contrast to TRPC4.

Figure 5. The effects of PKD1 on TRPC5 channels. (A) HEK 293 cells were co-transfected with TRPC5 and PKD(FL). The TRPC5 current was recorded without GTPγS, and the current amplitude was increased by 140 mmol/L Cs⁺. The current amplitudes at −60 mV and +60 mV were plotted against time (the left panel) in HEK cells expressing PKD1 and TRPC5 (red triangle) or TRPC5 only (black circle). The ramp pulses were applied every 10 sec. The I-V curves from HEK cells expressing PKD1 and TRPC5 (red line) or TRPC5 only (black line) showed a double-rectifying shape. (B) The bar graphs represent the means ± S.E.M of current density (pA/pF) at −60 mV in the absence of GTPγS infusion. (C) The current was recorded in TRPC5/PKD1 co-transfected HEK 293 cells infused with GTPγS. The I-V curves from HEK cells expressing PKD1 and TRPC5 (red line) or TRPC5 only (black line) showed a double-rectifying shape. (D) The bar graphs represent the means ± S.E.M of current density (pA/pF) at −60 mV in the presence of GTPγS infusion. (E) The effect of PKD1 on La³⁺-induced TRPC5 current. (F) The surface expression of TRPC5 in HEK cells coexpressing PKD1. Surface expression of TRPC5 was not increased by PKD1 as determined by co-expression and surface biotinylation. Immunoblots of surface and total were detected by anti-GFP antibody. Right panel: Bar graph showing the effect of PKD1 on the surface expression of TRPC5 (n = 3). *p < 0.05. PKD, polycystic kidney disease; TRPC, classical transient receptor potential, FL, full-length; CTF, C-terminal fragment; PM, plasma membrane; IB, immunoblot; n.s., not significant.

Second, we investigated the effects of PKD2 on TRPC4 and TRPC5 channels. PKD2 affected muscarinic stimulation of TRPC channels by carbachol (Fig. 6). When M₃R was co-expressed with PKD2, carbachol increased currents in both TRPC5 (from 33 ± 9 to 112 ± 33 pA/pF, n = 7) and TRPC4 channels (from 34 ± 4 to 77 ± 14 pA/pF, n = 7) (Fig. 6). These results suggest that muscarinic stimulation activates IP₃R and the activated IP₃R induces more calcium release via PKD2. Thus the released calcium potentiated TRPC5 currents.

Figure 6. The effects of PKD2 on TRPC4 and TRPC5 channels stimulated by carbachol. (A) HEK 293 cells were co-transfected with TRPC4 and PKD2, and M3 receptor was also expressed. TRPC4 current was activated by carbachol. Current amplitudes at −60 mV and +60 mV were plotted against time (left panel) in HEK cells expressing PKD2 and TRPC4 (red circles) or TRPC4 only (black circles). Ramp pulses were applied every 10 sec. I-V curves from HEK cells expressing PKD2 and TRPC4 (red line) or TRPC4 only (black line) showed a double-rectifying shape (right panel). Bar graphs represent the mean ± S.E.M of current density (pA/pF) at −60 mV. (B) HEK 293 cells were co-transfected with TRPC5 and PKD2, and M3 receptor was also expressed. TRPC5 current was activated by carbachol. Current amplitudes at −60 mV and +60 mV were plotted against time (left panel) in HEK cells expressing PKD2 and TRPC5 (red circles) or TRPC5 only (black circles). Ramp pulses were applied every 10 sec. I-V curves from HEK cells expressing PKD2 and TRPC5 (red line) or TRPC5 only (black line) showed a double-rectifying shape (right panel). Bar graphs represent the mean ± S.E.M of current density (pA/pF) at −60 mV. PKD, polycystic kidney disease; TRPC, classical transient receptor potential; CCh, carbachol.

Finally we investigated whether other stimulators like external cesium or La³⁺ have any effect on TRPC5 when PKD2 was co-expressed with TRPC5. TRPC5 has unique electrophysiological properties. As shown above, TRPC5 has a larger basal current than TRPC4 and responds well to external La³⁺ [5-9]. Without intracellular GTPγS, PKD2 decreased the basal Cs current of TRPC5 channels from 115.8 ± 29.8 (n = 7) to 13 ± 5 pA/pF (n = 7) (Fig. 7). However, with GTPγS, the basal Cs currents was not changed by PKD2 coexpression (99.3 ± 23.5 [n = 8] vs. 96.8 ± 27.0 pA/pF [n = 7]). Similar results were obtained in TRPC4 channels (97.2 ± 16.7 [n = 5] vs. 94.5 ± 11.1 pA/pF [n = 5]). La³⁺ induced TRPC5 currents was also decreased by PKD2 (from 208.0 ± 60.4 [n = 7] to 29.9 ± 11.9 pA/pF [n = 7]). We investigated whether the decrease of the basal or La³⁺ induced TRPC5 currents are due to the decreased surface expression of TRPC5 by PKD2. Contrary to our expectation, PKD2 increased the surface expression of TRPC5 at the plasma membrane (Fig. 3). These results suggest that PKD2 acts a negative regulator for basal and La³⁺ induced currents of TRPC5. There are two possibilities for such an effect of PKD2. First, PKD2 binds with endogenous PKD1 and decreases the activity of G_αi proteins. Second, PKD2 overexpression quenches the plasma membrane PIP₂ and decreases the level of PIP₂ for TRPC5 channels.

Figure 7. The effects of PKD2 on basal and La3+ induced TRPC5 currents. (A) TRPC5 basal current without GTPγS. HEK 293 cells were co-transfected with TRPC5 and PKD2. The basal current of TRPC5 channels was increased by external cesium. The current amplitudes at −60 mV and +60 mV were plotted against time (the left panel) in HEK cells expressing PKD2 and TRPC5 (red triangle) or TRPC5 only (black triangle). The ramp pulses were applied every 10 sec. The I-V curves from HEK cells expressing PKD2 and TRPC5 (red line) or TRPC5 only (black line) showed a double-rectifying shape. (right panel) The bar graphs represent the means ± S.E.M of current density (pA/pF) at −60 mV. (B) TRPC5 activated with GTPγS. HEK 293 cells were co-transfected with TRPC5 and PKD2. The TRPC5 current was activated by GTPγS and external cesium. The current amplitudes at −60 mV and +60 mV were plotted against time (the left panel) in HEK cells expressing PKD2 and TRPC5 (red triangle) or TRPC5 only (black triangle). The ramp pulses were applied every 10 sec. The I-V curves from HEK cells expressing PKD2 and TRPC5 (red line) or TRPC5 only (black line) showed a double-rectifying shape. (right panel) The bar graphs represent the means ± S.E.M of current density (pA/pF) at −60 mV. (C) La³⁺ induced TRPC5 current. The TRPC5 current was activated by external La³⁺. The current amplitudes at −60 mV and +60 mV were plotted against time (the left panel) in HEK cells expressing PKD2 and TRPC5 (red triangle) or TRPC5 only (black triangle). The ramp pulses were applied every 10 sec. The I-V curves from HEK cells expressing PKD2 and TRPC5 (red line) or TRPC5 only (black line) showed a double-rectifying shape. (right panel) The bar graphs represent the means ± S.E.M of current density (pA/pF) at −60 mV. PKD, polycystic kidney disease; TRPC, classical transient receptor potential.

The opposite effects of TRPC1 and TRPC5 on STAT

To see the combined effects of TRPC and PKD2 on cell signaling, we investigated which downstream effects occur when PKD2 and TRPC were co-expressed. In ADPKD pathogenesis, many signaling pathways are suggested, that is, 1) protein kinase C (PKC)- c-Jun N-terminal kinases (JNK)-p38-activating protein-1 (AP-1), 2) mechanistic target of rapamycin (mTOR)-AMP-activated protein kinase (AMPK)-extracellular signal–regulated kinase (ERK), 3) 3-phosphoinositide-dependent protein kinase-1 (PDK1)-Akt-GSK3, and 4) Src-STAT-p21 (Supplementary Fig. 5, see also [1,2,14,26,27]). Western blot analysis was performed to detect the phosphorylation of the proteins involved in the signaling pathways. There were no change of the phosphorylation of JNK, Jun, and p38 mitogen-activated protein kinase (MAPK) (Supplementary Fig. 6A). The phosphorylation of mTOR (Supplementary Fig. 6B) and ERK (Supplementary Fig. 6C) was not changed, either. There were no change of the phosphorylation of PDK1, Akt and GSK3, either (Supplementary Fig. 7).

The JAK-STAT signaling pathway is also upregulated in ADPKD, with cyst-lining cells showing increased levels of nuclear STAT-1, STAT-3, and STAT-6. This leads to enhanced STAT-dependent gene expression [27]. PKD1 has been implicated in a variety of intracellular signaling events, including JAK-STAT signaling. Overexpression of FL PKD1 can activate STAT1, STAT3 and STAT6 [28], which mediate signaling involved in proliferation, differentiation and death. We suggested that the C-terminal tail (CTT) of PKD1 activates TRPC4β via G_αi3 protein and that calcium influx through TRPC4β is required for the activation of STAT1. As PKD1 increased the phosphorylation of STAT [9], we investigated whether the co-expression of PKD2 and TRPC affects the phosphorylation of STAT. PKD2 increased STAT3 phosphorylation with TRPC5 whereas PKD2 decreased STAT1 phosphorylation with TRPC1 (Fig. 8). The effect of TRPC5 on STAT3 was not observed when PKD2 was co-expressed with TRPC1/5 heteromer (Supplementary Fig. 8). There were no change of the phosphorylation of STAT6 (Fig. 8). However, Src phosphorylation, upstream signaling molecule, was not changed when PKD2 and TRPC were coexpressed (Supplementary Fig. 6D).

Figure 8. STAT phosphorylation by coexpression of PKD2 with TRPC. (A) Effects of TRPC on STAT1 phosphorylation. HEK 293 cells were transfected with PKD2 and TRPC. Cell lysates were subjected to Western blot analysis using antibodies against total and phosphorylated STAT1. (B) Bar graphs showing mean levels of phosphorylated STAT1 relative to total STAT1 protein levels (n = 5). Statistical significance is denoted by an asterisk (*p < 0.05). The bar graphs represent the means ± S.E.M. (C) Effects of TRPC on STAT3 phosphorylation. HEK 293 cells were transfected with PKD2 and TRPC. Cell lysates were subjected to Western blot analysis using antibodies against total and phosphorylated STAT3. (D) Bar graphs showing mean levels of phosphorylated STAT3 relative to total STAT3 protein levels (n = 5). Statistical significance is denoted by an asterisk (**p < 0.01). (E) Effects of TRPC on STAT6 phosphorylation. STAT, signal transducer and activator of transcription; PKD, polycystic kidney disease; TRPC, classical transient receptor potential; n.s., not significant; IB, immunoblot.

Our findings indicate that 1) TRPC5 regulates the surface expression of PKD2 as well-known factors like PKD1, GSK3, Ca²⁺, ER retention signals of PKD2, 2) PKD2 regulates TRPC5 and TRPC4 channels by interacting with PKD1-G_α protein or ER calcium content, 3) PKD2 activates STAT3 signaling pathway with TRPC5 as PKD1-TRPC1/4-STAT1 signaling pathways, and 4) PKD1/2 and TRPC1/C4/C5 play the important roles in the regulation of calcium homeostasis independently, but interact each other via G protein or calcium dependent process.

PKD and TRPC channel play each role for regulation of calcium homeostasis. TRPC1 acts as a negative regulator for TRPC4 and TRPC5 after forming a heteromer channels. TRPC1 reduced calcium permeability and cellular excitability by modifying IV curve [29-33] (Fig. 9 blue dotted line). On the other hand, PKD2 needs PKD1 for trafficking and its function for ion channels. PKD1 acts a carrier for PKD2 from ER to the primary cilia as TRPC5 for TRPC1 from the ER to the plasma membrane. PKD1/PKD2 forms a complex and together play a very important role for anticystogenic activity at renal tubular epithelial cells. PKD1 also changes calcium permeability of PKD2 when they make PKD1/PKD2 complex [19]. Contrary to their independent roles in calcium regulation, TRPC and PKD have similar properties. First, both TRPC1 and PKD2 express at the ER and play a Ca²⁺ release channel at the ER maybe as a homotetramer (Fig. 9, see also [12,34]). Second, there is an intimate connection with ER calcium. TRPC channels are initially suggested as a store operated channels (SOCs). Regardless of TRPC as a SOC or not, TRPC seems very important for filling ER Ca²⁺, especially when GPCR induces Ca²⁺ release from the ER (Fig. 9 blue dotted line). PKD1 also acts as a GPCR and contributes to fill ER Ca²⁺ by activating TRPC4 or TRPC5. The depletion of ER calcium prevents the cleavage of PKD1 (Supplementary Fig. 1) and increase Ca²⁺ influx through TRPC at the plasma membrane. Thus, there is intimate interaction between ER calcium and PKD1 cleavage. In addition, the cleavage of PKD1 is needed for the trafficking of PKD2 to the primary cilia, thus the retention of PKD2 at the ER due to non-cleavage of PKD1 might exacerbate the ER Ca²⁺ depletion. When ER Ca²⁺ was modified with TG, the cleavage of PKD1 might not occur and bring PKD2 to the primary cilia (Supplementary Fig. 1). ER calcium content appears to modulate the response of PKD2 on the co-expression with TRPC channels. Thus, PKD2 increased carbachol-induced TRPC5 currents on the co-expression of PKD2 with TRPC channels (Figs. 6 and 9 (green line)). The sustained activation of the G_αq/11-PLCβ pathway results in ER stress and unfolded protein response (UPR) as demonstrated by the effect of the G_αq-blocker FR900359 on the transcriptome from patients [35]. Therefore intimate relationship between ER calcium and TRPC or PKD proteins are essential for cell calcium homeostasis as well as protein translation.

Figure 9. Schematic illustration of TRPC, PKD, and IP₃R interaction at the endoplasmic reticulum (ER) and their trafficking to the plasma membrane (PM). The schematic illustration of the interaction between TRPC and PKD proteins via the ER. TRPC1 and PKD2 function as calcium release channels in the ER. IP₃R interacts with both TRPC1 and PKD2 at the ER. The red arrow shows how PKD1 regulates TRPC function. The green arrow shows the process of regulating TRPC through PKD2. The blue arrow shows the signaling process through TRPC. PKD1 acts as a GPCR and activates G_αi proteins. The activated G_αi proteins induced TRPC4 and TRPC5 currents. On the other hand, PKD2 binds to PKD1 and inhibits the activity of PKD1, which lowers the basal activity of G protein and inhibits the basal current and La³⁺-induced current of TRPC5. Interestingly, when activated through M₃R, calcium release through IP₃R is added, and the function of PKD2 changes to activating TRPC5 current. This is because PKD2 acts as a calcium-releasing ion channel in the ER, and IP₃R further promotes PKD2 function, and vice versa. When TRPC4/5 is activated, calcium is supplied abundantly into the cell, and STAT1 (S701 phosphorylation) and STAT3 (S705 phosphorylation) is activated through the increased calcium and CaMK. STAT phosphorylation might increase the surface expression of PKD2 and TRPC5. TRPC, classical transient receptor potential; PKD, polycystic kidney disease; GPCR, G protein coupled receptor; STAT, signal transducer and activator of transcription.

Early studies have suggested that PKD2 plays an important role in intracellular Ca²⁺ homeostasis [10]. The loss of PC2 in cells results in defective ER Ca²⁺ release, which is believed to contribute to cAMP overproduction and cystogenesis, the foundation for treatment of ADPKD by tolvaptan [10,36]. Recently, Padhy et al. [37] showed that PKD2 in the ER appears to be critical for anticystogenesis and likely functions as a potassium ion channel to facilitate potassium–calcium counterion exchange for inositol trisphosphate–mediated calcium release. A recent study of patch-clamp recordings of ciliary membrane revealed that PKD2 conducts predominantly monovalent cations with approximately 40-fold more selectivity to K⁺ over Ca²⁺ [38]. The trimeric intracellular cation channels (TRIC) are ER-resident K channels that mediate K fluxes from cytosol into the ER lumen in exchange for IP₃R- and RyR-mediated Ca²⁺ release into the cytosol [39]. Padhy et al. [37] showed that function of PKD2 in ER is important for anticystogenesis and mediates K⁺-Ca²⁺ exchange in the ER to facilitate Ca²⁺ release like TRIC. TRPC1 itself shows less calcium permeability based on CryoEM structure of TRPC1/4 heteromer [32,33]. In addition, TRPC1 reduces the calcium permeability of TRPC4 and TRPC5 [29-33]. Thus both TRPC1 and PKD2 as ER calcium release channels should be reconfirmed considering that TRPC1 and PKD2 might be nonselective cation channel for coupled counter cation exchange to help calcium release from ER.

PKD2 increased M₃R-G_αq/11-PLCβ-induced TRPC5 currents because there is a calcium induced calcium release through PKD2 and IP₃R. PKD1 inhibits IP₃R whereas PKD2 activates IP₃R. When PKD1 and PKD2 were co-expressed, they enhanced IP₃R activity (Fig. 9, see also [40,41]). The sustained activation of the G_αq/11-PLCβ pathway results in ER calcium stress and UPR [35]. IP3 from M₃R-G_αq/11-PLCβ pathway usually binds with IP₃R and releases Ca²⁺ from the ER. IP3 also activates PKD2 and release Ca²⁺ from the ER via PKD2. Such a dynamic and large Ca²⁺ increase seems to facilitate TRPC5 channels. Uniquely, the speed of Ca²⁺ increase seems important because carbachol only increased the TRPC5 currents whereas GTPγS did not increase TRPC5 in PKD2 coexpressing HEK cells (Fig. 7). Because calcium itself also activates CaM-CaMKK-CaMK signaling pathways [42,43] and CaMKK is a final master for G_αs-cAMP, Src, GSK3 and calcium [44], CaMK might phosphorylates and activates TRPC5 currents. There are other possibilities for PKD2-induced changes in TRPC5 current activated by carbachol. First, as in leptin or insulin [45], PKD1/PKD2 activates JAK/STAT and increases TRPC5 current. The second possibility is increased surface expression of TRPC5 by PKD2. Stat binds the promoter region of TRPM3 and increases the surface expression of TRPM3. By chromatin immunoprecipitation (ChIP) assays, phospho-STAT3 direct binding to STAT3-binding sites near the TRPM3 promoter region, upstream of miR-204-5p, has been reported [46]. TRPM7 expression was increased by leptin-STAT pathway [47]. However, this is not the case for TRPC5 because there was no binding site for STAT in TRPC5.

For a role for polycystin dependent calcium signaling originating in the ER or at the plasma membrane, we suggested that the PKD1/TRPC4 signaling pathway controls the migration of endothelial cell monolayers, leading to endothelial permeability [9]. To determine whether PKD1 or TRPC4β affects vascular permeability, we investigated the distribution of an endothelial-specific cadherin, vascular endothelial (VE)-cadherin, and leakage of Evans blue dye in endothelial cells. We suggested that PKD1-mediated TRPC4β activation plays an important role in the stability of endothelial junctions and permeability [9]. TRPC4 was initially suggested to be involved in the regulation of vasorelaxation by acetylcholine in the endothelium-dependent manner [48] and lung microvascular permeability [49]. Following the publication of papers indicating that TRPC4 is involved in endothelial cell function, reports have subsequently emerged that TRPC5 and TRPC1/C5 heteromers are involved in NO-dependent endothelial cell function [50,51]. Recently, reports have been published that PKD2 and the PKD1/2 complex are also involved in mechanosensation by endothelial cells [52-55]. These results suggest that PKD1/2 and TRPC1/4/5 play important roles in the regulation of vascular tone and endothelial permeability by controlling ER calcium and Ca²⁺ influx across the plasma membrane as well as the primary cilia.

One function of the PKD1/PKD2 complex is to regulate the JAK/STAT pathway, providing insight into how mutations in either gene can lead to dysregulated cell growth. The unique channel properties that arise from the coassembly of polycystin-1 and polycystin-2 at the plasma membrane are believed to be responsible for the activation of JAK2 [28,56,57]. Initially, given the channel’s high permeability to calcium, an intriguing possibility was that calcium influx through the channel could trigger a signaling cascade, leading to the phosphorylation and activation of JAK2. PKD2 is essential for the proper function of PKD1, as neither protein alone can activate the JAK-STAT pathway. The involvement of PKD2 in PKD1-mediated JAK2 activation further supports that both proteins are key components of a shared signaling pathway, and disruptions in this pathway result in the indistinguishable phenotypes observed in PKD1 and PKD2 mutations [28]. IP₃R needs TRPC channels for STAT activation, especially for persistent STAT activation [58]. In hypoxic pulmonary vasoconstriction, JAK/STAT pathway needs TRPC1 and TRPC6 as SOC [59]. In our hands, TRPC4 channels are required for PKD1-STAT activation while TRPC5 and TRPC1 for PKD2-STAT signaling pathways.

PKD1/PKD2 are sensitive to ER calcium and PKD2 contributes to TRPC5 response via M₃R-G_q-ER calcium pathway. The reuptake of calcium ions into the ER by the sarcoplasmic/ER Ca²⁺ transporting ATPases (SERCAs or ATP2As) is ATP dependent. Mitochondrial dysfunction results in insufficient ATP production, which subsequently hinders calcium reuptake into the ER, setting off a cascade of stress responses [60]. Mechanistically, the resulting depletion of calcium ions in the ER lumen activates the UPR, also known as the ER stress response, by dissociating the chaperone-binding immunoglobulin protein (BiP) from ER stress sensors, including the PKC-related kinase–like ER kinase (PERK) [61-63]. ER calcium stabilizes the inhibitory interaction of the chaperone Bip with the ER stress-sensor PERK. A drop in calcium ion concentration in the ER activates PERK, which then activates the eukaryotic initiation factor-2a (eIF2A)-activating transcription factor 4 (ATF4) pathway and inhibits protein translation [64]. Most translocon pores are also blocked by BiP and/or ribosomes, maintaining the permeability barrier [63]. During ER stress, when protein translation is attenuated, BiP, sequestered by unfolded proteins, dissociates from the luminal domain of ribosome-free translocons. PKD1 regulates the ER stress response by preventing the cleavage of PKD1 and retaining PKD2 at the ER membrane, rather than transporting PKD2 to the primary cilia or plasma membrane.

In conclusion, we showed 1) a special role of TRPC5 for the expression of PKD2, 2) two PKD2-TRPC5-STAT3 and PKD2-TRPC1/C4 heteromer-STAT1 signaling pathways, and 3) PKD1-G protein TRPC4/C5 activation, PKD2-Ca²⁺-muscarinic activation of TRPC4/5 and PKD2-PKD1-G protein-inhibition of basal or La³⁺ activation of TRPC4/C5. Our findings thus provided a new potential therapeutic approach targeting TRPC1/C4/C5 channel in cerebral arterial aneurysm from polycystic kidney disease. Interestingly, while the PKD1/PKD2-STAT1 pathway exhibits an anticystogenic effect on tubular epithelial cells, the analogous PKD1/TRPC4/STAT1 pathway promotes proliferation and strengthens the tight junctions of endothelial cells.

This study was supported by National Research Foundation of Korea grant 2020R1A2C1012670 and 2021R1A4A2001857 (I.S.), Education and Research Encouragement Fund of Seoul National University Hospital (I.S.), Mid-Career Bridging Program through Seoul National University (800-20240545) (I.S.), Seoul National University Hospital Research Fund (03-2024-0440) (I.S.), BK21 FOUR education program scholarship (H.K., J.K., B.J.).

Human PKD1(FL) in pGFP-N1 and human PKD1(FL)-Flag in pCI-neo plasmids were kindly provided by Eric Honoré and Gregory Germino, respectively. HA-human PKD1(CTF)-Flag in pCI and EGFP-human PKD1(FL) in pCI were kindly provided by Feng Qian (Johns Hopkins University). Mouse PKD2 in pGFP-N1 were kindly provided by Yao Xiaoqiang (Hong Kong University). Human PKD2-myc AND Pet-15b mouse PKD2 were kindly provided by Yiqiang Cai (Yale University). Mouse TRPC4 and TRPC5 were kindly provided by Dr. M. Schaefer and cDNA human TRPC4 and TRPC5-GFP was kindly provided by Dr. Shuji Kaneko. cDNA of human TRPC1 isoform long tagged with CFP at N-terminus was kindly donated by Dr. Luis Vaca. Human G_αi1 Q204L, G_αi2 Q205L, G_αi3 Q204L, G_αs WT, G_αs Q277L, G_αo Q205L, human G_αq Q209L, and human M₃ receptor, were kindly provided by Dr. Yong-Sung Juhnn. Src Y527F and GSK3β were kindly provided by Juhong Jeon. Christine Haewon Park went through english proofreading.

The authors declare no conflicts of interest.

Supplementary data including eight figures can be found with this article online at https://doi.org/10.4196/kjpp.24.265