Human embryonic kidney (HEK293) cells (ATCC, Manassas, VA, USA) were maintained according to the supplier's recommendations. HEK293 cells were incubated in Dulbecco's Modified Eagle's Medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37℃ in a 5% CO₂ humidified incubator. Cells were seeded in confocal dish for recording images or a 12-well plate for whole-cell patch clamp recordings, and 6-well plate for western blot. The following day, transfection was carried out by using the FuGENE 6 Transfection Reagent (Promega, Madison, WI, USA) and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. All experiments were performed 20–30 h after transfection.

The patch pipettes were made from borosilicate glass and had resistances of 3–5 MΩ when filled with standard intracellular solution; 140 CsCl, 10 N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid (HEPES), 0.2 Tris-GTP, 0.5 ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 3 Mg-ATP, pH 7.3 with CsOH (in mM). Additional 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) concentrations in the pipette solutions are stated in figure legend. External solution was perfused constantly as follow; 135 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose,10 HEPES, pH 7.4 with NaOH (in mM).

The cells were transferred onto a solution chamber on the stage of an inverted microscope (IX70; Olympus, Tokyo, Japan). Whole cell configuration was used to measure TRPC channel currents in HEK293 cells as described previously [619]. Cells were attached to coverslip in the small chamber for 10 min prior for the patch recording. The currents were recorded using an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA, USA). Voltage ramp pulse from +100 mV to −100 mV over period of 500 ms was imposed with a −60 mV holding membrane potential. Experiments were performed at room temperature (18℃–22℃). The recording chamber was continuously perfused at a flow rate of 1–2 ml/min.

HEK293 cells were cultured in a 35-mm coverslip bottom dish or a 12-well plate to obtain. To obtain the image of a cell, we used an inverted microscope with 60× oil objective lens. Images were acquired on the cooled 3 MHz (14 bit) EMCCD camera (iXon Ultra 888; ANDOR, Belfast, UK) with 1 × 1, 2 × 2, or 3 × 3 binning under the control of MetaMorph 7.6 software (Molecular Devices, Sunnyvale, CA, USA). Each images were exposed over period of 100 ms.

The ratiometric measurement of [Ca²⁺]_i was performed using Fura-2, AM (Molecular Probes, Eugene, OR, USA). The cells were grown in a cover glass bottom dishes and loaded with 1 µM of Fura-2, AM, and incubated for 30 min at 37℃. The Fura-2 fluorescence was measured at a 510 nm emission with a 340/380 nm dual excitation using a PTi Sutter illuminator. Images were acquired on the cooled 3 MHz (14 bit) CCD camera (CoolSNAP HQ2; Photometrics, Tucson, AZ, USA) with 1 × 1, 2 × 2, or 3 × 3 binning under the control of MetaMorph 7.6 software. Each images were exposed over period of 100 ms.

The 0.5–2 µg/well of cDNA was transfected into cells. After transfection for 24 h, the cells were harvested as follows. Lysates were prepared in lysis buffer (0.5% Triton X-100, 50 Tris-Cl, 150 NaCl, 1 EDTA, pH 7.5, [in mM]) by being passed through a 26-gauge needle seven to ten times after sonication. Lysates were centrifuged at 13,000 × g for 10 min at 4℃, and the protein concentration in the supernatants was determined. The proteins extracted in sample buffer were loaded onto 8% Tris-glycine SDS-PAGE gels and then subsequently transferred onto a polyvinylidene fluoride membrane.

Phosphate buffered saline (PBS)-washed cells were incubated in 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce, Rockford, IL, USA) in PBS for 30 min on ice. Afterward, unreacted biotin was quenched by the addition of 100 mM glycine in PBS. The cells were then processed as described above to prepare cell extracts. Forty microliters of a 1 : 1 slurry of immobilized avidin beads (Pierce) were added to 300 µl of the cell lysate containing 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. Collected proteins were then analyzed by Western blot. The experiment was performed as previously described [20].

Data were analyzed using SPSS software (IBM SPSS Statistics 23; IBM Co., Armonk, NY, USA). Results are given as mean ± standard error of the mean (SEM). Error bars indicate SEM. Here, ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 were considered statistically significant, while n.s. indicates not statistically significant. Results were compared Student's t-test. All data were generated from cells pooled from at least two biologically independent experiments. No samples were excluded.

The TRPC1 subunit is not functional alone, but forms a heteromeric channel with TRPC4 or TRPC5. As a regulator, TRPC1 changes physiological properties. The current-voltage relationship of heteromeric channels shows an outward-rectifying shape, which is different from the double-rectifying shape of homomeric channels. Previously, we suggested that the Gα_q protein directly interacts with TRPC4 or TRPC5 heteromeric channels and activates heteromeric channels. After activation, breakdown of PI(4,5)P₂ by the Gα_q-PLCβ pathway inactivates heterotetramers [11]. While studying the inactivation mechanisms of heteromeric channels, we noticed that constitutively active Gα_q (Q209L) completely reduced the Englerin A (EA)-induced current to zero. EA is an agonist of TRPC4 and TRPC5 channels [21]. On the other hand, inositol polyphosphate 5-phosphatase (Inp54p) of the rapamycin-inducible system and Danio rerio voltage-sensitive lipid phosphatase (DrVSP) only partially reduced current. In other words, the channel currents were not inactivated to the zero level with maximum activation of these systems, which completely depletes the membrane PI(4,5)P₂. The physiological Gα_q-PLCβ pathway and these artificial lipid phosphatases hydrolyze PI(4,5) P₂ differently (Fig. 1A). In the Gα_q-PLCβ pathway, the guanosine triphosphate (GTP)-bound Gα_q subunit activates PLCβ, which in turn hydrolyzes PI(4,5)P₂ to DAG and IP₃ (Fig. 1A; right). DAG acts as a second messenger that activates PKC, and IP₃ releases Ca²⁺ from the ER. By contrast, activated DrVSP and Inp54p convert PI(4,5)P₂ to phosphatidylinositol 4-phosphate (PI(4)P) and inorganic phosphate (Fig. 1A; left) [2223]. Likewise, the physiological pathway and artificial PI(4,5)P₂ depletion system shows the difference in products after stimulation. We hypothesized that, if the Gα_q-PLCβ downstream products are involved in an inactivation process, these signals would cause the additional channel inhibition.

We first investigated the effect of IP₃-induced ER Ca²⁺ release on TRPC1α/4β and TRPC1α/5, considering that the level of cellular Ca²⁺ regulates TRPC4 and TRPC5 homomeric channels [124]. The cells co-expressed channel subunits and muscarinic receptor 3 (M₃R), and the calcium changes were monitored by Fura-2 calcium imaging. Application of a muscarinic receptor agonist, carbachol (CCh), activated the Gα_q-PLCβ pathway and markedly increased the intracellular free calcium ([Ca²⁺]_i) in cells expressing heteromeric channels (Supplementary Fig. 1A, B). On the other hand, there was no current inhibition in TRPC1α/4β or TRPC1α/5 when [Ca²⁺]_i was increased by an ER Ca²⁺ pump inhibitor, thapsigargin (TG) (Fig. 1B–D). We measured the [Ca²⁺]_i change in the cells expressing channel subunits and stimulated by EA (Supplementary Fig. 1C, D). The calcium change slightly increased compared to CCh stimulation. We also measured [Ca²⁺]_i change with the rapamycin-inducible system when Gα_q (Q209L, L254A) was expressed (Supplementary Fig. 1E, F). This mutant is GTPase deficient, which is constitutively active and impaired binding to PLCβ [25]. The calcium change was slightly increased compared to CCh stimulation.

To determine the [Ca²⁺]_i function on heteromeric channels, we buffered free Ca²⁺ levels in pipette solutions by a Ca²⁺ chelator, BAPTA (Fig. 2). HEK293 cells were co-transfected with channel subunits and M₃R, and the channels were stimulated by CCh. We recorded channel current changes in the whole-cell recording mode. Voltage ramp pulse from +100 mV to −100 mV over a period of 500 ms was imposed every 10 sec with a −60 mV holding membrane potential. With 0.5 mM of BAPTA, either the basal or CCh-evoked current increased in TRPC1α/4β-expressing cells (Fig. 1E). Furthermore, the currents were sustained longer than control in the presence of 0.5 mM of BAPTA. Strong titration of [Ca²⁺]_i with 5 mM of BAPTA remarkably inhibited the current amplitude activated by CCh in TRPC1α/4β-expressing cells (Fig. 1E), whereas, in cells expressing TRPC1α/5, calcium buffering with 0.5 mM of BAPTA showed similar current changes to control, 0 mM of BAPTA (Fig. 1G). Calcium buffering with 5 mM of BAPTA also did not enhance channel currents. We normalized the trace to identify the tendency of currents change depending on time. Fig. 1F and H show the normalized heteromeric currents for pipette solution containing 0 or 0.5 mM BAPTA. The normalized currents clearly shows the effect of BAPTA on the inactivation time course of heteromeric channels. The TRPC1α/4β currents were sustained longer in the presence of BAPTA (Fig. 1F), whereas the TRPC1α/5 currents were not (Fig. 1H). This is in line with the finding that TRPC4 channels were silent when the pipette solution contained a high EGTA concentration [1]. Collectively, the results of cytosolic calcium change support the hypothesis that calcium tonically inhibits TRPC1α/4β, but not TRPC1α/5.

To investigate the additional role of Ca²⁺ on TRPC1α/4β current inactivation, we depleted PI(4,5)P₂ first and then subsequently increased calcium. Increase of cytosolic calcium was induced by TG, a SERCA inhibitor, or ionomycin (iono) ionophore. The cells co-expressed TRPC subunits, Inp54p, and Lyn11. Channel currents were first activated by EA, and depletion of PI(4,5)P₂ was induced by Inp54p with the rapamycin-induced dimerization system. As shown in Supplementary Fig. 1C and D, EA increased intracellular Ca²⁺ slightly. Application of TG (1 µM) completely reduced current amplitude to the basal level and showed an outward rectifying I–V shape (Fig. 2A, B). Similar current inhibition was observed in cells stimulated by ionomycin (1 µM), which raises the intracellular calcium level by allowing calcium to cross biological membranes (Fig. 2C, D). In contrast, neither type of high calcium stimulation was sufficient to inactivate the TRPC1α/5 (Fig. 2E–H). These results suggest that Ca²⁺ inactivates the TRPC1α/4β heteromeric channel via the Gα_q-PLCβ-IP₃ pathway.

In our previous study [11], CCh stimulation induced the production of DAG and Ca²⁺. Since the inactivation of TRPC5 occurs via PKC-mediated phosphorylation [24], we examined the effect of the PLCβ-DAG-PKC pathway on heteromeric channels. To identify the effect of PKC on heteromeric channels, we stimulated channels by CCh (100 µM) repetitively with or without pretreatment of a PKC inhibitor, GF109203X, or chelerythrine. HEK293 cells were transfected with M₃R, TRPC1, and TRPC4 or TRPC5. To quantify the inactivation, we defined the change ratio of the current at the beginning of CCh application to the currents at the end of CCh application (2 / 1 and 2′ / 1′) as the inactivation ratio (Fig. 3). As a control, CCh was applied repetitively without PKC inhibitors (Supplementary Fig. 2A, B). Both control experiments showed transient current activation by the Gα_q-PLCβ pathway stimulation. The second peak current amplitude evoked by CCh stimulation was not as much as the first peak because the PI(4,5)P₂ resynthesis was insufficient (Supplementary Fig. 2C, D). There was no difference between the first application of CCh and the second application of CCh (Fig. 3C; control panel). With GF109203X, TRPC1α/4β showed a similar tendency of current inactivation as the first CCh stimulation (Fig. 3A), whereas, TRPC1α/5 currents that increased during the second CCh stimulation were reversed completely (Fig. 3B, C, GF109203X panel). Similar results were observed with chelerythrine pretreatment: the inactivation ratio (2 / 1 and 2′ / 1′) was only significantly increased in TRPC1α/5 (Fig. 3C; chelerythrine panel).

Next, we used a PKC-insensitive mutant of channels, TRPC4 (T887A) and TRPC5 (T972A) for further confirmation (Fig. 4) [24]. This mutation did not affect the expression and membrane localization (Supplementary Figs. 3, 4). In the cells transfected with M₃R and heteromeric channels together, we stimulated channels by CCh. Activity change of channel mutants corroborates the previous results. TRPC1α/4β (T887A) showed a similar level of enhancement to that of wild-type (Fig. 3D, E). In contrast, the TRPC1α/5 (T972A) mutant showed a large enhancement in both basal and peak current compared to wildtype TRPC1α/5 (Fig. 3G, H). To identify the tendency of current change depending on time, we normalized the current trace. We also noticed that the inactivation was prolonged in TRPC1α/5 (T972A) (Fig. 3I), but not in TRPCα/4β (T887A) (Fig. 3F). These results suggest the possibility of other downstream molecules of the Gα_q pathway, especially PKC involvement in TRPC1α/5 channel regulation.

Therefore, we also investigated whether the channel responds to direct PKC activation. To induce PI(4,5)P₂ depletion before PKC activation, we used a rapamycin-inducible system. HEK293 cells were transfected with channel subunits, Inp54p and Lyn11. First, we stimulated heteromers with channel agonist, EA. After that, we artificially depleted PI(4,5)P₂ by translocating inp54p to membranes with rapamycin. Then, DAG analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG) was added as a PKC activator. As expected, there was no further current inhibition in TRPC1α/4β after OAG stimulation (Fig. 4A, B), whereas the current amplitude of TRPC1α/5 decreased to the basal level (Fig. 4C, D). All heteromeric channels showed outward rectifying I–V curve with every drug treatment (Fig. 4A, C; left). Collectively, these results suggest that PKC via the PLCβ-DAG pathway inactivates the TRPC1α/5 heteromeric channel.