Canonical transient receptor potential channels and their modulators: biology, pharmacology and therapeutic potentials.

Some reports have shown that TRPCs are involved in other diseases, such as obesity, type II diabetes, and lung diseases (Yu et al. 2004 ; Krout et al. 2017 ). TRPC3/6 expression in the lung tissues and pulmonary artery smooth muscle cells of idiopathic pulmonary arterial hypertension patients was much higher than those normotensive or secondary pulmonary hypertension patients (Yu et al. 2004 ). It has been reported that hypoxia-inducible factor-1 boosts the expressions of TRPC1 and TRPC6 increasing intracellular Ca 2+ level in pulmonary artery smooth muscle cells (Wang et al. 2006 ). Another study suggested that TRPC1 plays a crucial role in adiposity via inducing autophagy and apoptosis; moreover, high-fat diet-induced obesity and type II diabetes were alleviated in TRPC1 knockout mice (Krout et al. 2017 ).

Ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate (Pyr3) is a specific and direct inhibitor of TRPC3 channel. Pyr3 ameliorates intracerebral hemorrhage-induced brain injury and could be used as a novel treatment strategy for TRPC3-mediated diseases, including pathological cardiac remodeling and heart failure (Glasnov et al. 2009 ; Munakata et al. 2013 ). Moreover, Pyr3 exerts an inhibitory effect on the growth of human glioblastoma cancer cells and triple-negative breast cancer cells in vitro and vivo through inducing apoptosis and inhibiting migration (Chang et al. 2018 ; Wang et al. 2019d ). The inhibitory effect of Pyr3 is dose-dependent in acute lymphoblastic leukemia cell lines (Abdoul-Azize et al. 2016 ). Pyr3 also inhibits smooth muscle proliferation and alleviates stent implantation-induced arterial injury (Álvarez-Miguel et al. 2017 ). Exposure to PM2.5 can increase the propensity to cardiac arrhythmias which could be attenuated with TRPC3 inhibition by Pyr3 (Cai et al. 2019 ). Pyr3 can also significantly decreases intracellular calcium concentrations, normalize resorptive activity and osteoclastic differentiation of TRPC6-deficient cells (Klein et al. 2020 ; Sato et al. 2020 ), and promotes systolic blood pressure in hypertensive patients (Hu et al. 2020d ). Galangin is a natural flavonoid compound from the ginger family that is used as a novel antidiabetic. It has been screened as a lanthanide-induced Ca 2+ entry antagonist in overexpressed TRPC5 HEK293 cells with IC 50 of 0.45 μM through a screen of natural compounds in traditional Chinese medicines (Naylor et al. 2016 ; Brás et al. 2020 ). Galangin also inhibits lanthanide-induced TRPC5-mediated current and calcium entry. TRPC5 is less inhibited by other related natural flavonols, such as kaempferol and quercetin, and is not affected by myricetin, luteolin, and apigenin. Galangin can attenuate isoproterenol-induced inflammation, cardiac fibrosis, pulmonary fibrosis and non-alcoholic fatty liver disease (Thangaiyan et al. 2020 ; Wang et al. 2020c ; Zhang et al. 2020 ). Galangin can also reduce cerebral ischemia–reperfusion injury by inhibiting ferroptosis and activating the SLC7A11/GPX4 axis (Guan et al. 2021 ). Additionally, galangin improves cardiac remodeling through the MEK1/2-ERK1/2 and PI3K-AKT pathways (Wang et al. 2019b ). Differentiation of dendritic cells hows tolerogenic properties in response to lipopolysaccharide stimulation during galangin treatment (Song et al. 2021 ). Also, galangin has a protective effect on ulcerative colitis induced by dextran sulfate sodium in mice and rats (Sangaraju et al. 2019 ; Fan et al. 2021 ). Galangin inhibits cell growth and metastasis of cholangiocarcinoma by downregulating the expression of microRNA-21 (Zou et al. 2020 ). Besides, galangin inhibits epithelial-mesenchymal transition and angiogenesis of glioma by downregulating CD44 (Chen et al. 2019a ). Simultaneously, galangin promotes apoptosis of diverse cancer cells, such as hepatocellular carcinoma cells, ovarian cancer cells and glioblastoma cells (Kong et al. 2019 ; Fang et al. 2019 ; Zhong et al. 2020 ; Huang et al. 2020 ). One study suggested that galangin, either alone or combined with insulin, can reduce glucose levels and improve skeletal muscle health in patients with diabetes (Kalhotra and Chittepu 2019 ). AC1903 is a specific TRPC5 inhibitor synthesized through experimentation. By comparison, AC1903 is nearly equipotent to ML204 in blocking riluzole-activated TRPC5-mediated whole-cell current, but AC1903 fails to inhibit carbachol (CCh)-evoked TRPC4 and OAG-induced TRPC6 currents in whole-cell patch recordings, with a half-maximal inhibitory concentration of 14.7 μM (Zhou et al. 2017 ). AC1903, as a TRPC5 inhibitor, can provide a therapeutic benefit to podocyte survivability in chronic kidney diseases, such as focal and segmental glomerulosclerosis (Zhou et al. 2017 ; van der Wijst and Bindels 2018 ; Sharma et al. 2019 ; Pablo and Greka 2019 ). NU6027 is a cyclin-dependent kinase inhibitor that inhibits the basal and zinc-augmented TRPC5 currents in TRPC5 overexpressed HEK293 cells. It has a neuroprotective effect on oxidative neuronal injury in prolonged seizures. NU6027 is considered a potent antagonist of the TRPC5 channel (Park et al. 2019 ). TRPC5 suppression using NU6027 reduces the neuronal death that may occur after traumatic brain injury (Park et al. 2020 ). Ribemansides A and B are two new acylated β-hydroxynitrile glycosides isolated from the aerial parts of Ribes manshuricum . They can inhibit the activity of the TRPC6 channel with IC 50 of 24.5 and 25.6 μM, respectively. These two compounds can suppress transforming growth factor β1 (TGF-β1)-induced fibrogenesis in HK-2 cells, similar to SAR7334, a TRPC6 inhibitor (Zhou et al. 2018 ). Through testing and identifying several preparations from plant extracts, larixol and larixyl acetate were identified as TRPC6 inhibitors of receptor-induced Ca 2+ entry (Urban et al. 2016 ). TRPC6 channel is inhibited by larixyl acetate in the DAG-stimulated rat’s pulmonary artery smooth muscle cells with IC 50 of 0.1–0.6 μM. Larixyl acetate can also prevent hypoxia-induced pulmonary vasoconstriction (HPV) (Urban et al. 2016 ) and improve the endothelial function after traumatic brain injuries in mice (Chen et al. 2019b ). TRPC3/7 and TRPC4/5 channels are weakly blocked by larixyl acetate, whereas no significant inhibition occurs in other related TRPV or TRPM channels (Urban et al. 2016 ). Moreover, larixyl acetate has analgesic and anti-inflammatory effects on neuropathic pain; therefore, it can be a novel therapy for intractable chronic pain (Wang et al. 2020b ). FK506 (Tacrolimus) is a potent immunosuppressive agent isolated from the filamentous bacteria fermentation broth, a strain of Streptomyces tsukubaensis No.9993. FK506 has been generally used in liver and kidney transplantation therapy (Kino et al. 1987 ; Dumont 2000 ; Tang et al. 2021 ). Studies have demonstrated that FK506 could suppress TRPC6 expression to ameliorate podocyte injury in T2DM cells (Chang et al. 2019 ); it also affects proteinuria and renal damage progression in renal tissues (Wei et al. 2017a ; Chen et al. 2021 ). Down-regulation of TRPC6 by FK506 can also be used to treat overactive bladder (Chang et al. 2019 ). Besides, FK506-binding protein 52, as a novel interaction partner of TRPC3, improves the hypertrophic growth of cardiomyocyte cultures (Bandleon et al. 2019 ). Also, FK506 induces the TGF-beta 1/Smad 3 pathway to prevent intervertebral disk degeneration independent of calcineurin inhibition (Ge et al. 2020 ). BI 749327 is an orally bioavailable TRPC6 antagonist that has been reported by Lin et al. ( 2019 ) with IC 50 of 13 nM; it can boost cardiac function and ameliorate renal disease fibrosis. DS88790512 is another orally bioavailable compound that is potent and a selective blocker of the TRPC6 channel; it is a icycle [4.3.0] nonane derivative with IC 50 of 11 nM. However, there have not been any reports of its efficacy in vivo (Motoyama et al. 2018 ) (Table 3 ). Table 3 TRPCs antagonists (1) Modulators Chemical structure Targeting channels and IC50 Predicted effects Reference Pyr3 TRPC3 Ameliorate severity of seizures and cardiac injury, inhibit human glioblastoma cancer and melanoma cells growth Álvarez-Miguel et al. ( 2017 ), Cai et al. ( 2019 ) Galangin 0.45 μM (TRPC5) Improve ulcerative colitis; apoptosis of diverse cancer cells Fan et al. ( 2021 ), Naylor et al. ( 2016 ), Zhong et al. ( 2020 ) AC1903 14.7 μM (TRPC5) Provide chronic kidney diseases Zhou et al. ( 2017 ) NU6027 TRPC5 Inhibit TRPC5 currents, and oxidative neuronal injury Park et al. ( 2019 ) Ribemansides A and B A: R = H B:R = OCH 3 24.5 μM (TRPC6, A) 25.6 μM (TRPC6, B) Suppress TGF-β1-induced fibrogenesis in HK-2 cells Zhou et al. ( 2018 ) larixyl acetate 0.1–0.6 μM (TRPC6) Prevent HPV, improved traumatic brain injuries Urban et al. ( 2016 ) FK-506 TRPC6 Ameliorate liver and renal damage Chang et al. ( 2019 ) BI 749327 13 nM (TRPC6) Support cardiac function and renal disease Lin et al. ( 2019 ) DS88790512 11 nM (TRPC6) Inhibit TRPC6 channel Motoyama et al. ( 2018 ) TRPCs antagonists (1) A: R = H B:R = OCH 3 24.5 μM (TRPC6, A) 25.6 μM (TRPC6, B) Richter et al. ( 2014a ) identified clemizole as a novel, reversible TRPC5 blocker that can efficiently inhibit TRPC5 currents and Ca 2+ entry with IC 50 ranging from 1.0 to 1.3 μM. In excised inside-out membrane patches, clemizole could still block TRPC5 current at single-channel level; it also affected TRPM3 and M8 and weakly impacted the TRPV1, V2, V3, and V4 channels at higher concentrations. Simultaneously, clemizole inhibits the heterologous TRPC1:C5 channel and the native TRPC5-like currents in the U-87 glioblastoma cell line. Studies have also shown that clemizole can have a certain therapeutic effect on Dravet syndrome (Strzelczyk and Schubert-Bast 2020 ). As a TRPC5 channel inhibitor, clemizole hydrochloride reduces electric field stimulation amplitude, which causes muscarinic receptor-induced contractions impairment of the detrusor and carotid arteries (Griffin et al. 2018 ; Liang et al. 2019 ). Through a cell-based high-throughput fluorescence assay for 305,000 compounds, ML204 has been reported as a novel, potent, and relatively selective antagonist of the TRPC4 and TRPC5 channels with IC 50 of 13.6 μM (Zhou et al. 2017 ), as well as weakly inhibiting TRPC6. ML204 has a direct inhibitory effect on TRPC4/5 channels; it also can affect other GPCR-independent receptors (Miller et al. 2011 ; Alom et al. 2018 ). ML204 can prevent pseudocyst formation; thus, podocyte numbers can be preserved by treatment with ML204 (Zhou et al. 2017 ). Also, ML204 reduces carotid arteries endothelium-dependent contractions in mice (Liang et al. 2019 ). The histamine-induced depolarization is significantly inhibited by ML204, which reduces neuronal excitation (Sato et al. 2020 ). Although ML204 cannot inhibit pacemaker activity, it modulates the depolarization of the membrane potential (Lee et al. 2020a ). Intradermal injections of ML204 in psoriasiform skin significantly reversed chronic pruritus and the inflammation induced by imiquimod (Lee et al. 2020b ). Recent studies have reported that ML204, as a selective TRPC4/5 blocker, can completely revert acetylcholine relaxations (Alom et al. 2018 ; Silva and Ballejo 2019 ). ML204 can also reduce electric-field stimulation and CCh-evoked contractions in TRPC4 −/− detrusor strips mice (Griffin et al. 2018 ). M084 is a 2-aminobenzimidazole derivative similar to ML204 reported to effectively block the TRPC4 and TRPC5 channels with IC 50 of 10.3 and 8.2 µM, respectively. On the other hand, M084 weakly inhibits TRPC3 and moderately inhibits TRPC6 (Zhu et al. 2015 ). Research has discovered that M084 also has antidepressant and anti-anxiety effects in mice experiments (Yang et al. 2015 ). Additionally, Zhu et al. ( 2015 ) proved that M084 effectively inhibits the plateau potential induced by TRPC4-containing channels through electrophysiological recording in the lateral septal neurons of mice. Through synthesizing and testing a total of 28 structural analogs of M084, it has been found that the 2-aminobenzimidazol skeleton is an essential requirement for blocking the TRPC4 channel (Zhu et al. 2015 ). Based on structure–activity relationship studies of the stated natural and other synthetic flavonols, 3,5,7-trihydroxy-2-(2-bromophenyl)-4H-chromen-4-one (AM12) was designed. AM12 can directly inhibit the activation of lanthanide-induced and (−)-englerin A -evoked TRPC5 channel with IC 50 of 0.28 μM. Likewise, it inhibits TRPC4 channels and weakly inhibits the TRPC1-TRPC5 channel (Naylor et al. 2016 ). Salvianolic acid B, extracted from the root of Salvia miltiorrhiza Bunge (Lamiaceae), is the most abundant bioactive compound that inhibits the expression of TRPC3 and TRPC6 channels. In the cardiovascular system, salvianolic acid B effectively counteracts doxorubicin (DOX)-evoked structural heart abnormalities and apoptotic damage, and inhibits the DOX-induced Ca 2+ overload and endoplasmic reticulum stress in cardiomyocytes via TRPC3 and TRPC6 inhibition (Chen et al. 2017 ; Li et al. 2020a ). Salvianolic acid B can also alleviate myocardial ischemic injury by inhibiting activation of the NLRP3 inflammasome and promoting mitophagy (Hu et al. 2020c ). Regarding its effects on the hepatic system, salvianolic acid B suppresses hepatic endoplasmic reticulum stress to improve insulin resistance in ob/ob mice (Shi et al. 2020 ). Salvianolic acid B also blocks the activation of hepatic stellate cells, protects against sepsis-induced liver injury, and prevents and alleviates liver, pulmonary and renal interstitial fibrosis (Su et al. 2020 ; Ma et al. 2020 ; Jiang et al. 2020a ; Hu et al. 2020b ; Tian et al. 2021 ; Gong et al. 2021 ). Furthermore, salvianolic acid B prevents high-fat diet-induced obese mice body weight gain and regulates gut microbiota (Li et al. 2020b ). Additionally, salvianolic acid B decreases mice interleukin-1 beta-induced colitis recurrence (Feng et al. 2020 ). Ragarding its effects on the skeletal system, salvianolic acid B promotes new bone formation and protects against oxidative damage (Kayalar et al. 2020 ; Xiao et al. 2020 ; Bian and Xiang 2020 ); it improves atherosclerosis by inhibiting the YAP/TAZ/JNK signaling pathway in endothelial cells and pericytes (Yang et al. 2020 ). In the nervous system, salvianolic acid B could improve the cognitive impairment of mice infected with Porphyromonas gingivalis by inhibiting neuroinflammation and reducing Aβ level (Liu et al. 2020 ). Salvianolic acid B can also improve chronic mild stress-induced depressive behaviors in rats via the AMPK/SIRT1 signaling pathway (Liao et al. 2020 ). As an otoprotective agent, salvianolic acid B inhibits ototoxic drug-induced ototoxicity by suppressing the apoptosis pathway (Zheng et al. 2020 ). Rox4560 is a TRPC3/6 channel antagonist that could suppress the elevation of thrombin intracellular calcium levels in podocytes (Guan et al. 2017 ). Sildenafil inhibits TRPC1 and TRPC6 expressions through PPARγ-dependent mechanisms counteracting podocyte injury and proteinuria (Sonneveld et al. 2017 ). Sildenafil also has potential cardioprotective effects (Santiago-Vacas et al. 2021 ). Pico145 (C31, HC-608) is a xanthine derivative that potently inhibits TRPC1/4/5 channels with IC 50 of 1.3 nM (TRPC5:C5), 0.349 nM (TRPC4:C4), 0.199 nM (TRPC5:C1, heteromers), and 0.033 nM (TRPC4:C1, heteromers) (Rubaiy et al. 2017b ). Pico145 can directly, reversibly, and competitively inhibits the AM237-mediated TRPC5:C5 channel activation (Rubaiy et al. 2017a ; Minard et al. 2019 ; Wright et al. 2020 ). The potency of Pico145 depends on the concentration of the (−)-englerin A (Rubaiy et al. 2017b ). Pico145 was also found to be highly similar to HC-070 (Just et al. 2018 ). Inhibition of TRPC1 by Pico145 causes group I metabotropic glutamate receptors-induced long-term depression and memory extinction in mice (Yerna et al. 2020 ). Additionally, Pico145 can prevent adverse reactions of the cancer cell cytotoxic agent (−)-englerin A (Cheung et al. 2018 ). HC070 is a highly potent, small molecule antagonist of TRPC4 and TRPC5 invented by Just et al. (Just et al. 2018 ). HC070 inhibits the homologous TRPC4:C4 (IC 50  = 46.0 nM) and TRPC5:C5 (IC 50  = 0.52 nM) channel, as well as the heterologous TRPC1:C5 (IC 50  = 1.4 nM, La 3+ -activated; IC 50  = 4.4 nM, M1 receptor-activated), and TRPC1:C4 (IC 50  = 1.3 nM) channel. HC070 also reversibly inhibits the lanthanum-induced mouse TRPC5 current with IC 50 of 0.55 nM. Additionally, HC070 and Pico145 showed anxiolytic and antidepressant effects on mice (Just et al. 2018 ; Rubaiy 2019 ). SAR7334 is a novel, highly potent, and bioavailable compound that inhibits TRPC6-, TRPC3-, and TRPC7-induced Ca 2+ influx with IC 50 of 9.5, 282, and 226 nM, respectively, whereas it does not affect TRPC4/5 channels (Maier et al. 2015 ). Furthermore, SAR7334 suppresses TRPC6-dependent acute HPV in the isolated perfused lungs of mice (Maier et al. 2015 ) and (O-3)-induced airway inflammatory responses (Chen et al. 2020a ). SAR7334 can also negate malignant hyperthermia hypersensitivity by blocking TRPC3/6 (Lopez et al. 2020a ). There is a strong possibility that SAR7334 improves cognitive deficits (Uryash et al. 2020 ). As for the cardiovascular system, SAR7334 dose-dependently attenuates systolic and diastolic blood pressure variability (Wang et al. 2020d ). Additionally, TRPC6 knockdown by SAR7334 inhibits renal tubular epithelial cells apoptosis upon oxidative stress through autophagy activation (Hou et al. 2018 ). Compound 14a is a novel TRPC6 antagonist that strongly inhibits 4o, TRPC3/6/7 agonist-induced, and receptor-operated activation of the TRPC6 channel. Compound 14a inhibits TRPC3/6/7 (TRPC6 > C7 > C3) with IC 50 of around 1 µM against gastric cancer cell growth and xenograft tumor formation. Compound 14a weakly affects the TRPC4 channel and doesn’t affect other TRP channels (Ding et al. 2018 ). Whole-cell voltage-clamp experiments confirmed that KB-R7943 could potently block TRPC3, TRPC6, and TRPC5 currents with IC 50 of 0.46, 0.71, 1.38 µM, respectively, and nearly fully suppress the OAG-induced Ca 2+ entry in HEK293 cells (Kraft 2007 ) (Table 4 ). Table 4 TRPCs antagonists (2) Modulators Chemical structure Targeting channels and IC50 Predicted effects Reference Clemizole 1.0–1.3 μM (TRPC5) TRPC1:C5 Inhibit TRPC1:C5 channel and TRPC5 currents in the U-87 glioblastoma cell line Richter et al. ( 2014a ) ML204 13.6 μM (TRPC4/5) Block riluzole-activated TRPC5-mediated current; reduce the mouse carotid arteries endothelium-dependent contractions Liang et al. ( 2019 ), Miller et al. ( 2011 ), Zhou et al. ( 2017 ) M084 10.3 µM (TRPC4), 8.2 µM (TRPC5) Antidepressant and anti-anxiety effects Yang et al. ( 2015 ), Zhu et al. ( 2015 ) AM12 0.28 μM (TRPC5) TRPC4 Inhibit the activation TRPC5 channel Naylor et al. ( 2016 ) Salvianolic acid B TRPC3, TRPC6 Counteracted DOX-evoked heart abnormalities and tissue injury Chen et al. ( 2017 ), Su et al. ( 2020 ) Rox4560 TRPC3/6 Suppress calcium levels in podocytes Guan et al. ( 2017 ) Sildenafil TRPC1/6 Counteracting renal injury; cardioprotective effects Lu et al. ( 2010 ), Santiago-Vacas et al. ( 2021 ), Sonneveld et al. ( 2017 ) Pico145 1.3 nM (TRPC5:C5), 0.349 nM (TRPC4:C4) 0.199 nM (TRPC5:C1), 0.033 nM (TRPC4:C1) Inhibit AM237-mediated TRPC5:C5 channel activation Minard et al. ( 2019 ), Rubaiy et al. ( 2017a , b ) HC070 46.0 nM (TRPC4:C4), 0.52 nM (TRPC5:C5) 1.4 nM (TRPC1:C5), 1.3 nM (TRPC1:C4) Anxiolytic and anti-depressant Just et al. ( 2018 ) SAR7334 9.5 nM (TRPC6), 282 nM (TRPC3) 226 nM (TRPC7) Suppress acute HPV; attenuate renal injury Hou et al. ( 2018 ), Maier et al. ( 2015 ) Compound 14a  ~ 1 µM (TRPC3/6/7) Inhibit gastric cancer cell growth Ding et al. ( 2018 ) KB-R7943 0.46 µM (TRPC3), 0.71 µM (TRPC6) 1.38 µM (TRPC5) Suppress Ca 2+ entry Kraft ( 2007 ) TRPCs antagonists (2) 1.0–1.3 μM (TRPC5) TRPC1:C5 0.28 μM (TRPC5) TRPC4 1.3 nM (TRPC5:C5), 0.349 nM (TRPC4:C4) 0.199 nM (TRPC5:C1), 0.033 nM (TRPC4:C1) 46.0 nM (TRPC4:C4), 0.52 nM (TRPC5:C5) 1.4 nM (TRPC1:C5), 1.3 nM (TRPC1:C4) 9.5 nM (TRPC6), 282 nM (TRPC3) 226 nM (TRPC7) 0.46 µM (TRPC3), 0.71 µM (TRPC6) 1.38 µM (TRPC5) Merritt et al. ( 1990 ) originally identified SKF96365 as a novel inhibitor of ROCE, (1-(β-[3-(4-methoxy-phenyl) propoxy]-4-methoxyphen-ethyl)-l H-imidazole hydrochloride), with IC 50 of around 10 μM. However, SKF96365 was not as potent or selective as desired because it could also block T-type calcium channels (Singh et al. 2010 ). A previous study indicated that SKF96365 inhibits TRPC channels, arrests the cell cycle in the G2/M phase, and suppresses cell growth in AGS or MKN45 human gastric cancer cell lines (Ge et al. 2018 ), as well as arresting the cell cycle in the S and G2 phases in glioblastoma cells (Song et al. 2014 ). SKF-96365 is proven effective in the therapy of primary thermal and mechanical hyperalgesia and persistent spontaneous nociception (Ding et al. 2011 , 2012 ). As a SOCE inhibitor, SKF-96365 also exhibits potent anti-neoplastic activity in non‑small cell lung cancer cells (Wang et al. 2018b ). Moreover, recent reports showed evidence that SKF-96365 induces reduction in cardiac conduction (He et al. 2017 ). Moreover, SKF96365 reduces Ca 2+ concentration and airway smooth muscle cell viability in asthmatic mice (Zhang et al. 2018a ). 2-Aminoethoxydiphenyl borate (2-APB) is an inositol 1,4,5-trisphosphate receptors (IP3Rs) antagonist and TRPC channel antagonist that blocks the expression of human TRPC5 and TRPC6 in HEK-293 cells. In the concentration–response curve, 2-APB has IC 50 of 20 μM (Diver et al. 2001 ; Xu et al. 2005 ; Sekaran et al. 2007 ). 2-APB can act directly on TRPC ion channels in melanopsin-expressing ganglion cells (Sekaran et al. 2007 ). TRPC1/3/6 inhibition by SKF96365 and 2-APB attenuates TGF-β1-induced epithelial-mesenchymal transition in gastric cancer via the Ras/Raf1/ERK signaling pathway (Ge et al. 2018 ) (Table 5 ). Table 5 TRPCs antagonists (3) Modulators Chemical structure Targeting channels and IC50 Predicted effects Reference SKF96365 TRPC, low-voltage-activated T-type calcium channels 10 μM suppress growth in human gastric cancer cell lines, glioblastoma cells, non‑small cell lung cancer and colorectal cancer cells, primary thermal and mechanical hyperalgesia and persistent spontaneous nociception, induce a reduction in cardiac condition Ding et al. ( 2011 ), Singh et al. ( 2010 ), Song et al. ( 2014 ), Ge et al. ( 2018 ) 2-APB TRPC, TRPM3 20 μM Act directly on a TRPC ion channel in melanopsin-expressing ganglion cells, block human TRPC5, TRPC6 and TRPM3 channels Diver et al. ( 2001 ), Ge et al. ( 2018 ), Sekaran et al. ( 2007 ), Xu et al. ( 2005 ) TRPCs antagonists (3) TRPC, low-voltage-activated T-type calcium channels 10 μM TRPC, TRPM3 20 μM All nonselective TRPCs antagonists have certain inhibitory effects on various cell types and can target a particular channel or disease through structural modification, which presents a novel TRPCs therapeutic target for drug discovery.

TRPC6 can accelerate the progression of many acquired glomerular diseases, such as glomerulosclerosis associated with autoimmune glomerulonephritis, primary and secondary focal and segmental glomerulosclerosis (FSGS), and type-1 diabetes (Dryer et al. 2019 ; Polat et al. 2019 ). Over-activation of the TRPC6 channel and Trpc6 gene mutations result in glomeruli injury (Dryer et al. 2019 ; Staruschenko et al. 2019 ). It is worth noting that reactive oxygen species and angiotensin II (AngII) are the two major factors that provoke dramatic increases of TRPC6-mediated Ca 2+ influx in diabetic kidney disease, resulting in hypertrophy and the death of podocytes (Ilatovskaya et al. 2015 ; Ma et al. 2016 ; Staruschenko et al. 2019 ). TRPC6 antagonist, BTP2, attenuates renal fibrosis and glomerulosclerosis in TRPC6-knockout mice (Wu et al. 2017a ). However, Wang et al. revealed that TRPC6 knockout reduces proteinuria and decreases tubule injury but increases mesangial expansion and promotes insulin resistance, causing exacerbation of diabetic kidney disease in Akita mice (Wang et al. 2019c ). AngII and endothelin-1-induced mesangial contraction are significantly decreased with the downregulation of TRPC1 in TRPC1 antibody-treated rats. Hence, TRPC1 could play a specific role in regulating mesangial cell contractility (Woudenberg-Vrenken et al. 2009 ). Studies have indicated that TRPC5 blocker, AC1903, could successfully protect podocytes injury in vitro and the kidney filter function in vivo; thus, TRPC5 could be a potential therapeutic target for FSGS (Pablo and Greka 2019 ).

TRPCs play a relevant role in the progression of different types of cancers, such as colorectal cancer, lung cancer, breast carcinoma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, and more (Wang et al. 2015b , 2018a ; Jardin et al. 2018 ). Multiple studies provided evidence that the homeostasis of intracellular Ca 2+ is disrupted in cancer cells and that the turbulence of Ca 2+ signaling is concerned with tumor proliferation, migration, and invasion contributing to the overall tumor progression (Yang et al. 2009 ; Wang et al. 2015b ; Jardin et al. 2018 ). One study reported that Trpc5-siRNA inhibits the Wnt/β-catenin signal pathway, reduces ABCA1 induction, and causes a prominent reversal resistance of 5-fluorouracil in human colorectal cancer cells (Wang et al. 2015b ). However, the role of TRPCs is still controversial in breast cancer. On the one hand, hyperforin depresses the breast cancer’s cell growth and viability in MDA-MB-231 cells, which may be due to over-activation of TRPC6, which disrupts Ca 2+ signaling, thereby affecting cell proliferation (Aydar et al. 2009 ). On the other hand, TRPC6 knockdown by shTrpc6 significantly attenuates MCF7 and MDA-MB-231 cell proliferation (Jardin et al. 2018 ). Liang Wen et al. demonstrated that calcium dependence is an essential mechanism for regulating multi-drug resistance via the TRPC6/calcium/STAT3 signal pathway, and silencing TRPC6 elevates the efficacy of doxorubicin in hepatocellular carcinoma cells (Wen et al. 2016 ). Hong-Ni Jiang et al. found that overexpressing TRPC1 and TRPC6 increases the proliferation of the A549 lung cancer cell (Jiang et al. 2013 ). Notably, the TRPC6-NFAT pathway is activated by WNK1 promoting clear-cell renal-cell carcinoma cell proliferation and migration (Kim et al. 2019 ).

TRPCs participate in the formation of synapses and the modulation of neurotransmitter release; this involvement of TRPCs plays a vital role in the neurological system’s functions, such as memory, movement, cognition, anxiety, and fear (Riccio et al. 2014 ; Hong et al. 2015 ). TRPCs are primarily expressed in the brain, which is susceptible to oxidative stress. The TRPC5-like current activated by oxidized glutathione increases Ca 2+ , which ultimately induces striatal neuronal cell death. TRPC5 inhibition by ML204 predominantly attenuates oxidation-provoked striatal neuronal cell death and improves motor and rearing behavior in Huntington’s disease transgenic mice (Hong et al. 2015 ). Many factors contribute to Parkinson’s disease, such as excitotoxicity, mitochondrial dysfunction, endoplasmic reticulum stress, reactive oxide species, and inflammation; almost all of them depend directly or indirectly on Ca 2+ signaling (Sukumaran et al. 2017 ). Reduction in apoptosis and Ca 2+ influx provides neuroprotection via activation of TRPC1 (Thapak et al. 2020 ). Studies have shown that TRPC6 specifically inhibits its cleavage by γ-secretase of amyloid precursor protein (APP) and reduces β-amyloid formation by preventing APP from interacting with presenilin 1, which could be a novel strategy for treating Alzheimer's disease (Wang et al. 2015a ). At the same time, neurotoxic TRPC6-mediated Ca 2+ entry is mediated by presenilin 2. Activation of TRPC6 increases adult hippocampal neurogenesis and long-term spatial memory but reduces Aβ accumulation due to increased cerebrovascular P-glycoprotein (Thapak et al. 2020 ). Antonio Riccio et al. reported that TRPC4 −/− mice displays decreased anxiety-like behavior and Gq/11-dependent responses (Riccio et al. 2014 ). Similarly, TRPC1/4/5 blocker, HC-070, alleviates anxiety and depression in mice (Just et al. 2018 ). Inhibition of TRPC6 degradation by calpain inhibitors prevents ischemic neuronal death, improves behavioral performance, and provides neuroprotection through the Ras/MEK/ERK/CREB pathway (Thapak et al. 2020 ).

Ions channels modulate the flow of ions through the plasma membrane through temperature, chemical signals, electrical signals, or mechanical signals. Determining the channels’ structure is crucial to understanding their molecular mechanisms (Moiseenkova-Bell and Wensel 2011 ). Along with the tremendous advancement of the electron cryo-microscopy (cryo-EM) technology, high-resolution structures have become an increasingly dominant method for determining the structure of many receptor complexes (Egelman 2016 ). This technological advancement has resulted in a revolution in the mammalian respiratory complex I structure (Agip et al. 2019 ). In the last few decades, the atomic models of macromolecular complexes were determined by X-ray crystallography (Egelman 2016 ). It was not until 2018 that the high-resolution structure of TRPCs was revealed (Li et al. 2019a ). Compared with classical X-ray crystallography, cryo-EM technology does not need to crystallize or diffract the protein/protein complex, and only uses a small number of samples. Owing to this advancing technology, membrane proteins' structure determination has become convenient, particularly for various ion channels (Li et al. 2019a ; Wang et al. 2020a ). The current TRPCs structures reported had TRPC3, TRPC4, TRPC5, and TRPC6 channels (Fan et al. 2018 ; Duan et al. 2018 , 2019 ; Azumaya et al. 2018 ; Vinayagam et al. 2018 ; Tang et al. 2018 ). The overall resolution of TRPCs in these reports is exceptionally similar, and they all showed that TRPCs are tetramer structures composed of homologous or heterologous monomers that contain six transmembrane spanning domains (Duan et al. 2018 , 2019 ). Many experiments showed that TRPC5 was closely related to TRPC4, with a 65% sequence identity (Duan et al. 2018 ). Mice TRPC4 and TRPC5 carry out protein purification (pH 7.5); then, they are used for the single-particle cryo-EM analysis of the overall resolution of 3.3 Å and 2.8 Å in its unliganded (apo) state, respectively. TRPC4 poor densities are due to disordered regions; it consists of 4 residues in the S1-S2 loop, 2 residues in the S3-S4 loop, 27 residues in the distal N terminus, and 28 residues in the truncated distal C terminus, whereas TRPC5 is composed of 7 residues in the S1-S2 loop, 28 residues in the distal N terminus, and 3 residues in the truncated distal C terminus (Duan et al. 2018 , 2019 ). The TRPC4 and TRPC5 structures are formed by a four-fold symmetric homotetramer with dimensions of 100 Å by 100 Å by 120 Å. Each of the four monomers consists of a unique compact cytosolic domain and a transmembrane domain (TMD), distinguishing it from other TRPs. The cytosolic domain is divided into two subdomains: the N terminus region with four ankyrin repeats domain of AR1 to AR4 and seven α-helices domains of H1 to H7 and the C terminus region with a connecting helix and a coiled-coil domain. The TMD is composed of six helices (S1 to S6), a TRP domain, and several small helices (Duan et al. 2018 , 2019 ). In the TRPC4 and TRPC5 channels, the extracellular third transmembrane helix S3 region is made up of four helical turns, limiting potential extracellular interactions (Duan et al. 2019 ). Compared with TRPC4 and TRPC5 channels, the extracellular S3 region in the TRPC3 and TRPC6 channels is a unique transmembrane domain that is remarkably long, constituting an extracellular region that can be used as a sensor of external stimuli (Fan et al. 2018 ; Duan et al. 2019 ). In the stable closed state, single-particle cryo-EM analyses revealed human TRPC6 (hTRPC6) channel with a newly identified high-affinity inhibitor [2-(benzo[d] [1, 3] dioxol-5-ylamino) thiazol-4-yl] [(3S, 5R)-3, 5-dimethylpiperidin-1-y-l] methanone (BTDM), which has an overall resolution of 3.8 Å; it also revealed human TRPC3 (hTRPC3) channel with a lipid-activator OAG of 4.4 Å resolution. The hTRPC3 and hTRPC6 channels tetramer possesses dimensions of 75 by 75 by 150 Å 3 . The TRPC3 channel has four elbow-like membranes prior to the first transmembrane helix, while the TRPC6 channel has only three. The TRP helix is perpendicular to the pore-lining S6, and the hTRPC6 intracellular cytoplasmic domain presents an inverted bell shape whose top is below the ion channel pore of TMD (Fan et al. 2018 ; Tang et al. 2018 ). The truncation of the N terminus 71 amino acids is dispensable for the hTRPC6 assembly and gating, and the amino acids of TRPC6 cytoplasmic C terminus fold into two long helices (Tang et al. 2018 ). Novel electron cryo-EM technology provides a structural basis for the function and gating mechanisms of TRPCs.

TRPCs are associated with the occurrence and development of many diseases. Studies have proved that they are linked with different crucial roles in the pathophysiological process of many diseases; thus, they present a novel target for the intervention and treatment of some diseases. Additionally, the emergence of TRPCs modulators can prompt an understanding of these channels in health and disease. At present, highly potent and highly selective TRPCs modulators, such as (−)-englerin A, hyperforin, ML204, Pico145, HC070, Pyr3, DS88790512, and AC1903 are still unprecedented opportunities for TRPCs research. However, some obstacles, such as the toxicity and instability of (−)-englerin A in the cardiac and respiratory systems limit their use (Wu et al. 2017b ; Minard et al. 2018 ; Rubaiy 2019 ). Additionally, hyperforin is also unstable and susceptible to oxygen, heat, and light. Pico145 and HC070 are suitable for in vivo studies, whereas ML204 and DS88790512 were only conducted in vitro studies; thus, there are no reports on their in vivo efficacy. AC1903 could cause a developmental defect in the amygdala. These problems are collectively obstructive to the usage of the current selective TRPCs modulators; thus, the development of new TRPCs modulators drugs that are more potent and more selective is needed. With the tremendous advancement of science and drug technologies, discovering the means to overcome the mentioned undesired effects becomes possible. In structural biology, high-resolution structures are increasingly becoming the dominant method for determining the structures of many receptor complexes through cryo-EM technology. In recent decades, rich combinatorial compound libraries provide an abundant material basis for high-throughput screening due to their vast quantities and diverse structures that have become a popular approach to discovering and developing new medications, offering possibilities for finding suitable TRPCs modulators. Furthermore, many reports revealed that antisense oligonucleotide therapies and small molecule peptides therapies have increasingly become a novel strategy for treating a variety of diseases (Yamamoto et al. 2016 ; Beekman and Howell 2016 ; Mignani et al. 2019 ; Chen et al. 2020b ; Tahirovic et al. 2020 ). Studying channel domains functions, especially the precise interpretation of modulators’ binding sites, helps discover highly selective TRPCs modulators. These new technologies permit a more comprehensive assessment of the structures and function of TRPCs in physiology and pathophysiology, thereby providing the means to find more potent and selective TRPCs modulators.

The primary Transient Receptor Potential (TRP) protein was initially discovered in the experiment of aberrant visual transduction of Drosophila melanogaster (Cosens and Manning 1969 ). TRP channels (TRPs) feasibly form tetramers, a six-transmembrane domain structure with a cation-permeable pore loop between the fifth and sixth transmembrane domains (Hofmann et al. 2017 ). It is worth noting that there are differential expressions of TRPs in many tissues and cells and that TRPs show functional diversity and different pathological characters (Zhu et al. 2011 ). So far, the TRPs superfamily in mammalian is composed of 28 nonselective cation channels, and consists of several subfamilies: TRPC (canonical), TRPM (melastatin), TRPA (ankyrin), TRPV (vanilloid), TRPML (mucolipin), and TRPP (polycystin) (Zhu et al. 2011 ; Zheng 2013 ; Minard et al. 2018 ). The TRPC channels (TRPCs) family is the closest homolog to Drosophila TRP channels (Zheng 2013 ; Li 2017 ). TRPCs are nonselective (Ca 2+ , K + , Na + ) and high calcium permeability cationic channels. They are extensively expressed in many cells and tissues, including the lung, heart, brain, placenta, adrenal gland, retinal endothelial, testis, and kidney; they play crucial roles in many human physiological and pathological processes and are involved in various pathogenesis (Tai et al. 2017 ; Li et al. 2019b ). TRPCs are activated by various chemical and physical stimuli through the phospholipase C (PLC) signaling pathway. For instance, TRPC1/4/5 homotetramers and heterotetramers channels are activated by Gq protein-coupled PLC and phosphatidylinositol 4,5-bi-sphosphate (PIP2) hydrolysis. This process generates inositol triphosphate (IP3) that binds to the endoplasmic reticulum IP3 receptor increasing extracellular Ca 2+ entry called store-operated calcium entry (SOCE). Also, TRPC3/6/7 channels are unique in being directly activated by diacylglycerol (DAG), a degradation product of PIP2, which is known as receptor-operated calcium entry (ROCE) (Minke and Cook 2002 ; Jeon et al. 2012 ; Zheng 2013 ; Li et al. 2019b ). The present review focuses on the classification, structures, and function of TRPCs, as well as TRPCs modulators and their desired therapeutic effects, highlighting examples and opportunities for the application of TRPCs modulators in the pathology of various diseases.

Ca 2+ plays a crucial role in maintaining physiological functions in the cardiovascular system, including diastolic cardiac function, cardiac contractility, and hemodynamic change (Tai et al. 2017 ; Wu et al. 2019 ). Several studies showed that TRPCs participate in cardiovascular system disease progression (Kitajima et al. 2016 ; Zhang et al. 2018b ; Falcon et al. 2020 ). Wu et al. found that Ca 2+ influx is increased in the hypertrophic myocytes of adult wild-type mice, while this phenomenon is not observed in the myocytes of dominant-negative (dn) TRPC3/4/6 adult mice (Wu et al. 2010 ). Additionally, the activity of the calcineurin-nuclear factor of activated T cells (NFAT) significantly decreases in dnTRPC3/4/6 mice (Wu et al. 2010 ). Over-expression of TRPCs could stimulate the activation of NFAT, which accelerates TRPCs expression through a positive feedback mechanism. The activation of this mechanism could contribute to the development of cardiac hypertrophy and hypertension (Watanabe et al. 2008 ; Wu et al. 2010 ). A previous study found that TRPC3 can form a complex with A2R and PDE1C, and PDE1C is activated by TRPC3-invoked Ca 2+ , thereby facilitating cardiomyocyte apoptosis (Zhang et al. 2018b ). Remarkably, a background Ca 2+ entry pathway mediated by TRPC1/C4 can regulate Ca 2+ cycling in cardiomyocytes. However, the pathological cardiac remodeling development is attenuated in transverse aortic constriction-induced TRPC1/4 double knockout mice (Camacho Londoño et al. 2015 ).

TRPs were discovered in 1969 when the founding member of this superfamily was studying the D. melanogaster visual nerve transduction system (Cosens and Manning 1969 ). However, it was not until 1975 that the channels were named (Minke et al. 1975 ). Since the detection of TRPs, TRPC1 was the first human homolog of TRPs reported in 1995, but no further study for its functionality had been conducted yet (Wes et al. 1995 ). Over the following 3 years, five more novel related mammalian homologs (TRPC2-6) were detected, in addition to the functional expression of TRPC1 and TRPC3 and the cloning and function of TRPC5 on the store- or receptor-operated Ca 2+ entry (Zhu et al. 1996 ; Boulay et al. 1997 ; Okada et al. 1998 ). Additionally, another novel Ca 2+ channel protein, TRPC7, was reported (Nagamine et al. 1998 ). At present, and considering the sequence homology, the seven mammalian TRPC subfamily members are subdivided into three subgroups: TRPC1/4/5, TRPC2, and TRPC3/6/7 (Zhu et al. 1995 ). Since human TRPC2 is most likely a pseudogene (Wissenbach et al. 1998 ), this review focuses on investigating TRPC1/4/5 and TRPC3/6/7 channels.