GPCR-G_i protein pathways are involved in the regulation of vagus muscarinic pathway under physiological conditions and are closely associated with the regulation of internal visceral organs. The muscarinic receptor-operated cationic channel is important in GPCR-G_i protein signal transduction as it decreases heart rate and increases gastrointestinal (GI) rhythm frequency. Among five muscarinic acetylcholine receptors (mAChRs)—M1 to M5—, M2 and M4 receptors primarily utilize G_i/o signaling. The M2 receptor, in particular, mediate the effects of parasympathetic stimulation on the heart and GI organs. The most significant involvement among receptor-operated ion channels in this G_i-related process is definitely that of TRPC channels, especially TRPC4/5 channels.

To begin with, the TRP channel superfamily, comprising 28 mammalian cation channels across seven subfamilies—TRPC, TRPV, TRPA, TRPM, TRPP, TRPN and TRPML (Minke et al., 1975; Zhang et al., 2003; Zhang et al., 2023). Within the subfamilies, TRPC is known to be activated by PLC signaling pathways that lead to membrane depolarization and the elevation in cytosolic Ca²⁺ concentration. Among the various kinds of PLC signaling pathways, the G_q/11-PLCβ and receptor tyrosine kinase (RTK)-PLCγ pathways are the most commonly known. TRPC ion channels are non-selective cation channels with variable ion selectivity and Ca²⁺ permeability. These receptor-operated channels affects membrane potential and Ca²⁺ signaling in different ways to regulate the physiological conditions (Jeon et al., 2020a). In addition to activation by PLC signaling, direct activation by Gα_i is known only for TRPC4/5 channels. Previous studies have shown that Gα_i2 prefers to bind with TRPC4 whereas Gα_i3 prefers TRPC5 (Jeon et al., 2008; Jeon et al., 2012). Recently, the dual activation of TRPC4 by both G_i and G_q signaling pathways has been recognized significant in brain (Yang et al., 2015; Jeon et al., 2020b; Tian et al., 2022). TRPC4 activation requires coincident G_i/o stimulation as well as PLC activity (Thakur et al., 2016). Neurons encode distinct messages that reflect the activation of two ion channels, TRPC4 and GIRK, through coincident G_q/11 and G_i/o signaling, transmitting the messages to downstream neurons (Tian et al., 2022).

As cryo-EM structure of TRPC4 and TRPC5 ion channels have been revealed, both channels came out to have similar binding sites−TRPC4/5 activators and inhibitors such as Pico145, Riluzole, HC-070, clemizole, PIP₂, etc (see also Figure 6)− as their structure significantly overlaps (Duan et al., 2018; Duan et al., 2019; Won et al., 2023). Moreover, several features in intracellular regions of TRPC channels are conserved: the pre-S1 elbow is situated in the N-terminal domain, and the connecting helix runs parallel to the membrane bilayer. However, the binding interface with Gα_i protein was conserved only in the N-terminal ankyrin repeat domain (ARD) of TRPC4 and TRPC5 channels, which means both channels may be the only direct modulators for Gα proteins in TRP subfamily (Won et al., 2023). The binding interface of Gα protein with its effector molecules was also found to be conserved in the Gα_i-bound TRPC5 cryo-EM structure (Lyon et al., 2013; Won et al., 2023). In conjunction with the electrophysiological result demonstrating that Gα_i3 increases the sensitivity of TRPC5 to phosphatidylinositol 4,5-bisphosphate (PIP₂), this structural discovery provides evidence that ion channel activity can be directly regulated by Gα protein following GPCR activation. This finding may offer a structural framework for unraveling the crosstalk between two major classes of transmembrane proteins: GPCRs and ion channels. In this review, we specify the G protein related pathway and the direct relationship with TRPC ion channels in various internal organs. Also, possible drug development and disease control studies are introduced by targeting the GPCR-G_i-TRPC4/5 pathway.

G proteins, also known as guanine nucleotide-binding proteins, are a family of proteins that act as molecular switches inside cells, and are involved in transmitting signals from a variety of stimuli outside a cell to its interior. The binding and hydrolysis of GTP to GDP, facilitated by specific regulatory factors, govern the activity of these molecules. When in the GTP-bound state, the switch turns on, and, when in the GDP-bound state, the switch turns off. The shutdown of the G protein cascade is possible due to the intrinsic GTPase activity of G proteins, as they belong to the larger group of enzymes called GTPases.

There are two classes of G proteins. The first class functions as monomeric small GTPases (small G proteins), while the second class functions as heterotrimeric G protein complexes. Small G proteins (also known as small GTPases, small GTP binding proteins and Ras protein superfamily) form an independent superfamily within the larger class of regulatory GTP hydrolases. This superfamily is made up of a diverse range of molecules that control a vast number of important processes and possess a common, structurally preserved GTP-binding domain (Agretti et al., 2007). The small G protein superfamily consists of Ras, Rho Rab, Rac, Sarl/Arf and Ran homologs. Within the family of small G proteins, Ras proteins are identified as the best-characterized members. Rasd1 belongs to the Ras superfamily of small GTPase, which is expressed in the brain, heart, liver, kidney, pancreas, skeletal muscle, and placenta (Tu and Wu, 1999; Bernal and Crespo, 2006; Bernal et al., 2021). Activation of Gα_i subunits by Rasd1 is known to be the primary mechanism for activating TRPC4 (Wie et al., 2015). Another small G protein that may be a novel target for TRPC5, Rac1, is known to mediate podocyte injury in focal segmental glomerulosclerosis. Studies showed Rac1-activating mutations are responsible for inherited cases of focal segmental glomerulosclerosis, leading to the stimulation of TRPC5 ion channel activity and cytoskeletal remodeling in podocytes (Zhou et al., 2017).

The larger type of G protein, heterotrimeric G proteins are the most commonly found signal transducers in eukaryotic cells, and they mediate the effects of many pharmaceutical products. Heterotrimeric G proteins are the molecular switches that turn on intracellular signaling cascades in response to the activation of GPCRs by extracellular stimuli. GPCRs belong to the largest family of transmembrane receptors and act as the most fundamental signals that are involved in the regulation of internal visceral organs (Kim et al., 2012). Therefore, G proteins have a crucial role in defining the specificity and temporal characteristics of the cellular response (Oldham and Hamm, 2008). The activation of GPCRs promotes an alpha subunit (Gα) of a heterotrimeric G protein to exchange a nucleotide from GDP to GTP inside its pocket, thereby triggering the dissociation of a heterotrimeric G protein (Gαβγ) into Gα and Gβγ. Once activated, Gα proteins amplify the initial signal from the switch by activating effector molecules such as adenylyl cyclase, phospholipase C (PLC), and protein kinases (Liu et al., 2021). Due to the comparable density of ion channels in the plasma membrane (Clapham, 1994), various lines of evidence suggest that not only membrane-bound enzymes but also ion channels could serve as direct effectors of Gα and Gβγ proteins. There is a possibility that ion channels and GPCRs may coexist in close proximity, forming a signaling cluster within a specific region of the plasma membrane (Neves et al., 2002). The recent cryo-EM structure demonstrated that Gα_i3 could directly activate the TRPC5 channels, and the channel requires both Ca²⁺ and PIP₂ as essential cofactors for the complete activation of Gα_i3 (Won et al., 2023).

Neural circuits regulate organ function to stabilize physiological conditions, providing homeostasis to the body’s internal environment (Figure 1). The vagus nerve travels to the internal visceral and cardiovascular organs, where it regulates physiological responses to environmental changes and damages (Rosas-Ballina et al., 2011). ACh released from the vagus nerve binds to the muscarinic receptors. mAChRs comprise a family of five GPCRs, M1 to M5. Three of these receptor subtypes (M1, M3, and M5) have been shown to mainly couple to G proteins of the G_q/11 family, whereas the remaining two subtypes (M2 and M4) preferentially signal through the G_i/o family of G proteins (Hulme et al., 1990). The most well-known example of regulating effects on organs by mAChRs is in the heart, where the activation of M2 receptor results in the activation of Gβγ-dimer, thereby stimulating the GIRK channel to causing membrane hyperpolarization, ultimately slowing pacemaker depolarization (Harvey and Belevych, 2003).

In the GI tract and many other visceral organs, release of ACh from autonomic nerves triggers excitation and contraction of smooth muscle by activating mAChRs. Although various types of mAChRs contribute to concurrent signals for mIcat generation, the activation of M2 receptors predominantly induces the opening of cationic channels. These channels are also subject to modulation by M3 receptors (Bolton and Zholos, 1997; Zholos and Bolton, 1997). Only M2 and M3 receptors mediate contraction in all studied visceral smooth muscles, and M2 receptors contribute to contraction by inhibiting relaxation caused by agents that increase cAMP (Tanahashi et al., 2021). However, some evidences suggest that increase in intracellular Ca²⁺ concentration eliminates the influence of Ca²⁺ release, leading to 1) mIcat inhibition and 2) Gα_o-regulated depression (Zholos and Bolton, 1997; Yan et al., 2003). In the smooth muscles of various visceral organs, ACh serves as the primary neurotransmitter for excitation (Beech, 1997). It is released from short postganglionic nerves providing parasympathetic innervations to the smooth muscles of organs such as urinary bladder or myometrium (Zholos et al., 2024). The GI tract is equipped with inherent neural plexuses, where ACh is discharged by stimulating motor neurons within the enteric nervous system (Zholos et al., 2024).

Moreover, in the lingual artery, peripheral nerve stimulation resulted in relaxation and membrane hyperpolarization, which inhibitory responses were hindered by atropine (Bevan and Brayden, 1987). ACh plays an important role of endothelium dependent vascular relaxation in the aorta tissue preparation (Freichel et al., 2001). The relaxation was partially blocked in TRPC4 knockout mice (Freichel et al., 2001). On the other hand, Mori group showed that TRPC5 could be nitrosylated by G protein-coupled ATP stimulation in the endothelium. In addition, TRPC1/5 heteromer perform a major role on the NO formation from eNOS in the endothelium via a physical interaction of TRPC5 with eNOS (Yoshida et al., 2006). Interestingly, PKD1 activates TRPC4 in the endothelium through the G_i/o protein activation and controls endothelial cell migration and proliferation (Kwak et al., 2018).

TRPC4/5 channel is a molecular candidate for mIcat (Zhu et al., 2003; Lee et al., 2005) and can be directly activated by constitutively active Gαi^QL proteins. TRPC4 and TRPC5 belong to the same subfamily and both are activated by G_i/o proteins. G_i2 prefers to bind with TRPC4 whereas G_i3 prefers TRPC5 (Jeon et al., 2008; Jeon et al., 2012). Initial studies suggested that the binding sites for G protein exist at the rib helix of TRPC4/5 channels or the CIRB domain (Jeon et al., 2012). However, recent cryo-EM structure showed that IYY^58-60 amino acids at ARD bind with G_i3 protein (Won et al., 2023). Main debate concerns with the role of PIP₂. We showed that PIP₂ is essential for maintaining TRPC5 channel activity. Recently, we directly applied PIP₂ with inside-out patch mode and activated TRPC5 channels. When the binding sites was mutated, the mutants did not respond to intracellularly applied PIP₂. The role of Gα_i protein was to enhance the affinity of TRPC5 channels to PIP₂ at the physiological PIP₂ range (Won et al., 2023). Other research groups showed that PIP₂ inhibited TRPC5 tonically at the basal level, and depletion of PIP₂ decreased the activation time constant and rapidly increased the TRPC5 current (Thakur et al., 2016). Furthermore, Gudermann group suggest that DAG is a real activator for TRPC4 and TRPC5 channels because PIP₂ depletion cause TRPC4/5 to respond to DAG (Storch et al., 2017). Another important point is the roles of PLCδ1. We showed that PLCδ1 was activated by Ca²⁺ influx through TRPC4 and played a negative role on TRPC4 currents (Ko et al., 2023). Zhu group showed the contrary results. They needed G_i protein and PLCδ1 to activate TRPC4 channels (Thakur et al., 2016). When PLCδ1 was inhibited, TRPC4 was not activated by agonists, even in the presence of G_i proteins. Ca²⁺ and H⁺ ion were suggested as activators (Jeon et al., 2020a; Thakur et al., 2020). The exact roles of PIP₂ would be revealed when the cryo-EM structure of the PIP₂-bounded TRPC5 channel is obtained.

Recent cryo-EM structure supports the fact that PIP₂ binding site on TRPC5 is located near the S2-S3 linker, S4-S5 linker, TRP helix, and helix-loop-helix region. As Gα_i protein binds to TRPC5, increase in PIP₂ affinity leads to the increase as well. This means that Gα_i protein is not necessary to open the TRPC5 channel, but the intracellular Ca²⁺ concentration and PIP₂ affinity (or binding) may be the direct trigger for opening the TRPC5 channel. Both Ca²⁺ and PIP₂ have the potential to serve as a cofactor in the activation of the channel at the intracellular leaflet, consistent with findings from previous studies (Ningoo et al., 2021). Full activation of Gα_i3 to the channel may require the involvement of all three factors: Ca²⁺, PIP₂, and Gα_i3 (Figure 4). Also, PLCδ1 does not bind to TRPC5 unlike it does with TRPC4, which causes TRPC5 to have basal current with a high concentration of PIP₂.

We summarizes the complex interaction of G protein, DAG, PIP₂ and calcium as in Figure 4. First, to indicate that the channel is not open without PIP₂, the side consisting of the Gα and Ca²⁺ axes is darkened and points on the side set to be closed (C₀, C₁). In the presence of PIP₂ and Ca²⁺, channels are partially open (O_PIP2+Ca). As Gα_i activity increases and Gα_i bind directly to channels (O_PIP2+Ca→O_Gi), the PIP₂ sensitivity of the channel increases. In the diagram, this change is depicted by the increase in PIP₂ concentration, although it does not imply the actual elevation in PIP₂ concentration. G_q activity also opens TRPC4/5 channels potently (O_PIP2+Ca→O_Gq). Both open states induced by G_q and G_i reach to closed state through activation of PLCβ (O_Gq or O_Gi→C₁). However, the transition from G_i-open state is not powerful, as depicted. This process is accompanied by an increase in Ca²⁺ and a decrease in PIP₂. TRPC4/5 channels can open by under specific conditions when diacylglycerol (DAG) is generated from PIP₂ molecule (O_DAG). PIP₂ hydrolysis occurs in TRPC4β by PLCδ1. At this time, PLCδ1 becomes active due to an increase in Ca²⁺ independent of any Gα activities. Therefore, the process is drawn at the bottom (O_PIP2+Ca→O_DAG’). Since PLCβ is activated by G_q, it is plotted diagonally to reflect the Ca²⁺ increase and PIP₂ depletion at the time point in which G_q activity has progressed to some extent along the vetical O_PIP2+Ca–O_Gq line (middle of O_PIP2+Ca–O_Gq line→O_DAG). DAG-induced open states become closed when the C-terminus of the channel is phosphorylated by PKC, followed by binding with PDZ motif of Na⁺/H⁺ exchanger regulatory factor (NHERF). The process occurs concurrently with the advancement of G_q activity, depletion in PIP₂, and an increase in Ca²⁺ levels, reaching the dark side mentioned first and entering a closed state (O_DAG→C₁). But Gα activity is not needed in the case of TRP4β (O_DAG’→C₁).

GPCR-Gi/o protein signaling pathway in neurons is an essential component of the complex network of signaling mechanisms that regulate neuronal function (Figure 5). Recent studies indicated the direct relationship between neurological disorders and TRPC4/5 channels. Increased TRPC5 S-glutathionylation by oxidative stress contribute to neuronal damage in striatum that may result in Huntington’s disease (Hong et al., 2015). Dysfunction in TRPC4 may lead to epilepsy or autism spectrum disorder (Zheng, 2017; Gupta et al., 2023; Zhou et al., 2023). Freichel group showed that heteromeric TRPC1/4/5 channels are involved in depression and anxiety (Broker-Lai et al., 2017; Chu et al., 2020) TRPC1/4/5 channels play a role in the development of morphine tolerance and hyperalgesia. Prolonged exposure to morphine results in an increase in the expression of TRPC1/4/5 channels in the spinal cord (Chu et al., 2020). TRPC1/4/5 channels also possess developmental functions in neurons. TRPC5 regulates hippocampal neurite development (Greka et al., 2003), and dendrite patterning (Puram et al., 2011). TRPC4 in rat dorsal root ganglion neurons are known to be necessary for neurite outgrowth. Suppression of TRPC4 immuno-reactivity resulted in decrease in the length of neurites in cultured dorsal root ganglion neurons, confirming the necessity of TRPC4. Nerve injury causes increase in TRPC4 as well (Wu et al., 2008). Later research reported activation of TRPC4β, TRPC4 splice variants, through Gα_i regulates the morphogenesis of dendrites in cultured hippocampal neurons (Jeon et al., 2013).

TRPC4 as well as TRPC1 support the repetitive neural spiking in brain, confirming the various functions on the neuronal pathway. Relatively high expression level of TRPC4 in lateral septum promotes firing rate. Lateral septum receives signals from various brain regions, extending from hippocampus to amygdala, where diverse neurotransmitters such as ACh, dopamine, glutamate, GABA, and serotonin converge. The depolarization of plateau potential, responsive to electrical stimulation in the presence of blockers for inotropic GABA and glutamate receptors, were shown to be mediated by the G_q/11-coupled group 1 metabotropic glutamate receptors (Gallagher et al., 1995). Later, Zhu group showed that both G_i- and G_q-coupled signaling pathways are important for the spike firing in lateral septal nucleus and the response differs from G_q-only or G_i-only signaling (Yang et al., 2015; Jeon et al., 2020b; Tian et al., 2022). Two interconvertible depolarization responses (below-threshold-depolarization and above-plateu-depolarization) of TRPC4-group 1 metabotropic glutamate receptor activation contribute to patterns in lateral septal neuron firing activities (Phelan et al., 2012; Tian et al., 2014; Phelan et al., 2023). Another research has shown that not only TRPC4 but also TRPC1 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons (Phelan et al., 2012).

Flockerzi group elaborately showed that heteromeric TRPC1/4/5 channels are the major functional channels in the brain using multiple specific antibodies for TRPC1/4/5, multi-epitope affinity purifications, and high resolution liquid mass spectrometry (nano-LC-MS/MS) (Kollewe et al., 2022). The amount of TRPC proteins determined in each sample by nano-LC-MS/MS were finally combined to deduce the abundance of each isoform in all possible tetrameric configurations. The importance of hetero-tetramers is rising, given that only minor portions of the TRPC1, TRPC4, and TRPC5 proteins in the brain are present in homomers (13%, 6%, 9%, respectively). The majority is incorporated into three categories of heteromers: TRPC1/C4, TRPC1/C5, and TRPC1/C4/C5 (Kollewe et al., 2022). These findings are notable since homo- or hetero-tetramers modify channel properties significantly, such as Ca²⁺ permeability, PIP₂ sensitivity, and I-V curve. Further studies of the heteromeric TRPC1/4/5 channels in GPCR signaling pathway are necessary to understand their activity under physiological conditions.

Shapiro group put an emphasis on the role of TRPC in the G_i signaling pathway in the brain, as well as M channels and GIRK channels (Carver and Shapiro, 2019; Carver et al., 2020; Carver et al., 2021). Sohn group also suggested that TRPC5 mediates the effects of leptin and serotonin via POMC neurons (Gao et al., 2017), and this effect is independent of altering GIRK channel activity (Sohn et al., 2011). Recently, G_i/o-coupled GPCR in the paraventricular nucleus of the hypothalamus was found to antagonize the anorexic effect of serotonin agents via K_ATP channels (Yoo et al., 2021). Melanocortin 4 receptors (MC₄Rs) in parasympathetic preganglionic neurons activate K_ATP channels via G_s signaling, but in sympathetic preganglionic neurons, they activate putative nonselective cation channels (Sohn et al., 2013). In case of this neuronal circuit regulating feeding behavior and energy metabolism (involving POMC or NPY/AgRP neurons), TRPC5 channels are more crucial than GIRK and Ca_V channels. Most importantly and recently, Zhu group suggested that the lateral septal nucleus utilizes a minimum of two channels, TRPC4 and GIRK, both of which are modulated by G_i/o and G_q/11 pathways. While the G_i/o and G_q/11 pathways compete in their effects on GIRK, they cooperate in producing a self-propagating all-or-none activation of TRPC4. Zhu group emphasized that these nonlinear interactions allow for the encoding of coincident signaling, particularly the relative degrees to which the 2 G protein pathways are being activated, resulting in discernible action potential firing patterns (Tian et al., 2022).

In case of TRPC5, it exhibits the highest expression in the brain, mostly in CA1 pyramidal cell, amygdala, cingulate gyrus, and cerebellar nuclei (Riccio et al., 2002). M2R and M4R, the G_i/o-coupled GPCRs, are localized to both presynaptic and postsynaptic terminals, where they inhibit neuronal excitation with the coupled-G_i/o proteins. G_i/o-coupled GPCRs mediate inhibitory signals. For example, activation of these receptors can lead to a decrease in cAMP levels, which, in turn, can modulate ion channel activity and neurotransmitter release. This inhibition is crucial for maintaining the balance of excitatory and inhibitory signals in various processes in nervous system, such as synaptic transmission and neuronal excitability. Knockout mice of M4R, not M2R, show increased basal ACh release in the hippocampus (Tzavara et al., 2003). Considering with the recent finding that TRPC5 is the direct effector of G_i/o protein triggered by GPCR activation (Won et al., 2023), researchers should particularly consider the novel GPCR-Gα_i-TRPC5 pathway, especially in studies related to neuronal diseases.

TRP channels are known to be transducers of exogenous and endogenous noxious cues. The last decade has been superb with dramatically high resolution of molecular structures that have allowed us to learn the molecular intricacies of TRP channels using cryogenic electron microscopy. These findings, in combination with functional studies, have provided insights into the role played by these channels in the generation and maintenance of pain (Rosenbaum et al., 2022). The expression pattern in brain nuclei of TRPC4 and TRPC5 also show possibilities to become a novel TRP targets involved in pain processing. While the emphasis on the generation of pain has traditionally been centered on sensory neurons, there is a high possibility of discovering new drugs based on non-neuronal cell types, which can also impact pain perception (Rosenbaum et al., 2022). Research on TRPC4 knockout rats showed tolerance to visceral pain responses, whereas somatic pain responses were uninfluenced (Koivisto et al., 2022). In addition, the non-selective TRPC4/5 antagonist, 4-methyl-2-(1-piperidinyl)quinoline (ML-204), inhibited visceral pain responses in wild-type rats, confirming the role of TRPC4 in visceral pain. The application of ML-204 to amygdala results in suppression of mechanical hypersensitivity and attenuated neuropathic pain behavior in rats with spared nerve injury (Wei et al., 2015). Another TRPC4/5 antagonist HC-070 developed by Hydra and Boehringer Ingelheim is currently in clinical trial for the treatment of anxiety disorder and depression (Wulff et al., 2019). HC-070 also had a significant anti-hypersensitivity effect in the established phase of the chronic constriction injury model (Jalava et al., 2023). TRPC5 inhibitor GFB-887, currently in phase 2 clinical trial, is being developed by Goldfinch Bio for the treatment of kidney disease. GFB-887 was first developed as a treatment for diabetic nephropathy but GFB-887 showed the best result in patients with focal segmental glomerulosclerosis, a rare kidney disease marked by blood vessel scarring in the glomerulus (NCT number: NCT04387448). These findings suggest that centrally mediated TRPC4 and TRPC5 antagonists could relieve visceral and neuropathic pain (Blum et al., 2019). On the other hand, TRPC4 and TRPC5 activator Englerin A has been developed for cancer therapy as it can inhibit growth of tumor cell lines at nanomolar concentrations (Carson et al., 2015). Englerin A is a selective inhibitor of renal cancer cell growth compared to normal kidney cells and cancer cell lines of different origin (Akbulut et al., 2015). Selectivity turns out to be one of the most important factors in drug development, meaning that a discovery of a precise structure of Englerin A binding site in TRPC4/5 would be crucial for further research (Jeong et al., 2019; Kim et al., 2019).

Recent TRPC4/5 inhibitors block both TRPC4/5 with relatively similar potency, which confirms that they are not ready for pharmaceutical use (Zheng, 2022). The zinc binding site and CIRB site in the more variable cytosolic domain may also be promising for developing drugs that can differentiate TRPC4 and TRPC5 (Zheng, 2022). Furthermore, the PIP2 binding site may be a novel site to target as well. Activators or inhibitors for TRPC4/5 can be classified into three types, extracellular type, transmembrane (TM) type and cytosolic type (Figure 6). Ions like H⁺ ion, La³⁺ or Gd³⁺, binds to extracellular sites and activates TRPC4/5 channels (Jung et al., 2003; Semtner et al., 2007). This sites might be suitable for drugs which are water soluble and have charges. Many drugs, like riluzole, pico145, HC-070 or clemizole, binds to TM area (Wright et al., 2020; Song et al., 2021; Yang et al., 2022). Physiological modulators like Ca2⁺, DAG, or PIP₂ also binds to TM domain. The posttranslational modification, like PKA or PKC phosphorylation and glutathionylation, occurs on cytosolic area. We showed that G protein binds to cytosolic ARD and activates TRPC5 channels. For specific effect of drugs, multiple sites should be considered like G protein binding and PKA phosphorylation sites, PIP₂ and NHERF binding sites, or DAG binding and PKC phosphorylation sites (Zhu et al., 2005; Sung et al., 2011; Storch et al., 2017). Focusing only on the TM sites might not be enough for visualizing specific effect of drugs on TRPC4/5. Recent genetic study showed that R175C gain of function mutation in TRPC5 cause an impaired intellectual ability (Leitao et al., 2022). In this case, the drug affecting glutathionylation might improve said symptoms.

Ion channels, especially TRPC channels are now considered as novel target to be directly regulated by Gα_i proteins. GPCR-G_i protein pathways are involved in the regulation of vagus muscarinic pathway under physiological conditions and are closely associated with the regulation of internal visceral organs. The direct and indirect modulations of TRPC channel by G protein play an important role in the muscarinic stimulation that is known to involve the GPCR-G_i protein pathway including dopamine, μ-opioid, serotonin, glutamate, GABA, and the complex interaction between GIRK and TRPC4/5 should be considered in the field of neuroscience. However, two big questions need to be further addressed: the structure and functional involvement of heteromeric TRPC channels and the PIP₂ binding site regarding the TRPC channel and G protein complex. Heteromeric TRPC channels are naturally expressed at relatively high levels in the brain, which may be a key for a drug development in the field.