TRPC4 as a coincident detector of G_i/o and G_q/11 signaling: mechanisms and pathophysiological implications

Abstract

TRPC channels are Ca²⁺-permeable nonselective cation channels activated downstream from phospholipase C (PLC). Although most TRPC channels can be activated by stimulating G_q/11-coupled receptors, TRPC4 requires simultaneous stimulation of G_i/o-coupled receptors, making it a perfect detector of coincident G_i/o and G_q/11 signaling. Evidence shows that activated Gα_i/o proteins work together with PLCδ1 to induce robust TRPC4 activation and the process is accelerated by stimulation of other PLC isozymes, such as PLCβ through G_q/11 proteins. Mechanistically, G_q/11-PLCβ activation produces triggering proton and calcium signals to initiate self-propagating PLCδ1 activity, crucial for G_i/o-mediated TRPC4 function. Thus, TRPC4-containing channels are activated under conditions not only when coincident G_i/o and G_q/11 stimulation occurs, but also when G_i/o stimulation coincides with proton and Ca²⁺ signals. The resulting cytosolic Ca²⁺ rise and membrane depolarization switch the inhibitory G_i/o response to excitation. The conditions and implications of G_i/o-mediated TRPC4 activation in physiology and pathophysiology warrant further investigation.

1. Introduction

TRPC4 is a member of the canonical subfamily of Transient Receptor Potential (TRP) channels. TRP superfamily contains 28 mammalian members of related proteins that share homology with a Drosophila protein named as TRP due to the transient receptor potential phenotype found in the loss-of-function mutations that affected phototransduction of fruit flies ¹,². All TRPs function as cation channels with variable selectivities and Ca²⁺ permeabilities and thus affecting membrane potential and Ca²⁺ signaling in different ways to contribute to physiology and pathophysiology.

Structurally, they share a common tetrameric assembly with each subunit containing 6 transmembrane (TM) segments (S1-S6) and the intervening pore loop between the S5 and S6 segments, as well as the cytoplasmically localized amino-termius and carboxyl-terminus (Fig. 1A). The S5-pore-S6 domains from the four subunits form the ion conducting pore, encircled by the S1-S4 TM regulatory domains. This architecture resembles that of classical voltage-gated K⁺, Na⁺, and Ca²⁺ channels, to which TRP channels display identical membrane topology and some sequence homology ³, except for the more extended lengths and functional motifs found in the cytoplasmic termini of TRP channels. Based on sequence homology, the mammalian TRPs are divided into six subfamilies, TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin), and TRPML (mucolipin), among which the seven TRPC members are closest to the original Drosophila TRP protein, sharing 30-35% sequence identity at the protein level ².

Figure 1. Structural organization of TRPC4 channel.

A. Diagram of main structural features of TRPC4 protein single subunit. Cylinders highlight α-helices in the transmembrane domain (S1-S6), amino (N) and carboxyl (C) terminal domains. AR1-AR4 indicate ankyrin-like repeats and H1-H7 indicate helix-loop-helix domains in the N-terminus. TRP helix or TRP domain (TRP) is present in most TRP channels. The regulatory domain formed by S1-S4 and pore domain formed by S5-pore loop-S6 are common for all TRP channels and most voltage-gated cation channels ³. B. Assembly of the TRPC4 channel with four subunits. For the cytoplasmic area, only two subunits are shown. Note the crossover of the C-terminal coiled-coil domain downstream from the connecting helix. The disordered region between TRP re-entrant (labeled in A) and the connecting helix is highlighted by the red chain. The calmodulin and IP₃ receptor binding (CIRB) motif that encompasses parts of the connecting helix and the disordered region is indicated by the oval shade. The CIRB motif has been implicated to be critical for TRPC4 interaction with and/or regulation by calcium calmodulin (Ca²⁺-CaM), IP₃ receptors (IP₃R), SEC14 domain and spectrin repeat-containing protein 1 (SESTD1), and Gα_i2-GTP. C-E. Ribbon diagrams of closed mouse TRPC4 channel structure based on single particle cryogenic electron microscopy ³², with side views for a single subunit (C) and all four protomers (D), as well as the top and bottom views of the whole channel (E). Adapted from ref. 28 with modifications. Key structural regions are labeled in (C). CIRB motif and the disordered region (missing in the resolved structures ^32,33) are indicated as yellow ovals and red chains in (C) and (D). Arrowheads in (E) indicate that the CIRB motif is largely exposed in the periphery of the cytoplasmic area of the channel.

TRPCs form Ca²⁺-permeable nonselective cation channels activated downstream of phospholipase C (PLC) signaling ^4,5. Of the many mechanisms that activate PLC, the G_q/11-PLCβ and receptor tyrosine kinase (RTK)-PLCγ pathways are the most commonly known, and both have been shown to induce robust TRPC channel activation, resulting in membrane depolarization and cytosolic Ca²⁺ concentration ([Ca²⁺]_c) elevation. Therefore, TRPC channels have been referred to as receptor-operated channels and because of their pronounced effects on [Ca²⁺]_c, they are also frequently studied in the context of receptor-operated Ca²⁺ entry and/or store-operated Ca²⁺ entry. The latter designation implies a mechanism of activation by Ca²⁺ depletion from the endoplasmic reticulum (ER) store. Whether or not TRPC channels are directly activated in response to ER Ca²⁺ store depletion in the absence of receptor activation is still a subject of debate. Evidence exists for TRPC channels or their mediation of Ca²⁺ influx to respond to store depletion, perhaps through interaction with STIM1 ^6-8, an ER membrane protein that transmits store depletion signal to plasma membrane channels ⁹,¹⁰. However, the TRPC currents exhibit distinct Ca²⁺ selectivity, unitary conductance, and voltage dependence from the most commonly known store-operated current, namely Ca²⁺-release activated Ca²⁺ current ¹¹. Here, we focus on receptor-operated TRPC activation, giving specific attention to channels formed by TRPC4 because of its unique requirement on pertussis toxin (PTX)-sensitive G_i/o proteins.

2. PLC dependence of TRPC channel activation

Nearly all constituents of PLC signaling have been implicated in the activation of TRPC channels⁵. These include diacylglycerols (DAGs), inositol 1,4,5-trisphosphate (IP₃) or IP₃ receptors, Ca²⁺, and phosphatidylinositol 4,5-bisphosphate (PIP₂). DAGs are thought to act as direct ligands of TRPC channels; however, the binding site on the channel remains undefined ¹², although recent evidence suggests that DAGs may gain access to TRPC3 channels through a subunit-joining fenestration behind the selectivity filter ¹³. Besides DAG, IP₃ receptors can bind to a conserved C-terminal motif, known as Calmodulin IP₃ Receptor Binding (CIRB) site (Fig. 1B), found in all TRPC proteins; whereas the binding by IP₃ receptors promoted, that by Ca²⁺-calmodulin inhibited the channel activation ¹⁴. Moreover, Ca²⁺ and PIP₂ exert both positive and negative effects on TRPC activities ⁵. The dependence on PIP₂ may reflect the needs for the PIP₂ hydrolysis products, e.g. DAGs and protons, to support TRPC activation ^15-17, and in the case of TRPC4, it may also reflect the need for plasma membrane targeting of PLCδ1, an isozyme specifically required for the activation of TRPC4 channels ¹⁸ (see later). On the other hand, phosphoinositides, including PIP₂, may exert a tonic inhibition on TRPC channels ⁵,¹⁶,¹⁸. The PIP₂ concentrations optimal for the activation and inhibition of different TRPC channels, including both homomeric and heteromeric ones ¹⁹, and under different stimulation conditions still remain to be elucidated. Generally, TRPC channel activation is regulated by multiple factors associated with PLC activation. Additionally, there are other modulatory factors, such as nitric oxide, reactive oxygen species and thioredoxin ²⁰-²², indicating complex mechanisms of regulation.

Most studies have relied on overexpression of TRPC proteins in heterologous cells, which enhances the chance of spontaneous channel activation likely due to high protein abundance and/or uncontrolled endogenous factors. As a result, it is not easy to distinguish modulation from true activation mechanisms. Examining TRPC4 activation through G_q/11-PLCβ signaling, Thakur et al¹⁸ discovered that while the muscarinic receptor agonist, carbachol, caused robust [Ca²⁺]_c rise in parental HEK293 cells, the same treatment only elicited very weak, if any, TRPC4 current, unless a G_q/11-coupled muscarinic receptor, M1, M3, or M5, was also overexpressed in these cells. By contrast, there was no difficulty in inducing TRPC3, C5, C6, and C7 currents via carbachol stimulation of the endogenously expressed muscarinic receptors in the HEK293 cells ¹⁸,²³,²⁴. Since overexpressed receptors can promiscuously couple to multiple G protein subtypes ²⁵, PTX was tested and shown to be able to abolish the carbachol-evoked TRPC4 currents no matter if M1, M3, or M5 receptors were overexpressed. Therefore, unlike other TRPCs, the receptor-operated activation of TRPC4 shows an absolute dependence on PTX-sensitive G_i/o proteins. This is in line with the findings that stimulating G_i/o-coupled receptors, such as M2 and M4 muscarinic receptors, μ opioid receptors (μOR), and receptors for sphingosine-1-phosphate (S1P) or oxidized phospholipids, also activate TRPC4 or TRPC5 channels ¹⁸,²⁶-²⁹. Notably, although TRPC5 is closely related to and shares many common features and regulatory mechanisms with TRPC4, PTX only inhibited ~50% of TRPC5 currents elicited by either endogenous or overexpressed M3 receptors, indicative of partial dependence on G_i/o signaling ¹⁸.

3. G_i/o-mediated TRPC4 activation and its dependence on PLCδ1

Receptor-mediated activation of G_i/o proteins produces two transducers, the activated Gα_i/o-GTP and free Gβγ dimer, between which the latter could directly activate certain PLCβ subtypes ³⁰ and thereby induce TRPC activation similarly as G_q/11. However, this mechanism does not seem to work in the HEK293 cells used, as stimulating the heterologously expressed μOR barely elicited any Ca²⁺ transients; nor it evoked detectable responses of the co-expressed TRPC3, C6, or C7 ¹⁸. Thus, at least in HEK293 cells, G_i/o stimulation is not efficiently coupled to PLC signaling. On the other hand, in experiments where different G protein subunits were coexpressed with TRPC4 or TRPC5, the constitutively active Gα_i2, Gα_i3, and Gα_o, but not Gα_i1 or various combinations of Gβγ, were found to support TRPC4/C5 activation in the absence of any stimulus ³¹. Also, the activated Gα_i2 physically interacts with TRPC4 carboxyl terminus and the CIRB motif of TRPC4 is important for Gα_i2-mediated channel activation ³¹.

Interestingly, the CIRB motif is located in an exposed region at the end of a long α-helix that extends all the way to the center core of the cytoplasmic assembly in recently reported high resolution TRPC4 cryogenic electron microscopy structures ³²,³³. A long stretch of unresolved structure of ~40 residues, including a short amino-terminal portion (~6 residues) of the CIRB motif, exists upstream from CIRB, outlining the widest area of the channel complex (Fig. 1B-E). This suggests flexibility of the pre-CIRB region for binding to different protein partners, whereby variable conformations may be adopted for specific functional outcomes by pulling or pushing the upstream TRP re-entrant loop connected to the TRP helix (Fig. 1A-C). The long α-helix initiated from the CIRB motif is also perfectly situated to transmit the protein-protein interaction signals allosterically to channel gating through its central access and perhaps interactions with TRP helix, S4-S5 linker, and/or the C-terminal end of the S6 TM segment (Fig. 1A-C).

Although the stimulation of G_i/o-coupled μOR did not cause obvious PLC activation in the absence of TRPC4, μOR-mediated TRPC4 activation was strongly dependent on PLC, as it was completely prevented by the PLC inhibitor, U73122. Through a number of complementary approaches, RNA interference, dominant negative suppression and inhibition by RhoA, it was determined that a specific PLC isozyme, PLCδ1, underlies this absolute dependence on PLC ¹⁸. PLCδ1 is likely the simplest and most ancient form of all thirteen mammalian PLC isoforms known to date ³⁴. Unlike other PLC isoforms that have developed sensing mechanisms for various extra- and intracellular signals, it is only known to be activated by Ca²⁺, with PIP₂ also required as both the substrate and membrane anchor. Therefore, PLCδ1 may partially explain the Ca²⁺ and PIP₂ dependence of TRPC4 activation. Consistent with the functional coupling, the levels of both TRPC4 and PLCδ1 were increased in surgical specimens of epilepsy patients with either cortical lesions of focal cortical dysplasia II or tuberous sclerosis complex as compared to controls ³⁵.

Notably, although PLCβs are not absolutely required for G_i/o-mediated TRPC4 activation, costimulating a G_q/11-coupled receptor or an RTK (e.g. EGF receptors) facilitated TRPC4 current development. Because of the Ca²⁺ dependence of PLCδ1, it was thought that G_q/11-PLCβ or RTK-PLCγ signaling probably provided the initial [Ca²⁺]_c elevation that was propagated to PLCδ1, which then further amplified the signal to induce robust channel activation. However, substituting the receptor agonist with the Ca²⁺ ionophore, ionomycin, only increased the probability but not the rate of G_i/o-mediated TRPC4 activation ¹⁸. More recently, this missing component of G_q/11-PLCβ pathway is found to be protons ¹⁷, a by-product of PIP₂ hydrolysis ¹⁶. Indeed, intracellular acidification resulting from PIP₂ hydrolysis had been shown to facilitate activation of TRP and TRP-like channels in Drosophila photoreceptors ¹⁶. It was shown that intracellular protons act at PLCδ1, rather than TRPC4, to accelerate Ca²⁺-dependent PLCδ1 activation via a positive-feedback mechanism ¹⁷. Such a self-propagating capability of PLCδ1, supported by H⁺ and Ca²⁺ signals generated from its own action on PIP₂ hydrolysis, likely also underpins its function in amplifying signaling initiated by G_q/11-PLCβ, RTK-PLCγ, and other PLC isozymes. Interestingly, for Drosophila TRP, intracellular protons also affected the channel through an indirect effect³⁶. Thus, although G_i/o signaling is crucial for receptor-operated TRPC4 activation, it requires coincident PLCδ1 activity, which can be triggered through ligand stimulation of G_q/11-PLCβ or RTK-PLCγ, or intracellular acidification and [Ca²⁺]_c rise (Figs. 2, 3A).

Figure 2. Mechanism of TRPC4 channel activation by coincident G_i/o and G_q/11 signaling.

Schematic drawing of factors critical for receptor-operated TRPC4 channel activation in response to agonists that simultaneously activate both G_i/o-coupled and G_q/11-coupled receptors. The light yellow and light pink circles highlight the propagating H⁺ and Ca²⁺ signals, respectively, that support continued PLCδ1 activation via positive feedback mechanisms, which can be initiated by stimulating G_q/11-PLCβ with a G_q/11-coupled receptor agonist (G_q/11-agonist). The G_q/11-PLCβ requirement may be bypassed by intracellular acidification in combination with an increase in cytosolic Ca²⁺ level. The propagating PLCδ1 activation needs simultaneous G_i/o activation, most likely Gα_i/o-GTP although the contribution of Gβγ cannot be completely ruled out at this point (pink dashed arrow), in order to support robust TRPC4 activation. Red arrows, protein-based regulation; blue arrows, regulation by ions and small diffusible messengers. ± indicate that the effects (of PIP₂ and Ca²⁺) on TRPC4 activation can be both positive and negative. Abbreviations are given in the text.

Figure 3. Physiological and pathophysiological functions mediated by TRPC4 in response to receptor-operated activation of G_q/11 and/or G_i/o proteins. A & B.

Possible differences in the mechanisms of activation of homotetrameric TRPC4 channel (A) from TRPC1-TRPC4 heterotetrameric channels (B) in response to coincident G_q/11 and G_i/o signaling. For homomeric TRPC4, the G_q/11-PLCβ pathway mainly serves to initiate the self-propagating and H⁺- and Ca²⁺-dependent positive feedback activation of PLCδ1, and the channel opening requires coincident G_i/o input (A). For heteromeric TRPC1-C4 channels, Gα_q/11-GTP might exert a direct stimulatory effect through TRPC1 ⁴⁸, which might obliviate the requirement for PLCδ1 and/or G_i/o (B). However, these possibilities have not been directly tested. C. Physiological and pathophysiological functions demonstrated to require TRPC4 based on studies using Trpc4 knockout mice. Among them, gastrointestinal motility and bladder detrusor contraction regulations are cholinergic and require M2 (G_i/o-coupled) and M3 (G_q/11-coupled) muscarinic receptors ^39,41,⁴². Lung vascular permeability regulation ⁴⁶ likely requires both G_q/11 and G_i/o when it is induced via thrombin receptors. The depolarizing plateau that underlies epileptiform burst firing and neurodegeneration of lateral septal neurons⁴³, is triggered by stimulating group 1 metabotropic glutamate receptors ^43,47, but the response is inhibited by pertussis toxin (unpublished). For fear related-behavior and serotonergic itch, only G_q/11 signaling has been suggested ^44,45; however, no report has ruled out the involvement of G_i/o signaling at this point.

4. Outstanding questions on G_i/o-mediated TRPC4 activation

Mechanism of TRPC4 activation by G_i/o remains undefined. Experimental evidence supporting the involvement of Gα_i/o but not Gβγ includes a) coexpression of constitutively active Gα_i2, Gα_i3, or Gα_o, but not Gα_i1, Gα_q or Gβγ supported spontaneous TRPC4 activation ³¹, b) selectively inhibiting Gα_i/o-GTP while enhancing free Gβγ production through GoLoco domain proteins suppressed TRPC4 activation ³⁷, and c) intracellular pipette dialysis of antibodies for Gα_o, but not that for Gβ, suppressed the development of muscarinic cation current (m/_CAT) ³⁸, an endogenous current found in gastrointestinal smooth muscle cells mediated mainly by TRPC4 ³⁹. However, unequivocal evidence ruling out the involvement of Gβγ has not been obtained. Secondly, although a direct interaction between TRPC4 carboxyl-terminus (residues 621-890) and Gα_i2 has been demonstrated by glutathione S-transferase pull-down using purified proteins, other experimental support of the interaction had come from co-immunoprecipitation of cell lysates and Forster resonance energy transfer (FRET) in intact cells ³¹,⁴⁰. It remains to be elucidated whether Gα_i/o or other Gα or Gβγ also binds to TRPC4 and where exactly they bind or whether other proteins are involved in facilitating the interaction. More importantly, solving structures of TRPC4 in complex with G_i/o subunit(s) will reveal molecular details on how G_i/o proteins gate this channel.

Physiological relevance of G_i/o-mediated TRPC4 activation is another important question. The best example is m/_CAT being mostly mediated by TRPC4 and responsible for neurogenic cholinergic contraction of gastrointestinal smooth muscles ³⁹. m/_CAT is dependent on both M2 and M3 muscarinic receptors ⁴¹, and is suppressed by PTX treatment⁴², suggesting a codependence on G_i/o and G_q/11 pathways. However, in other systems where TRPC4 function has been implicated, the studies have only considered G_q/11-PLCβ signaling (Fig. 3C)⁴³-⁴⁵. It would be important to examine the unintended involvement of G_i/o proteins in these functions. In some cases, for instance thrombin-induced endothelial permeation ⁴⁶, this would be obvious as thrombin receptors are coupled to both G_q/11 and G_i/o. In other cases, the coregulation might involve a different transmitter or receptor subtype, which would require more careful examinations. Indeed, we found that PTX treatment completely eliminated the large depolarizing plateau in lateral septal neurons induced by (S)-3,5-dihydroxyphenylglycine (DHPG) (unpublished), a group 1 metabotropic glutamate receptor agonist that induces epileptiform burst firing through TRPC4-containing channels ⁴³,⁴⁷. Alternatively, the G_i/o requirement might be abrogated by including TRPC1 in the heteromeric TRPC1-C4 channels (Fig. 3A-B)⁴³,⁴⁸,⁴⁹. However, since neither the native nor heterologously expressed TRPC1-C4 channels had been tested for PTX sensitivity, the involvement of G_i/o proteins cannot be completely rule out. The likelihood of TRPC4 to function as a detector of coincident G_i/o and G_q/11 signaling in various physiological settings should not be overlooked. Given that in the nervous system, the activation of G_q/11 is typically associated with excitation whereas that of G_i/o is accompanied with inhibition ⁵⁰, the consequence and functional significance of G_i/o and G_q/11 costimulation, which likely happens often and with G_i/o and G_q/11 inputs having different strengths, warrant further investigation.

Although stimulation of G_i/o-coupled receptors evoked TRPC4 currents in heterologous systems ^18,31, no similar response has been demonstrated in native systems. Therefore, the physiological activation of TRPC4 most likely requires coincident inputs of G_i/o and G_q/11, with the latter probably substitutable by RTK or other means that triggers PLC activation. The latest finding, however, suggests that tissue acidosis, which occurs commonly under pathological conditions and lowers not only extracellular but also intracellular pH, can facilitate G_i/o-dependent TRPC4 activation through sensitizing PLCδ1 ¹⁷. This could underlie the mechanism by which TRPC4 contributes to neurodegeneration under pathological conditions ⁴³

In summary, as a nonselective cation channel codependent on G_i/o and PLCδ1 for activation, TRPC4 is perfectly suited in integrating metabotropic signaling through the G_i/o and G_q/11 pathways. The G_q/11 pathway may be substituted by stimulating other PLC isozymes or intracellular acidification to give greater versatility of this coincident sensor for signaling. Although the physiological and pathological implications remain to be fully elucidated, the detailed regulatory mechanisms revealed thus far have tremendously enriched our knowledge on receptor-operated channels and their functional significance.