The structural basis of the G protein–coupled receptor and ion channel axis

Abstract

Sensory neurons play an essential role in recognizing and responding to detrimental, irritating, and inflammatory stimuli from our surroundings, such as pain, itch, cough, and neurogenic inflammation. The transduction of these physiological signals is chiefly mediated by G protein-coupled receptors (GPCRs) and ion channels. The binding of ligands to GPCRs triggers a signaling cascade, recruiting G proteins or β-arrestins, which subsequently interact with ion channels (e.g., GIRK and TRP channels). This interaction leads to the sensitization and activation of these channels, initiating the neuron's protective mechanisms. This review delves into the complex interplay between GPCRs and ion channels that underpin these physiological processes, with a particular focus on the role of structural biology in enhancing our comprehension. Through unraveling the intricacies of the GPCR-ion channel axis, we aim to shed light on the sophisticated intermolecular dynamics within these pivotal membrane protein families, ultimately guiding the development of precise therapeutic interventions.

Graphical abstract

Highlights

• •Sensory neurons transduce harmful stimuli via GPCRs/ion channels with ligand-GPCR binding triggers signaling cascade.
• •Structural study of GPCR-G protein-ion channel interactions provides understanding of the activation/sensitization.
• •Elucidating GPCR-channel axis structure enhances understanding on biomolecular complexes and targeted therapies.

1. Introduction

Transmembrane (TM) proteins, including G protein-coupled receptors (GPCRs) and ion channels, play a pivotal role in signal transduction. It is well-recognized that numerous intracellular compounds facilitate the communication between GPCRs and ion channels. These compounds act as triggers for vertical signaling, such as the secondary messenger-downstream kinase-physiological process axis in GPCRs and ion channels. Conversely, they also facilitate horizontal signaling through conformational changes in GPCRs, which in turn influences cellular potential and ion concentrations by activating ion channels. Recent advancements in structural biology, particularly with the advent of Cryo-electron microscopy (Cryo-EM), have enhanced our understanding of these vertical signaling processes. This technology has allowed us to capture the complex structures involving G proteins and β-arrestin at atomic resolution. Additionally, current studies have increasingly shown that at the horizontal level, membrane proteins can transiently interact or even form large-scale complexes regularly at the cellular membrane, such as the complexes formed between G proteins and ion channels (Whorton and Mackinnon, 2013a; Zhao and Mackinnon, 2023; Behrendt et al., 2020a). In this review, we focus on the interactions between GPCRs and ion channels and their roles in mediating physiological responses, discussing developments from the perspective of structural biology in recent years.

1.1. GPCR signaling transduction on membrane vertical axis

The intricate nature of downstream regulation observed in GPCRs is due to their ability to interact with different types of receptors. These receptors can engage with various members within the G protein or arrestin families. This diversity in coupling mechanisms enables GPCRs to participate in a wide range of physiological responses by selectively activating distinct signaling pathways based on the specific ligand-receptor interactions. Consequently, the engagement of a particular GPCR with a specific G protein or arrestin variant can trigger the activation of distinct series of intracellular events. These events result in the modulation of cellular functions through the alteration of gene expression, enzyme activity, or ion channel conductivity, among other effects. Therefore, the versatility and specificity in the coupling of GPCRs with their effectors underpin the complexity of their downstream regulatory roles in cellular signaling networks (Davies et al., 2023) (see Fig. 1).

Fig. 1.

Schematic representation of the canonical GPCR-mediated transmembrane signaling. When GPCRs are activated by specific ligands, they undergo conformational changes that promote the binding of heterotrimeric G proteins. This binding results in the dissociation of the G protein into Gα and Gβγ subunits. The Gα subunit can activate downstream effectors such as PKA (protein kinase A) or PKC (protein kinase C), as well as other secondary messengers. Following this activation, G protein-coupled receptor kinases (GRKs) migrate towards the activated GPCR, leading to receptor phosphorylation. This phosphorylation induces further conformational changes, facilitating the recruitment and binding of arrestin to the phosphorylated GPCRs. Arrestin binding can result in receptor internalization or the initiation of alternative signaling pathways.

1.1.1. The structural basis of GPCR- G protein complex

In a canonical signaling pathway, GPCR activation leads to the dissociation of the heterotrimeric G protein into G_α and G_βγ subunits, which subsequently affects the downstream partners and triggers a cascade of signaling events (Ben-Chaim et al., 2006). Recently, a study utilizing nanodisc technology to assemble a D2 dopamine receptor (DRD2)-G_i heterotrimeric protein complex shed light on the coupling mechanism of class A receptors to G proteins in a phospholipid environment. Key regions of interaction were identified between transmembrane (TM) segments TM5, TM6, TM7, and the intracellular loop 2 (ICL2) of the receptor and the α5 and αN helices of the G protein. Notably, the ICL2 of the receptor interacts prominently with the αN helix of the G_αi protein. Similar class A GPCR-heterotrimeric G protein complexes partially revealed a model in which the G protein interacts with various TM domains including TM3, TM5, TM6, and TM7, forming a cavity into which the α5 helix is inserted. This insertion allows specific side chains in the core of the G protein to interact with the receptor. Additionally, a conserved ICL2 motif of the GPCR forms a compact helix, which contributes to the mutual stabilization of the interface structure (Yin et al., 2020; Kim et al., 2020; Alegre et al., 2021; Liu et al., 2023; Suzuki et al., 2023). Furthermore, the coupling of G proteins to receptors is flexible rather than rigidly coordinated, and agonist binding alone does not fully activate the receptor. This implies that the interaction between G proteins and receptors allows for different conformations, or that binding does not occur in a single, uniform step, but instead may involve multiple steps or states that contribute to the final activated conformation of the GPCR. The wide range of intermediate states and conformational changes forms the foundation of the complex mechanism of GPCR activation (Weis and Kobilka, 2018).

1.1.2. The structural basis of GPCR-arrestin complex

The arrestin family comprises four members: arrestin-1 (visual arrestin), arrestin-2 (β-arrestin-1), arrestin-3 (β-arrestin-2), and arrestin-4 (cone arrestin) (Gurevich et al., 2006). Typically, arrestin facilitates the internalization of activated GPCRs, occurring at different timescales depending on the receptor activation (Moo et al., 2021). Additionally, arrestin also serves as a scaffold protein, crucial for transmitting signals through cascades (Beaulieu and Gainetdinov, 2011). Yang et al. elucidated the structure of the β1 adrenergic receptor (β1AR)–βarr1 complex bound to the biased agonist formoterol within lipid nanodiscs using Cryo-EM, offering insights into the classical GPCR–β-arrestin complex model (Wang et al., 2024). The arrestin family typically exhibits highly conserved conformations and domains for binding onto GPCRs, featuring a notable transition from an unstructured region in inactive state arrestin to an active state finger loop (s5s6). This loop inserts into the receptor with varying secondary structures and penetration depths in different GPCR-arrestin complexes. Moreover, diverse binding orientations were observed in analyzed GPCR-arrestin models. Almost all of the cytoplasmic structural elements of β1AR engage with βarr1, with the exception of TM1 and ICL3. The βarr1 elements contacting β1AR include several β-sheets and loops, notably the finger loop, which accounts for the majority of these interactions, particularly with TM6, TM2, and ICL2 on β1AR. ICL2 of β1AR also plays a significant role in interacting with βarr1, constituting nearly half of the contact points between the two structures. As the G protein dissociates and arrestin binds, the intracellular ends of helices 5 and 6 move closer to the receptor core, away from the extracellular ligand-binding site, allowing extracellular loop 2 (ECL2) to enter the binding pocket. These altered interactions with the agonist may explain the observed decrease in affinity in the G protein-isolated state, as previously mentioned (Lee et al., 2020). In other GPCR-arrestin complexes, the interactions between ICL2 and the finger loop continue to demonstrate a conservative effect (Staus et al., 2020; Huang et al., 2020).

1.2. GPCR-ion channel signaling transduction on membrane horizonal level

The downstream regulatory mechanisms of GPCRs have been firmly established. Nevertheless, a growing evidence suggests that signaling transduction can be mediated directly on or near the membranous spatial layer. Such process often coincides with the co-localization of proteins, including the formation of extensive super-complexes within the membrane. This is particularly evident in the interactions between GPCRs and G proteins, as well as between GPCRs and arrestins, in concert with ion channels. Upon ligand stimulation, GPCRs engage G proteins, leading to a series of interactions that modulate ion channel function, affecting cellular processes such as ion flux, electrical excitability, and second messenger cascades. Similarly, the binding of arrestins to activated GPCRs not only regulates receptor desensitization and internalization but may also facilitate the assembly of signalosomes. These multi-molecular complexes can recruit and modulate ion channels, thereby influencing cell signaling and physiology in a spatially and temporally coordinated manner.

1.2.1. The interaction between GPCR-ion channel via G protein

1.2.1.1. G protein-GIRK

Through electrophysiological studies, as well as fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) analyses, it has been established that the G protein-gated inwardly rectifying potassium channel (GIRK) is selectively activated by the G_βγ subunits, specifically upon their release triggered by G_αi/o subtype actions in response to GPCR-mediated cellular signaling. Around the turn of the new millennium, crystallography techniques succeeded in resolving the initial structure of the ion channel and specifically of GIRK, capturing mainly the cytoplasmic pore's framework. Despite this achievement, comprehensive biochemical and biophysical experimentation remains necessary to delineate the full scope of interactions and conformational changes undergone by G protein subunits upon activation. These conformational rearrangements directly activate GIRK following the activation of GPCRs and the subsequent dissociation of the G protein into its constituent subunits. This critical sequence of events allows GIRK to regulate membrane potential and contributes to the slowing of the heart rate by modulating the flow of potassium ions into cells, demonstrating the intricacies of signal transduction mechanisms at a molecular level (Mackinnon et al., 1998; Nishida and Mackinnon, 2002; Mase et al., 2012; Yokogawa et al., 2011; Whorton and Mackinnon, 2011; Riven et al., 2006) (see Table 1).

Table 1.

Summary of elucidated complexes between GPCRs and ion channels.

Target complex	Analysis assay	Resolution (PDB ID)	Reference
TRPC5-Gαi3-μOR	Cryo-EM	3.93 Å (7X6I)	Won et al. (2023)
GIRK2-Gβ1γ2-M2R/β2AR	X-ray crystallography	3.45 Å (4KFM)	(Whorton and Mackinnon, 2013b; Touhara and Mackinnon, 2018)
TRPM3-Gβ1γ2-M2R/μOR	Cryo-EM	3.80 Å (8DDX) 1.94 Å (6RMV, peptide)	(Zhao and Mackinnon, 2023; Behrendt et al., 2020a)
TRPC3-PLCγ-βarrestin1-AT1R	confocal microscopy for immunofluorescent co-localization; immunoprecipitate; Sequential immunoprecipitation	–	Liu et al. (2017)
Cav1.2-PKC-Pyk-Src-α1AR	confocal microscopy for immunofluorescent co-localization; immunoprecipitate	–	Man et al. (2023)

The current understanding of macromolecular complexes, which include GIRK channels, G proteins, regulator of G protein signaling (RGS) proteins, and GPCRs, has evolved considerably. Recent support from X-ray crystallography has depicted the GIRK-G_βγ structure, revealing a configuration wherein four G protein units couple with an integral channel. This assembly is stabilized in an intermediate state that bridges between the inactive and active forms. Within this complex, the partners generate a substantial contact surface of approximately 700 Å². This surface involves the interaction of β-sheet regions K, L, M, N from the intracellular cytoplasmic domains (CTCDs) of one channel subunit with β-sheet regions D, E from the CTCDs of an adjacent subunit to form the GIRK interaction interface. On Gβ, which adopts a β-propeller structure including blades 1 and 7, a set of smaller residues—such as Q248/F254 on the βL-βM loop and the L342-T/S343-L344 motif—facilitate close-range interactions with G_βγ. For instance, residue Q248 forms contacts with Q75, S98, and W99 on Gβ, while L55 and K78 on Gβ interact with the L-T/S-L region on GIRK (Whorton and Mackinnon, 2013b).

Electrostatic interactions serve as long-range forces, and the presence of numerous acidic amino acids on the βL-βM loop of GIRK creates a positively charged surface crucial for binding affinity and specificity. This region acts as a ‘beacon’ that guides the G_βγ subunits directly to their binding locations. G_β’s expansive, sticky surface, structured by the β-propeller, permits a multitude of unique interactions essential for the signaling function of the complex. These structural insights have galvanized further research into the G protein subunits and their interactions with ion channels, deepening our understanding of signal transduction mechanisms (Whorton and Mackinnon, 2013a; Behrendt et al., 2020b; Won et al., 2023). Moreover, certain elements located beyond the immediate interaction interface may indirectly affect the interactions. Significantly, the observed co-localization of Muscarinic acetylcholine receptor (M2R) and GIRK supports the hypothesis that a complex involving GIRK, GPCR, and the G_i/oαβγ proteins can indeed form, providing a more nuanced understanding of the potential interactions within this molecular assembly (Lüscher and Slesinger, 2010; Kano et al., 2019; Mathiharan et al., 2021a).

1.2.1.2. Gα_i3- TRPC5 ion channel

Over the last two decades, whole-cell patch clamp studies have determined that the dissociated Gα_i subunit can directly regulate transient receptor potential canonical (TRPC) 4/5 channels, inducing membrane depolarization and excitation in gastrointestinal smooth muscle cells or systems with heterologous overexpression (Benham et al., 1985; Logothetis et al., 1987; Zholos et al., 2004). The structural analysis of the G protein-channel complex has revealed that Gα_i3 induces a conformational change in TRPC5. Notably, the interacting interface deviates 50 Å from the region stabilized by nanodiscs, bearing partial resemblance to the interface domains observed in the TRPV4 (vanilloid)-RhoA complex. The binding mechanism primarily involves the ankyrin repeat domain (ARD) helix pairs 1–2 of TRPC5 fitting into a shallow groove between helices α2 and α3 of Gαi₃, which typically interfaces with downstream effectors of the GPCR. Additionally, the loop that connects ARD regions 3–2 and 4–1 is anchored by its contact with Gα, a structural feature suggesting the specific active capabilities of the channel-G protein interaction. Significantly, residues I57, Y58, and Y59 on TRPC5 are critical for binding: I57 interacts with K205 and K208 on Gα_i3, Y58 forms a hydrogen-bonding network with Gα_i3 R205 and R245, and Y59 engages in hydrophobic interactions with W211, I212, and F215 where the α subunit meets the downstream effector. In the TRPC channel family, the IYF motif, conserved in TRPC4, suggests that both TRPC4 and TRPC5 can be activated by Gα_i subunit, while other channel members do not interact with G proteins in this manner. This implies that similar motifs may govern interactions with G_α subunits. Externally, the positively charged cavity on G_αi3 neutralizes electrons to stabilize the loop connecting ARD regions 3–2 and 4–1, creating a distinct interface. Within these critical areas, the IYY motif directs electrostatic attractions, drawing the interaction partners sufficiently close to establish contact (Won et al., 2023; Nadezhdin et al., 2023) (see Fig. 2A and Table 1).

Fig. 2.

Proposed Mechanism of Horizontal GPCR-Channel Signaling.

A. The Gα_i3 is shown forming a complex with the TRPC5 channel (PDB ID: 7X6I) in a 4:4 stoichiometry, indicating a potential mechanism for GPCR-mediated modulation of TRPC5 channel activity.

B. The Gβγ protein dimer binds to the GIRK2 channel (PDB ID: 4KFM) in a 4:4 stoichiometry or to the TRPM3 channel (PDB ID: 8DDX) in a 1:4 stoichiometry, demonstrating diverse potential interactions and signaling outcomes via different stoichiometric assemblies.

C. Arrestin mediates the linkage between the angiotensin II type 1 receptor (AT1R) and the TRPC3 ion channel, which may facilitate acute catecholamine secretion. This interaction suggests a model where arrestin acts as a scaffold to organize GPCR and ion channel complexes.

D. Intracellular kinases and scaffold proteins can potentially bridge the ion channel and GPCR, forming a larger functional complex that is crucial for efficient signal transduction within the cell membrane environment.

1.2.1.3. G_βγ- TRPM3 ion channel

When the G protein subunit is isolated, the diffused G_βγ can bind to the ion channel and act as an antagonist to suppress channel activation, particularly in heterotrimeric G_i/o systems like the μ-opioid receptors (μORs). Upon stimulation by endogenous or exogenous opioids, the released G_βγ targets bind to nociceptor-related channels at the peripheral terminals of nociceptive neurons. Numerous coimmunoprecipitation experiments have confirmed the formation of a multiprotein complex. Due to the presence of splice variants, specific fragments are considered to be crucial for interacting with G proteins, and the target exon 17 peptide at the cytosolic N terminus is particularly significant, as demonstrated by various truncated versions in structure patch clamp experiments. Subsequently, the 1.94 Å resolution structure reveals that the exon 17-encoded segment binds into the crater-like surface of the G_β protein, forming extensive contacts that stabilize the peptide into a helical formation sufficient for retention on an affinity chromatography column, though not with G_γ. The peptide forms a seven amino acid-long α-helix that relies on the G_β protein, with channel residues K595, A596, L599, and L600 interacting with Gβ residues Y59, W99, M101, L117, Y145, M188, and W332 through salt-bridges, hydrophobic interactions, and hydrogen bonds. Mutation of W99 does not reduce ion current, while mutations at nearby residues like K57 significantly diminish inhibition, highlighting the critical roles that these residues play in mediating interactions and affecting inhibition (Behrendt et al., 2020a).

In subsequent experiments, the entire TRPM3 (Melastatin) channel complexed with the Gβγ model was analyzed. The 3.8 Å resolution Cryo-EM structure showed the channel primarily interacts with G_βγ through an ICL that contains the exon 17-encoded region. There are also minor contacts in other domains, where two other loops contact the platform side of G_β. This suggests that TRPM3 has a higher affinity for G_βγ than the exon 17-encoded peptide alone. More specifically, residues L549, P550, F589, A596, L599, and L600 on the channel form hydrophobic interactions with residues P94, M101, L117, and M188 on the Gβ protein. These interactions account for more than half of the interface interactions. Additionally, residues N548, D552, K592, R593, K598, L600, and G601 on the channel form hydrogen bonds and salt-bridges with residues K57, Y59, A92, R96, N119, T143, G162, D186, and D246 on G_β, with many of these interactions mirroring those observed in earlier findings. It is worth noting that only one G_βγ, rather than four, was detected in the density map, suggesting that the current model of TRPM3-G_βγ might be incomplete. Moreover, residue I80 on G_β, identified through alanine scanning mutation, despite being distant from the interface, showed a reduction in inhibitory effect on TRPM3 when mutated to alanine. Yet, no further progress has been made in elucidating its mechanistic role (Zhao and Mackinnon, 2023; Behrendt et al., 2020a; Yuan et al., 2023) (see Fig. 2B and Table 1).

1.2.2. GPCR-arrestin-channel

Traditionally, GPCRs are known to initiate acute physiological responses in cells through G protein signaling, a process often referred to as first-wave signaling. This involves the recognition of conformationally changed or phosphorylated receptors by arrestin, leading to either receptor desensitization or prolonged GPCR function. A recently identified third-wave signaling involves the interaction of the receptor-G protein complex within endosomes, serving as a prolonged signal for downstream G protein activities. However, an evolutionary comparison of the emergence of arrestin and G proteins suggests that the pathway involving acute arrestin-mediated regulation of GPCR functions could have evolved prior to the conventional second-messenger signaling associated with GPCRs, albeit lagging in some biological phenomena. Initial investigations have reported numerous interactions between arrestin and ion channels, indicating that ion channel activation or trafficking can be regulated by this signaling partner (Hermosilla et al., 2017; Rowan et al., 2014; Yang et al., 2016; Kashihara et al., 2020; Sangoi et al., 2017; Por et al., 2012). Furthermore, a number of models have emerged depicting the assembly of GPCRs and ion channel macromolecules facilitated by arrestin, following in-depth studies of physiological events and their molecular mechanisms. Specifically, during studies mimicking clinical conditions induced by angiotensin II receptor type 1 (AT1R) activation, the application of biased agonists revealed β-arrestin-dependent catecholamine secretion, a process that occurs independently of Gq-phospholipase C β (PLCβ)-mediated signaling. To decode this unexpected biological process, an array of molecular biology techniques was employed—including knockout models, recombinant systems, electrophysiological assays, and sequencing co-immunoprecipitation (Co-IP)—to demonstrate the sequence of interactions that begin with the formation of the AT1R-β-arrestin-1 complex, leading to the recruitment of PLCγ and TRPC3 by essential β-arrestin. This recruitment is attributed to β-arrestin's role in facilitating the rapid and specific conformational change in TRPC3, mediated indirectly by PLCγ and through direct contact. In particular, the interaction within the C-terminal region of the partners is crucial; residues 759 to 790 of TRPC5 are vital for interacting with soluble downstream kinases, while only one or two key residues within β-arrestin-1 appear to be instrumental in these interactions—truncation of the last 45 residues of the β-arrestin-1 C-terminus does not lessen the contact. With regard to allosteric modulation, BRET experiments have been critical in characterizing the allosteric state during β-arrestin binding (Liu et al., 2017; Chai et al., 2017). In another instance involving GPCR and β-arrestin interactions, the adhesion G-protein coupled receptor G2 (ADGRG2) forms a complex with the cystic fibrosis transmembrane conductance regulator (CFTR) channel, employing β-arrestin as a structural scaffold. Although the Gq protein remains active within the ADGRG2-CFTR complex, the involvement of non-canonical pathways reveals nonselective regulatory effects. Importantly, the coupling ability of downstream CFTR is compromised due to specific mutations, including Y708A on the ICL2 and R803E/K804E, which disrupt Gq's coupling efficiency (Zhang et al., 2018). Additionally, β-arrestin sometimes acts as a scaffold for ubiquitin ligase, facilitating the ubiquitination and subsequent internalization of channels, highlighting its versatile role in channel regulation beyond traditional GPCR signaling pathways (Shukla et al., 2010; Zaccor et al., 2020) (see Fig. 2C and Table 1).

1.2.3. Kinases collective protein ‘bridge’

Due to the extensive interactions within the GPCR signaling network involving G proteins, kinases, and other effectors, a fast-acting regulatory pathway is formed, spanning from adrenergic receptors to their ultimate downstream targets. This leads to the creation of a multiprotein ‘super-complex,’ akin to a highly organized ‘highway,’ consisting of numerous partners. Such a compartment typically incorporates a variety of kinases that initiate phosphorylation cascades ultimately affecting ion channels and governing distinct biological processes. The family of A-kinase anchoring proteins (AKAPs) holds motifs that bind to PKA regulatory subunits and serve as intermediaries in diverse G protein-dependent kinase signaling pathways, operating within these subcellular signaling domains. Moreover, AKAPs are often reported to function independently of GPCRs, acting to direct kinases towards specific target channels (Davare et al., 2001; Man et al., 2023; Man et al., 2020; Sanderson and Dell'acqua, 2011; Prada et al., 2020; Flynn and Altier, 2013; Esseltine and Scott, 2013; Zou et al., 2023) (see Fig. 2D and Table 1).

1.2.4. The lipid and its derivatives membrane structure influence on potential macromolecule complex

Lipids play a crucial role in the functionality of transmembrane proteins, acting both as endogenous activators and external ligands. They bind to active sites on GPCRs and ion channels, activating and sustaining their functions to respond to extracellular signals (Suh and Hille, 2008; Hansen, 2015; Uchida et al., 2016; Duncan et al., 2020; Thompson and Baenziger, 2020; Retamal et al., 2021; Krishna et al., 2021; Hu et al., 2024). Among these lipids, phosphatidylinositol-(4,5)-bisphosphate (PIP2), a substrate of phospholipase C (PLC), is particularly significant in ion channel research due to its unique binding characteristics and its integral role in functional activation (Whorton and Mackinnon, 2011, 2013a; Uchida et al., 2016; Zakharian et al., 2010; Sun and Zakharian, 2015; Mathiharan et al., 2021b).

The presence of PIP2 in TRP, Kir, and GIRK channels emphasizes its fundamental role in modulating channel behavior and responsiveness. PIP2's interaction with these channels stabilizes their structures and modulates their activity, illustrating its function as a key modulator in ion channel gating and signaling pathways. Its absence significantly affects the stability and activity of certain complexes, such as TRPM3-Gβγ, underscoring its essential agonistic role (Zhao and Mackinnon, 2023).

PIP2 contributes to membrane curvature through mechanisms like asymmetric desorption in biomimetic vesicles and interactions with actin-related proteins, which help maintain this curvature. Cholesterol also plays a critical role in membrane curvature control, and together with PIP2 and other lipids, it supports the formation of lipid rafts (Levental and Lyman, 2023; Ferré et al., 2022; Shukla et al., 2019; Tsai et al., 2022; Prakash et al., 2023; Gov and Gopinathan, 2006; Ivankin et al., 2012; Martyna and Rossman, 2014). These lipid rafts organize membrane components, influencing the function and distribution of GPCRs and transmembrane proteins. The structural arrangement and clustering of PIP2 can be regulated by transmembrane proteins, with cholesterol filling spaces between PIP2 molecules to stabilize and form lipid rafts. These lipid structures maintain specific membrane curvatures and can enrich certain protein species, including GPCRs, thus facilitating the formation and function of membrane protein complexes (Fotiadis et al., 2003; Kaneshige et al., 2020; Zhao et al., 2019; Park, 2019; Neri et al., 2010; Periole et al., 2007; Gahbauer and Böckmann, 2020). Computational simulations and emerging evidence suggest that lipid rafts and lipid properties contribute to organizing transmembrane proteins into ordered structural complexes, such as rhodopsin dimers (Yin et al., 2020).

The membrane structure not only includes lipid nanodomains but also involves scaffold proteins and their complex arrangements, which are integral to maintaining normal physiological processes (Ferré et al., 2022). During experimental observations such as patch-clamp recordings, the TRPM3 channel exhibits a higher binding affinity for G protein subunits compared to reconstituted complexes, suggesting that certain aspects of native membrane environments may facilitate this interaction. Despite high concentrations of ions used in patch-clamp studies, some G protein binding models remain elusive. Moreover, data collection from detergent micelles containing TRPM3 often reveals severe structural disorder, which limits the resolution of the complex. This limitation allows only one of the possible four Gβγ units to be resolved, even with the aid of nanobody techniques (Zhao and Mackinnon, 2023). In contrast, the intact TRPC5 channel demonstrates a stoichiometry of 1:4 or 4:4 when coupled to Gαi subunits within a lipid nanodisc environment. These nanodiscs mimic the native phospholipid bilayer and the typical negative charge milieu, which helps prevent distortion and static of the biomolecules during sample preparation, allowing for a more accurate reconstruction of membrane proteins (Won et al., 2023). The mentioned DRD2, along with other class A GPCRs, features ionic residues that form electrostatic rings at the ends of TM domains. These rings interact antagonistically with the negatively charged phospholipid headgroups, contributing to the membrane architecture required for optimal receptor function (Heijne, 1986; Okamoto et al., 2013; Dorairaj and Allen, 2007). Additionally, covalent lipidation of the C-terminal end of GPCRs plays a critical role during X-ray crystallography analyses, as these lipid anchors help to integrate the receptor into the lipid-like layers, thus facilitating the formation of complexes between the heterotrimeric G protein and the GPCR or channel. Therefore, the charged lipid membrane environment is crucial for the stabilization and proper function of these complexes (Whorton and Mackinnon, 2013a; Rasmussen et al., 2011) (see Fig. 3A).

Fig. 3.

Mechanisms Driving the Formation of Horizontal GPCR-Channel Complexes.

A. Lipid-Induced Modulation: Lipid components can function as allosteric modulators or activators of membrane curvature, facilitating the hydrophobic mismatch that drives the assembly of GPCR-channel complexes. This process underscores the role of lipids in promoting and stabilizing interactions within the membrane environment.

B. Ligand-Induced Clustering: Natural or synthetic ligands can induce nanoclustering of their target receptors, such as GPCRs. This clustering effect enhances receptor density and facilitates the formation of receptor-channel complexes within localized membrane domains.

C. Influence of Membrane Voltage and Ion Pocket Effects: Variations in membrane voltage and interactions within the GPCR ion pocket can modulate the assembly and stability of GPCR-channel super-complexes. These electrical and structural factors contribute to the dynamic regulation of signal transduction pathways.

1.2.5. Ligands have potential assemble effect on membrane protein

In living cells, the spatial restriction and increased local concentration provided by membranes, the cytoskeleton, or other cellular frameworks enhance the likelihood of protein-protein interactions, facilitating molecular encounters necessary for complex formation (Behrendt et al., 2020a).

Bivalent ligands, emerging as potent tools in the study of multi-pocket protein complexes—particularly dimeric receptors—can induce positive cooperativity akin to GPCR dimerization and cross-talk. This effect, exemplified in the assembly of DRD2-dimers, DRD3-Neurotensin receptor type 1 (NTSR1), and μOR-C-X-C chemokine receptor type 4 (CXCR4) complexes through synthesized bivalent compounds, has been primarily observed in molecular dynamics (MD) simulations and awaits further experimental validation (Qian et al., 2023; Huang et al., 2021; Pulido et al., 2018; Budzinski et al., 2021; Ma et al., 2020). Therefore, bivalent ligands and their function in simultaneously engaging partner proteins may catalyze interactions within the TM protein superfamily. Notably, certain endogenous metabolites or even the normal secretion ligand have been reported to play a role in triggering activities in GPCRs and ion channels alike. For example, glucosylsphingosine has dual effects on evoking the 5-HT2A receptor and the TRPV4 channel. Subsequently, active state transmembrane proteins would colocalize and elicit pruritus (Sanjel et al., 2022). In terms of single ligand regulation, CXCL12 induces the formation of nanoclusters for its receptor CXCR4, and this clustering is essential for the potent response elicited by chemokines (Martínez-Muñoz et al., 2018). These findings suggest that ligands, especially bivalent ones, play a crucial role in promoting the assembly and function of complex protein structures, illustrating their potential in both understanding and manipulating cellular signaling pathways (see Fig. 3B).

1.2.6. GPCR ion pocket

Substantial research has confirmed that ions can specifically bind to pockets within class A GPCRs, particularly highlighting numerous instances where sodium ions interact with the TM helical bundle at allosteric sites to influence the conformational restructuring of receptors. Sodium ions typically engage with several highly conserved residues and are often coordinated with surrounding water molecules. For instance, in the high-resolution crystallographic structure of β1AR, a sodium ion is coordinated by a salt-bridge at the D2.50 residue in the inactive site (Katritch et al., 2014). It has also been observed that the binding of agonists occurs independently of high sodium concentrations, indicating that sodium's role is distinct from ligand activation. Upon activation of the GPCR, the conformational rearrangement facilitates the release of the sodium ion from its binding site toward the cytoplasm. This ion binding and subsequent release play critical roles in stabilizing the receptor's inactive state and triggering the structural changes necessary for activation, underscoring the importance of ions in the functional regulation of GPCR complexes (Vickery et al., 2018).

Ion channels often demonstrate selectivity for certain ions (e.g., potassium (K⁺), sodium (Na⁺), calcium (Ca²⁺)). The flow of these ions across the membrane can further affect GPCR signaling. The ionic gradient maintained by these channels contributes to the cell's resting membrane potential, which can influence GPCR activity by modulating the membrane environment. This interaction between ion gradients and GPCR function illustrates a feedback mechanism where ion channel activity can feedback and affect GPCR behavior. In summary, ions contribute significantly to the stability and functionality of GPCR-ion channel complexes. They do so by directly modulating receptor and channel activity, influencing the signaling cascade through allosteric effects or secondary messenger roles, and possibly through direct receptor-channel interactions affecting the cellular responses precisely (see Fig. 3C).

1.2.7. Membrane voltage effects on receptors-ligand affinity

Twenty years ago, researchers discovered that membrane depolarization induces a reverse affinity state in GPCRs, a physiological feature governed by a select few residues involved in voltage modulation. This voltage-dependent phenomenon operates independently of G proteins, yet is associated with the region where GPCRs and G proteins interact. Further research could unravel new mechanisms by which ion channels regulate GPCRs, considering the intrinsic properties of the channels themselves. This suggests an intricate interplay between membrane potential changes and GPCR activity, potentially unveiling unique regulatory pathways influenced by the ionic environment surrounding these receptors (Ben-Chaim et al., 2006; Rozenfeld et al., 2021) (see Fig. 3C).

2. Discussion

The intricate interactions between GPCRs and ion channels underscore a fundamental and complex aspect of membrane signaling mechanisms (Clapham, 1994; Garcia et al., 1994). Advances in biochemical and imaging techniques have illuminated the ways in which GPCRs and ion channels cluster and directly interact within localized membrane domains, as evidenced by Co-IP and fluorescent imaging studies. Such interactions, exemplified by associations between many transmembrane proteins (see Table 2) suggest a nuanced and direct regulatory relationship between these two protein families. This further proposes that these contacts might be a general feature of GPCR-mediated signaling. The persistence of these interactions, even in native apo forms of the proteins, indicates a structurally inherent propensity for GPCRs to associate closely with ion channels. This opens new avenues for understanding the molecular basis of signaling complexes that transcend traditional, separate investigations of GPCRs and ion channels. However, despite significant progress in resolving the structures of these complexes, especially through Cryo-EM, challenges remain. High-resolution structural analysis of these membrane protein super-complexes is often hindered by technical limitations and the complex's dynamic nature (see Table 2).

Table 2.

Summary of potential bioactive complexes between GPCRs and ion channels.

Target complex	Analysis assay	Reference
AT1R-TRPV4	immunoprecipitate; confocal microscopy for immunofluorescent	(Shukla et al., 2010; Zaccor et al., 2020)
DRD1-CaV2.2	immunoprecipitate; confocal microscopy for label-fluorescent	Kisilevsky et al. (2008)
β2AR-voltage-dependent L-type Ca2+ channel 1 (Cav1) functional subunit	immunoprecipitate; confocal microscopy for immunofluorescent	Davare et al. (2001)
DRD2-TRPC1	Immunolabeling; immunoprecipitate	Hannan et al. (2008)
β1AR-inwardly rectifying potassium channel 3.1/3.2 (Kir3.1/3.2) DRD2/DRD4-Kir3.1/3.2/3.3/3.4	immunoprecipitate	Lavine et al. (2002)
Gamma-aminobutyric acid type B receptor subunit 1 (GABAB1R)-Kir3	confocal microscopy for immunofluorescent; immunoprecipitate	David et al. (2006)
nociceptin receptor (ORL1R)-Cav2.2	immunoprecipitate; confocal microscopy for label-fluorescent	Beedle et al. (2004)
M1R-TRPC6	immunoprecipitate	Kim and Saffen (2005)

Moreover, while the tendency of GPCRs to assemble and possibly form dimers has been observed, such events are rare and suggest that the architecture of membrane protein super-complexes may have nuanced regulatory implications. The exploration of GPCR dimers as a simpler model to study the mechanisms behind larger membrane protein aggregations could provide valuable insights. The development of bivalent ligands and other assembling-inducing methodologies offer promising tools to further investigate the formation and function of these complexes.

The dynamic interplay between GPCRs and ion channels, facilitated by scaffold proteins like arrestins and influenced by the membrane microenvironment, represents a key frontier in cell signaling research. Understanding these interactions at an atomic level could significantly advance our knowledge of cellular communication networks and potentially uncover novel therapeutic targets. As technical barriers are overcome and more detailed structures of these super-complexes become available, we can anticipate deeper insights into the intricacies of membrane signaling pathways (Zhao et al., 2019; Velazhahan et al., 2022).

The dynamic nature of biomolecular interactions, particularly protein-protein interactions within cellular environments, presents significant challenges in structural characterization and data acquisition. To overcome these challenges, developing and employing advanced methodologies have become essential to deciphering complex interactions and stabilizing transient or weak associations between proteins. For instance, the use of nanobodies in stabilizing the TRPM3-Gβγ structure exemplifies a strategy to limit the diffusion of G protein complexes, affording more precise structural characterization (Zhao and Mackinnon, 2023); Advancements in dimerization techniques, such as the chemical-induced dimerization (CID) brought forth by FK506 binding protein/rapamycin-binding fragment (FKBP/FRB) systems, have profoundly impacted the study of receptor oligomerization, pushing the boundaries of what can be achieved in receptor biology (Wang et al., 2023; Fegan et al., 2010); Moreover, Duan et al.'s approach employing a multifaceted method to assemble stable GPCR complexes underscores the complexity and necessity of integrated methodologies in modern protein analysis. Despite successful attempts at stabilizing complexes with innovative binding enhancers and crosslinkers, challenges in resolving full densities of G protein subunits in membrane environments indicate that further technological advancements are needed. The limitations observed in detergent-based solutions for maintaining stable protein complexes highlight the need for novel formulations or robust membrane mimetics, such as nanodiscs and in situ Cryo-EM approach, to stabilize intricate TM super-complex structures effectively (Whorton and Mackinnon, 2013a; Zhao and Mackinnon, 2023; Won et al., 2023; Zheng et al., 2024).

These technological advancements and innovative approaches not only facilitate a deeper understanding of GPCR and ion channel interactions but also highlight the critical need for continued development in experimental tools and techniques. The push towards resolving the complexities of super-complex structures is essential for unlocking the full potential of membrane protein research, which holds significant implications for drug discovery and the broader field of molecular biology. These endeavors to stabilize and analyze protein complexes will undoubtedly usher in new insights and breakthroughs in understanding cellular signaling mechanisms (Duan et al., 2023; Dixon et al., 2016; Dewey et al., 2023).

The integration of advanced artificial intelligence (AI) technologies, such as AlphaFold and RosettaFold, has revolutionized the field of structural biology, particularly in the prediction and analysis of complex structures like GPCR-ion channel supercomplexes. AlphaFold, with its latest multimer and AlphaFold 3 versions, has notably extended its capabilities to predict not only individual biomolecules like proteins and nucleic acids but also their interactions with metal ions and chemical compounds. This enhancement facilitates a more comprehensive understanding of molecular systems and their intricate interplays (Evans et al., 2021; Jumper et al., 2021; Abramson et al., 2024). Similarly, Rosetta has evolved with innovations like RosettaFold all-atoms (RFAA), RosettaFold diffusion (RFdiffusion), and the integrative RFdiffAA model, progressively refining the framework for predicting biomolecular complexes (Baek et al., 2021; Krishna et al., 2024; Watson et al., 2023). Furthermore, increased hardware capabilities enhance the feasibility of MD simulations, such as Docking Assay for Transmembrane Components (DAFT) based on MD trajectories, enable precise predictions of binding conformations and orientations even for loosely bound compounds, thus providing critical insights into the dynamic interactions within membrane proteins (Gahbauer and Böckmann, 2020; Wassenaar et al., 2015). The synergy of these tools not only improves accuracy in model building but also aids in simulating near-native structures that can predict ligand docking, identify interaction hot-spots, and suggest potential cross-linking sites. These predictions are valuable for guiding experimental strategies such as protein thermostability enhancements, choosing fusion partners, or deciding on crosslink site locations. By reducing reliance on experimental trial-and-error, these in silico methods streamline the research process and make the daunting task of deciphering complex protein interactions more feasible (Ma et al., 2014; Wood et al., 2020; Li et al., 2024; Weng et al., 2021). The role of AI-powered technology in predicting structures of GPCR and ion channel complexes highlights a transformative shift in how researchers approach structural biology, offering deeper insights and more precise control over molecular modeling and experimental planning. This advancement holds significant promise for accelerating discoveries in molecular biology and facilitating the development of targeted therapies based on intricate protein interactions.

CRediT authorship contribution statement

Yulin Luo: Writing – original draft, Writing – review & editing. Liping Sun: Writing – review & editing. Yao Peng: Investigation, Writing – original draft, Writing – review & editing, Supervision, Funding acquisition.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Yao Peng reports article publishing charges was provided by iHuman Institute of ShanghaiTech. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Dr. N Strynadka

Data availability

No data was used for the research described in the article.