Gβγ Protein Research Articles

Structure and dynamics of a pentameric KCTD5/CUL3/Gβγ E3 ubiquitin ligase complex

Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3KCTD5) that ubiquitylates Gβγ and reduces Gβγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/Gβ1γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB/CUL3NTD and KCTD5CTD/Gβγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gβγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gβ and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/Gβγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex.

Tuft cells utilize taste signaling molecules to respond to the pathobiont microbe Ruminococcus gnavus in the proximal colon

Tuft cells are a type of rare epithelial cells that have been recently found to utilize taste signal transduction pathways to detect and respond to various noxious stimuli and pathogens, including allergens, bacteria, protists and parasitic helminths. It is, however, not fully understood how many different types of pathogens they can sense or what exact molecular mechanisms they employ to initiate targeted responses. In this study, we found that an anaerobic pathobiont microbe, Ruminococcus gnavus (R. gnavus), can induce tuft cell proliferation in the proximal colon whereas the microbe’s lysate can stimulate these proximal colonic tuft cells to release interleukin-25 (IL-25). Nullification of the Gng13 and Trpm5 genes that encode the G protein subunit Gγ13 and transient receptor potential ion channel Trpm5, respectively, or application of the Tas2r inhibitor allyl isothiocyanate (AITC), G protein Gβγ subunit inhibitor Gallein or the phospholipase Cβ2 (PLCβ2) inhibitor U73122 reduces R. gnavus-elicited tuft cell proliferation or IL-25 release or both. Furthermore, Gng13 conditional knockout or Trpm5 knockout diminishes the expression of gasdermins C2, C3 and C4, and concomitantly increases the activated forms of caspases 3, 8 and 9 as well as the number of TUNEL-positive apoptotic cells in the proximal colon. Together, our data suggest that taste signal transduction pathways are not only involved in the detection of R. gnavus infection, but also contribute to helping maintain gasdermin expression and prevent apoptotic cell death in the proximal colon, and these findings provide another strategy to combat R. gnavus infection and sheds light on new roles of taste signaling proteins along with gasdermins in protecting the integrity of the proximal colonic epithelium.

Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors.

G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such “invasive” techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood. To address this issue, we established a non-invasive dynamic mass redistribution (DMR) assay for the human H1–4Rs expressed in HEK cells, showing excellent signal-to-background ratios above 100 for histamine (HIS) and higher than 24 for inverse agonists with pEC50 values consistent with literature. Taking advantage of the integrative nature of the DMR assay, the involvement of endogenous Gαq/11, Gαs, Gα12/13 and Gβγ proteins was explored, pursuing a two-pronged approach, namely that of classical pharmacology (G protein modulators) and that of molecular biology (Gα knock-out HEK cells). We showed that signal transduction of hH1–4Rs occurred mainly, but not exclusively, via their canonical Gα proteins. For example, in addition to Gαi/o, the Gαq/11 protein was proven to contribute to the DMR response of hH3,4Rs. Moreover, the Gα12/13 was identified to be involved in the hH2R mediated signaling pathway. These results are considered as a basis for future investigations on the (patho)physiological role and the pharmacological potential of H1–4Rs.

Expression and Purification of Yeast-derived GPCR, Gα and Gβγ Subunits for Structural and Dynamic Studies.

In the last several years, as evidence of a surged number of GPCR-G complex structures, the expressions of GPCRs and G proteins for structural biology have achieved tremendous successes, mostly in insect and mammalian cell systems, resulting in more than 370 structures of over 70 GPCRs have been resolved. However, the challenge remains, particularly in the conformational transition and dynamics study area where a much higher quantity of the receptors and G proteins is required even in comparison to X-ray and cryo-EM (5 mg/ml, 3 μl/sample) when NMR spectroscopy (5 mg/ml, 250 μl /sample) is applied. As a result, the expression levels of the insect and mammalian systems are also difficult to meet this demand, not to mention the prohibitive cost of producing GPCRs and G proteins using these systems for a vast majority of laboratories. Therefore, exploration of an effective, affordable, and practical approach with broad applicability is demanded. Pichia pastoris expression system has shown its promise in the GPCR preparation with many merits that other eukaryotic expression systems can't compete with. GPCRs expressed in this system are inexpensive, easy-to-manipulate, and capable of isotopically labeling. Herein, we present related protocols recently developed and upgraded in our lab, including expressions and purifications of P. pastoris derived GPCR along with Gα and Gβγ proteins. We anticipate that these protocols will advance the conformational transition and dynamics studies of the GPCR and its complexes.

Stereospecific Inhibition of Ethanol Potentiation on Glycine Receptor by M554 Stereoisomers.

Blocking the interaction between the Gβγ protein and the glycine receptor (GlyR) has emerged as a promising pharmacological strategy to treat acute alcohol intoxication by inhibiting ethanol potentiation on GlyR. M554 is a recently discovered small molecule capable of binding to Gβγ with potent in vitro and in vivo inhibitory activity. This compound has been tested as a mixture of diastereomers, and no information is available concerning the stereospecific activity of each species, which is critical to pursue efforts on lead optimization and drug development. In this work, we explored the differential activity of four M554 stereoisomers by in silico molecular dynamics simulations and electrophysiological experiments. Our results revealed that the (R,R)-M554 stereoisomer is a promising lead compound that inhibits ethanol potentiation of GlyR.

The structural basis for an on–off switch controlling Gβγ-mediated inhibition of TRPM3 channels

TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gβγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gβγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gβγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gβ1 that are individually necessary for TRPM3 inhibition by Gβγ. The 10-amino-acid Gβγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gβγ proteins exert on TRPM3 channels.

The first DEP domain of the RhoGEF P-Rex1 autoinhibits activity and contributes to membrane binding

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gβγ subunits, PIP3, and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module.

Variable-Length Ester-Based Staples for α-Helical Peptides by Using A Double Thiol-ene Reaction.

A novel peptide stapling method effected by a double thiol-ene reaction between two cysteine residues and a divinyl diester to access stapled peptides with enhanced cell permeability is reported. This diverse chemical tool kit provides facile access to stapled peptides with varying bridge lengths. Stapled Axin mimetics were synthesised by using this stapling method resulting in improved α-helicity relative to the unstapled peptide. Cell penetrating stapled analogues of the SIGK peptide that targets the protein-protein interaction hotspot of Gβγ proteins were also synthesised that exhibited a moderate increase in α-helicity and were cell permeable. This chemoselective peptide stapling method is highly amenable as a facile method to easily modify synthetic α-helical peptides to target intracellular proteins.

Gi-coupled receptor activation potentiates Piezo2 currents via Gβγ.

Mechanically activated Piezo2 channels are key players in somatosensory touch, but their regulation by cellular signaling pathways is poorly understood. Dorsal root ganglion (DRG) neurons express a variety of G-protein-coupled receptors that modulate the function of sensory ion channels. Gi-coupled receptors are generally considered inhibitory, as they usually decrease excitability. Paradoxically, activation of Gi-coupled receptors in DRG neurons sometimes induces mechanical hypersensitivity, the mechanism of which is not well understood. Here, we find that activation of Gi-coupled receptors potentiates mechanically activated currents in DRG neurons and heterologously expressed Piezo2 channels, but inhibits Piezo1 currents in heterologous systems in a Gβγ-dependent manner. Pharmacological inhibition of kinases downstream of Gβγ, phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) also abolishes the potentiation of Piezo2 currents. Local injection of sumatriptan, an agonist of the Gi-coupled serotonin 1B/1D receptors, increases mechanical sensitivity in mice, and the effect is abolished by inhibiting PI3K and MAPK. Hence, our studies illustrate an indirect mechanism of action of Gβγ to sensitize Piezo2 currents and alter mechanosensitivity after activation of Gi-coupled receptors.

Ligand-dependent spatiotemporal signaling profiles of the μ-opioid receptor are controlled by distinct protein-interaction networks

Ligand-dependent differences in the regulation and internalization of the μ-opioid receptor (MOR) have been linked to the severity of adverse effects that limit opiate use in pain management. MOR activation by morphine or [d-Ala2,N-MePhe4, Gly-ol]enkephalin (DAMGO) causes differences in spatiotemporal signaling dependent on MOR distribution at the plasma membrane. Morphine stimulation of MOR activates a Gαi/o–Gβγ–protein kinase C (PKC) α phosphorylation pathway that limits MOR distribution and is associated with a sustained increase in cytosolic extracellular signal-regulated kinase (ERK) activity. In contrast, DAMGO causes a redistribution of the MOR at the plasma membrane (before receptor internalization) that facilitates transient activation of cytosolic and nuclear ERK. Here, we used proximity biotinylation proteomics to dissect the different protein-interaction networks that underlie the spatiotemporal signaling of morphine and DAMGO. We found that DAMGO, but not morphine, activates Ras-related C3 botulinum toxin substrate 1 (Rac1). Both Rac1 and nuclear ERK activity depended on the scaffolding proteins IQ motif-containing GTPase-activating protein-1 (IQGAP1) and Crk-like (CRKL) protein. In contrast, morphine increased the proximity of the MOR to desmosomal proteins, which form specialized and highly-ordered membrane domains. Knockdown of two desmosomal proteins, junction plakoglobin or desmocolin-1, switched the morphine spatiotemporal signaling profile to mimic that of DAMGO, resulting in a transient increase in nuclear ERK activity. The identification of the MOR-interaction networks that control differential spatiotemporal signaling reported here is an important step toward understanding how signal compartmentalization contributes to opioid-induced responses, including anti-nociception and the development of tolerance and dependence.

Promiscuous G-Protein-Coupled Receptor Inhibition of Transient Receptor Potential Melastatin 3 Ion Channels by Gβγ Subunits.

Transient receptor potential melastatin 3 (TRPM3) is a nonselective cation channel that is inhibited by Gβγ subunits liberated following activation of Gαi/o protein-coupled receptors. Here, we demonstrate that TRPM3 channels are also inhibited by Gβγ released from Gαs and Gαq Activation of the Gs-coupled adenosine 2B receptor and the Gq-coupled muscarinic acetylcholine M1 receptor inhibited the activity of TRPM3 heterologously expressed in HEK293 cells. This inhibition was prevented when the Gβγ sink βARK1-ct (C terminus of β-adrenergic receptor kinase-1) was coexpressed with TRPM3. In neurons isolated from mouse dorsal root ganglion (DRG), native TRPM3 channels were inhibited by activating Gs-coupled prostaglandin-EP2 and Gq-coupled bradykinin B2 (BK2) receptors. The Gi/o inhibitor pertussis toxin and inhibitors of PKA and PKC had no effect on EP2- and BK2-mediated inhibition of TRPM3, demonstrating that the receptors did not act through Gαi/o or through the major protein kinases activated downstream of G-protein-coupled receptor (GPCR) activation. When DRG neurons were dialyzed with GRK2i, which sequesters free Gβγ protein, TRPM3 inhibition by EP2 and BK2 was significantly reduced. Intraplantar injections of EP2 or BK2 agonists inhibited both the nocifensive response evoked by TRPM3 agonists, and the heat hypersensitivity produced by Freund's Complete Adjuvant (FCA). Furthermore, FCA-induced heat hypersensitivity was completely reversed by the selective TRPM3 antagonist ononetin in WT mice and did not develop in Trpm3-/- mice. Our results demonstrate that TRPM3 is subject to promiscuous inhibition by Gβγ protein in heterologous expression systems, primary neurons and in vivo, and suggest a critical role for this ion channel in inflammatory heat hypersensitivity.SIGNIFICANCE STATEMENT The ion channel TRPM3 is widely expressed in the nervous system. Recent studies showed that Gαi/o-coupled GPCRs inhibit TRPM3 through a direct interaction between Gβγ subunits and TRPM3. Since Gβγ proteins can be liberated from other Gα subunits than Gαi/o, we examined whether activation of Gs- and Gq-coupled receptors also influence TRPM3 via Gβγ. Our results demonstrate that activation of Gs- and Gq-coupled GPCRs in recombinant cells and sensory neurons inhibits TRPM3 via Gβγ liberation. We also demonstrated that Gs- and Gq-coupled receptors inhibit TRPM3 in vivo, thereby reducing pain produced by activation of TRPM3, and inflammatory heat hypersensitivity. Our results identify Gβγ inhibition of TRPM3 as an effector mechanism shared by the major Gα subunits.

Mechanical stress impairs pheromone signaling via Pkc1-mediated regulation of the MAPK scaffold Ste5.

Cells continuously adapt cellular processes by integrating external and internal signals. In yeast, multiple stress signals regulate pheromone signaling to prevent mating under unfavorable conditions. However, the underlying crosstalk mechanisms remain poorly understood. Here, we show that mechanical stress activates Pkc1, which prevents lysis of pheromone-treated cells by inhibiting polarized growth. In vitro Pkc1 phosphorylates conserved residues within the RING-H2 domains of the scaffold proteins Far1 and Ste5, which are also phosphorylated in vivo. Interestingly, Pkc1 triggers dispersal of Ste5 from mating projections upon mechanically induced stress and during cell-cell fusion, leading to inhibition of the MAPK Fus3. Indeed, RING phosphorylation interferes with Ste5 membrane association by preventing binding to the receptor-linked Gβγ protein. Cells expressing nonphosphorylatable Ste5 undergo increased lysis upon mechanical stress and exhibit defects in cell-cell fusion during mating, which is exacerbated by simultaneous expression of nonphosphorylatable Far1. These results uncover a mechanical stress-triggered crosstalk mechanism modulating pheromone signaling, polarized growth, and cell-cell fusion during mating.

Development of Gαi protein selective μ opioid receptor analgesics to limit side‐effects

The clinical utility of opioids as analgesics is limited by their serious side effects, which include respiratory inhibition, gastrointestinal motility disorders, tolerance and addiction. Opioids act by binding to μ opioid receptors (MORs) to trigger G‐protein and β‐arrestin‐mediated signaling cascades. The Gαi class of G‐protein is thought to play an important role in transducing the analgesic actions of opioids. Gαi subunits include Gi1, Gi2, Gi3, Go and Gz. In addition, Gβγ subunits also contribute to the actions of opioids, and the many subtypes of Gβγ subunits adding yet more complexity to MOR‐mediated signaling.Gαi proteins show discrete tissue and brain distributions, each subunit has discrete enzymatic kinetics, and these subunits are differentially regulated by GTPase‐activating proteins (GAPs), Regulator of G protein (RGS) proteins, and other effectors. Moreover, findings from genetically modified mice suggest that disruption of specific Gαi proteins results in discrete patterns of altered responses to opioids. Based on these observations, we hypothesize that compounds can be developed to modulate specific Gi protein coupling to μ receptors, which will result in analgesic actions with less pronounced side‐effects.Currently, novel opioids are developed by structure‐based or Gqi5‐mediated calcium readout. Consequently, Gqi5 biased opioids are likely to be preferred. To identify functionally novel opioids, we constructed MOR/Gqi15, i35, o5 and z5 in HEK 293 cells. We profiled ten reference compounds: DAMGO, β‐endorphin, met‐enkephalin, morphine, hydrocodone, oxycodone, TRV130, PZM21, SR17018 and fentanyl in these cell lines. We found that the majority of opioids signal through all Gi proteins with similar potency and efficacy. However, a minority showed strong Gi protein bias. Finally, because neuronal excitability is mediated by Gβγ subunits acting at N‐type CaV and G protein gated inwardly‐rectifying potassium (GIRK) channels, we generated MOR/GIRK1/2 and MOR/CaV2.2 α1/α2δ1/β1 in HEK 293 cells. These lines will be used to identify molecules that influence Gβγ actions on the activity of these effector channels.These preliminary data demonstrate that opioids are likely signal in manner that can be biased to specific Gi proteins. This offers an opportunity to differentiate/discover compounds with unique pharmacodynamic profiles. Novel opioids that act through discrete Gi protein, or on Gβγ proteins, will facilitate greater understanding about the relationship between selective G‐protein coupling and opioid‐related side‐effects. We aim to leverage this new information to develop novel opioid analgesics with limited side‐effects.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular mechanisms involved in epidermal growth factor receptor-mediated inhibition of dopamine D3 receptor signaling

The phenomenon wherein the signaling by a given receptor is regulated by a different class of receptors is termed transactivation or crosstalk. Crosstalk between receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) is highly diverse and has unique functional implications because of the distinct structural features of the receptors and the signaling pathways involved. The present study used the epidermal growth factor receptor (EGFR) and dopamine D3 receptor (D3R), which are both associated with schizophrenia, as the model system to study crosstalk between RTKs and GPCRs. Loss-of-function approaches were used to identify the cellular components involved in the tyrosine phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which is responsible for EGFR-induced regulation of the functions of D3R. SRC proto-oncogene (Src, non-receptor tyrosine kinase), heterotrimeric G protein Gβγ subunit, and endocytosis of EGFR were involved in the tyrosine phosphorylation of GRK2. In response to EGF treatment, Src interacted with EGFR in a Gβγ-dependent manner, resulting in the endocytosis of EGFR. Internalized EGFR in the cytosol mediated Src/Gβγ-dependent tyrosine phosphorylation of GRK2. The binding of tyrosine-phosphorylated GRK2 to the T142 residue of D3R resulted in uncoupling from G proteins, endocytosis, and lysosomal downregulation. This study identified the molecular mechanisms involved in the EGFR-mediated regulation of the functions of D3R, which can be extended to the crosstalk between other RTKs and GPCRs.

Biochemical Characterization of the Interaction of TRPM3 with Gβγ Proteins

Biochemical Characterization of the Interaction of TRPM3 with Gβγ Proteins

G-Protein Beta-Gamma Subunits Inhibit the Heat-Sensitive TRPM3 Ion Channels

Transient Receptor Potential Melastatin 3 (TRPM3) is a heat-activated non-selective, Ca2+ permeable cation channel also stimulated by chemical agents such as pregnenolone sulphate and CIM0216. Here we show that activation of Gi-coupled cell surface receptors inhibits TRPM3 currents in a mammalian expression system, which was alleviated by co-expression of proteins that bind βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβ1γ2, Gβ3γ2, Gβ4γ2, but not Gβ5γ2 or constitutively active mutants of Gαo or Gαi, inhibited pregnenolone sulphate-induced TRPM3 currents. Purified Gβγ proteins applied to excised inside out patches also inhibited TRPM3 activity, indicating a direct effect. Baclofen, somatostatin, and DAMGO, agonists of Gi coupled receptors, inhibited Ca2+ signals induced by pregnenolone sulphate and CIM0216 in dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited CIM0216-induced currents in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in vivo. Our data show that Gβγ inhibits TRPM3 channels upon Gi-coupled receptor activation.

Prolonged seizure activity causes caspase dependent cleavage and dysfunction of G-protein activated inwardly rectifying potassium channels

Recurrent high-frequency epileptic seizures cause progressive hippocampal sclerosis, which is associated with caspase-3 activation and NMDA receptor-dependent excitotoxicity. However, the identity of caspase-3 substrates that contribute to seizure-induced hippocampal atrophy remains largely unknown. Here, we show that prolonged high-frequency epileptiform discharges in cultured hippocampal neurons leads to caspase-dependent cleavage of GIRK1 and GIRK2, the major subunits of neuronal G protein-activated inwardly rectifying potassium (GIRK) channels that mediate membrane hyperpolarization and synaptic inhibition in the brain. We have identified caspase-3 cleavage sites in GIRK1 (387ECLD390) and GIRK2 (349YEVD352). The YEVD motif is highly conserved in GIRK2-4, and located within their C-terminal binding sites for Gβγ proteins that mediate membrane-delimited GIRK activation. Indeed, the cleaved GIRK2 displays reduced binding to Gβγ and cannot coassemble with GIRK1. Loss of an ER export motif upon cleavage of GIRK2 abolishes surface and current expression of GIRK2 homotetramic channels. Lastly, kainate-induced status epilepticus causes GIRK1 and GIRK2 cleavage in the hippocampus in vivo. Our findings are the first to show direct cleavage of GIRK1 and GIRK2 subunits by caspase-3, and suggest the possible role of caspase-3 mediated down-regulation of GIRK channel function and expression in hippocampal neuronal injury during prolonged epileptic seizures.

Anti-nociceptive action of peripheral mu-opioid receptors by G-beta-gamma protein-mediated inhibition of TRPM3 channels

Opioids, agonists of µ-opioid receptors (µORs), are the strongest pain killers clinically available. Their action includes a strong central component, which also causes important adverse effects. However, µORs are also found on the peripheral endings of nociceptors and their activation there produces meaningful analgesia. The cellular mechanisms downstream of peripheral µORs are not well understood. Here, we show in neurons of murine dorsal root ganglia that pro-nociceptive TRPM3 channels, present in the peripheral parts of nociceptors, are strongly inhibited by µOR activation, much more than other TRP channels in the same compartment, like TRPV1 and TRPA1. Inhibition of TRPM3 channels occurs via a short signaling cascade involving Gβγ proteins, which form a complex with TRPM3. Accordingly, activation of peripheral µORs in vivo strongly attenuates TRPM3-dependent pain. Our data establish TRPM3 inhibition as important consequence of peripheral µOR activation indicating that pharmacologically antagonizing TRPM3 may be a useful analgesic strategy.

Inhibition of Transient Receptor Potential Melastatin 3 ion channels by G-protein βγ subunits.

Transient receptor potential melastatin 3 (TRPM3) channels are activated by heat, and chemical ligands such as pregnenolone sulphate (PregS) and CIM0216. Here, we show that activation of receptors coupled to heterotrimeric Gi/o proteins inhibits TRPM3 channels. This inhibition was alleviated by co-expression of proteins that bind the βγ subunits of heterotrimeric G-proteins (Gβγ). Co-expression of Gβγ, but not constitutively active Gαi or Gαo, inhibited TRPM3 currents. TRPM3 co-immunoprecipitated with Gβ, and purified Gβγ proteins applied to excised inside-out patches inhibited TRPM3 currents, indicating a direct effect. Baclofen and somatostatin, agonists of Gi-coupled receptors, inhibited Ca2+ signals induced by PregS and CIM0216 in mouse dorsal root ganglion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited inward currents induced by CIM0216 in DRG neurons, and nocifensive responses elicited by this TRPM3 agonist in mice. Our data uncover a novel signaling mechanism regulating TRPM3 channels.

Dynamic role of the tether helix in PIP2-dependent gating of a G protein-gated potassium channel.

G protein-gated inwardly rectifying potassium (GIRK) channels control neuronal excitability in the brain and are implicated in several different neurological diseases. The anionic phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) is an essential cofactor for GIRK channel gating, but the precise mechanism by which PIP2 opens GIRK channels remains poorly understood. Previous structural studies have revealed several highly conserved, positively charged residues in the "tether helix" (C-linker) that interact with the negatively charged PIP2 However, these crystal structures of neuronal GIRK channels in complex with PIP2 provide only snapshots of PIP2's interaction with the channel and thus lack details about the gating transitions triggered by PIP2 binding. Here, our functional studies reveal that one of these conserved basic residues in GIRK2, Lys200 (6'K), supports a complex and dynamic interaction with PIP2 When Lys200 is mutated to an uncharged amino acid, it activates the channel by enhancing the interaction with PIP2 Atomistic molecular dynamic simulations of neuronal GIRK2 with the same 6' substitution reveal an open GIRK2 channel with PIP2 molecules adopting novel positions. This dynamic interaction with PIP2 may explain the intrinsic low open probability of GIRK channels and the mechanism underlying activation by G protein Gβγ subunits and ethanol.