The Nematode Pristionchus pacificus Requires the Gβ and Gγ Proteins for Light Adaptation But Not For Light Avoidance

ZOOLOGICAL SCIENCE, Journal Year: 2025, Volume and Issue: 42(1)

Published: Jan. 29, 2025

Most organisms can sense and adapt to a wide range of light intensities. Although animals commonly use opsins for detection, the nematode Pristionchus pacificus lacks conserved photoreceptors. The cyclic GMP signaling pathway G protein-coupled receptor kinase are essential light-avoidance behavior in P. pacificus. mechanism sensing has been partially elucidated, it remains unclear whether, how, adapts light. Here, we found that prior exposure reduced frequency pacificus, indicating its ability To reveal adaptation used CRISPR/Cas9 genome editing generate Gβ Gγ subunit mutants, as these subunits involved chemosensory Caenorhabditis elegans. mutants exhibited similar wild type, but was impaired mutants. Similarly, arrestin showed minor abnormalities adaptation. These findings suggest proteins play role sensory beyond chemosensation could contribute response mechanisms nematodes.

Language: Английский

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Multiple potassium channel tetramerization domain (KCTD) family members interact with Gβγ, with effects on cAMP signaling

Journal of Biological Chemistry, Journal Year: 2023, Volume and Issue: 299(3), P. 102924 - 102924

Published: Feb. 1, 2023

G protein–coupled receptors (GPCRs) initiate an array of intracellular signaling programs by activating heterotrimeric proteins (Gα and Gβγ subunits). Therefore, protein modifiers are well positioned to shape GPCR pharmacology. A few members the potassium channel tetramerization domain (KCTD) family have been found adjust through interaction with Gβγ. However, comprehensive details on KCTD remain unresolved. Here, we report that nearly all 25 interact In this study, screened capacity across entire using two parallel approaches. a live cell bioluminescence resonance energy transfer–based assay, find roughly half in agonist-induced fashion, whereas except were coimmunoprecipitation. We observed was dependent amino acid hot spot C terminus KCTD2, KCTD5, KCTD17. While KCTD2 KCTD5 require both Bric-à-brac, Tramtrack, Broad complex C-terminal regions for interaction, uncovered KCTD17 is sufficient interaction. Finally, demonstrated functional consequence KCTD–Gβγ examining sensitization adenylyl cyclase–cAMP pathway cells. Gβγ-mediated cyclase 5 blunted KCTD. conclude broadly engages signal transmission. represent one most prominent mechanisms cellular communication, controlling key physiological processes almost every mammalian tissue type (1Wettschureck N. Offermanns S. Mammalian their specific functions.Physiol. Rev. 2005; 85: 1159-1204Crossref PubMed Scopus (870) Google Scholar). prototypic series events, ligand-activated GPCRs induce mobilization obligatory dimers) engage effector molecules triggering downstream events (2Lambert N.A. Dissociation g cells.Sci. Signal. 2008; 1: re5Crossref (69) Scholar, 3Pierce K.L. Premont R.T. Lefkowitz R.J. Seven-transmembrane receptors.Nat. Mol. Cell Biol. 2002; 3: 639-650Crossref (2163) Thus, modulation active lifetime critically dictates magnitude duration (4Anderson G.R. Posokhova E. Martemyanov K.A. The R7 RGS family: multi-subunit regulators neuronal signaling.Cell Biochem. Biophys. 2009; 54: 33-46Crossref (114) particular, host effectors (5Dupre D.J. Robitaille M. Rebois R.V. Hebert T.E. role Gbetagamma subunits organization, assembly, function complexes.Annu. Pharmacol. Toxicol. 49: 31-56Crossref (211) 6Smrcka A.V. Fisher I. G-protein betagamma as multi-functional scaffolds transducers G-protein-coupled receptor Life Sci. 2019; 76: 4447-4459Crossref (43) 7Tennakoon Senarath K. Kankanamge D. Ratnayake Wijayaratna Olupothage et al.Subtype-dependent regulation signalling.Cell 2021; 82109947Crossref (18) Scholar) spatiotemporal scale (8Masuho Skamangas N.K. Muntean B.S. Diversity complexes defines spatial temporal bias Syst. 12: 324-337.e325Abstract Full Text PDF (29) This exemplified case gating K+ flux protein–gated inwardly rectifying (GIRK) channels (9Luscher C. Slesinger P.A. Emerging roles protein-gated health disease.Nat. Neurosci. 2010; 11: 301-315Crossref (454) GPCR–GIRK axis finely tuned either promote or reduce availability (10Luo H. Marron Fernandez de Velasco Wickman Neuronal K(+) channels.Am. J. Physiol. 2022; 323: C439-C460Crossref (11) Scholar), example pertaining binding certain (11Turecek R. Schwenk Fritzius T. Ivankova Zolles G. Adelfinger L. al.Auxiliary GABAB uncouple from desensitization.Neuron. 2014; 82: 1032-1044Abstract (79) 12Zheng Abreu Levitz Kruse A.C. Structural basis KCTD-mediated rapid desensitization signalling.Nature. 567: 127-131Crossref (48) consists contain great diversity outside structurally similar (BTB) domain, which organizes oligomerization (13Schwenk Metz Turecek Bildl W. al.Native GABA(B) heteromultimers auxiliary subunits.Nature. 465: 231-235Crossref (248) 14Teng X. Aouacheria A. Lionnard Soane Kamiya al.Kctd: new gene involved neurodevelopmental neuropsychiatric disorders.CNS Ther. 25: 887-902Crossref (55) remained relatively obscure, despite numerous ties pathophysiological conditions (14Teng 15Angrisani Di Fiore De Smaele Moretti emerging cancer.Cell Commun. 19: 56Crossref (24) addition regulating GABAB–Gβγ–GIRK KCTD12 KCTD16 there growing appreciation KCTDs serve adapters scaffold Cullin3 mediate ubiquitination target (16Azizieh Orduz Van Bogaert P. Bouschet Rodriguez Schiffmann S.N. al.Progressive myoclonic epilepsy-associated KCTD7 regulator conductance neurons.Mol. Neurobiol. 2011; 44: 111-121Crossref (53) 17Bayon Y. Trinidad A.G. la Puerta M.L. Del Carmen Bogetz Rojas al.KCTD5, putative substrate adaptor cullin3 ubiquitin ligases.FEBS 275: 3900-3910Crossref (67) 18De Marcotullio Pelloni Occhione M.A. Infante al.Identification characterization KCASH2 KCASH3, 2 novel adaptors suppressing histone deacetylase Hedgehog activity medulloblastoma.Neoplasia. 13: 374-385Crossref (75) 19Kasahara Kawakami Kiyono Yonemura Kawamura Era al.Ubiquitin-proteasome system controls ciliogenesis at initial step axoneme extension.Nat. 5: 5081Crossref (106) 20Smaldone Pirone Balasco Gaetano Pedone E.M. Vitagliano Cullin 3 recognition not universal property among proteins.PLoS One. 2015; 10e0126808Crossref (39) Several targets KCTD-dependent described (19Kasahara 21Cho H.J. Ryu K.J. Baek K.E. Lim Kim Song C.Y. al.Cullin 3/KCTD5 promotes Ubiqutination rho guanine nucleotide dissociation inhibitor 1 regulates its stability.J. Microbiol. Biotechnol. 2020; 30: 1488-1494Crossref 22He Peng Fan Chen Zheng Li Cullin3/KCTD5 induces monoubiquitination DeltaNp63alpha impairs activity.FEBS Lett. 2018; 592: 2334-2340Crossref (8) Curiously, best characterized (23Brockmann Blomen V.A. Nieuwenhuis Stickel Raaben Bleijerveld O.B. al.Genetic wiring maps single-cell states reveal off-switch 2017; 546: 307-311Crossref (77) mediated (24Young B.D. Sha Vashisht A.A. Wohlschlegel J.A. Human multisubunit E3 ligase required beta-subunit signaling.J. Proteome Res. 20: 4318-4330Crossref (4) Indeed, loss (as KCTD17) leads enhanced Gβγ-dependent second messenger cAMP (25Muntean Marwari Sloan D.C. Young al.Members major signaling.Proc. Natl. Acad. U. 119e2119237119Crossref Despite substantial information resulting interactions between (12Zheng molecular determinants underpinning such still unclear. Moreover, investigation into remaining toward has yet be defined. utilized independent approaches screen profiles family. considerable KCTDs, interacts though immunoprecipitation (IP), about fashion transfer (BRET)–based assay. then subset further investigate rules engagement selectivity demonstrate how these principles enable KCTD2/5/9/17 modulate transmission various efficacy primary striatal neurons. Combined structural data strongly support following activation 23Brockmann 26Zuo Glaaser Zhao Kurinov Mosyak Wang al.Structural subunit receptor.Proc. 116: 8370-8379Crossref (28) began unbiased evaluation determine could dimers. devised BRET assay monitor agonist–induced For donor, fused Nanoluciferase (Nluc) each full-length Upon transfection human embryonic kidney 293 (HEK293) cells exposure Nano-Glo substrate, Nluc construct yielded intensity suggesting equal expression level (Fig. 1A). well-characterized bimolecular fluorescence complementation Venus fluorophore split Gβ1 (Venus 156-239-Gβ1) Gγ2 1-155-Gγ2) acceptor strategy (27Hollins B. Kuravi Digby G.J. Lambert c-terminus GRK3 indicates heterotrimers.Cell 21: 1015-1021Crossref (105) our HEK293 transiently transfected KCTD-Nluc, Gβγ–Venus, D2 dopamine (D2R), GαoA 1B). initiated D2R dopamine. recorded response after min compared basal readings 1C). As reference, performed control experiments (GRK3ct), exhibits nanomolar affinity (28Pitcher Inglese Higgins J.B. Arriza J.L. Casey P.J. al.Role beta gamma targeting beta-adrenergic kinase membrane-bound receptors.Science. 1992; 257: 1264-1267Crossref (640) readily reports association Gβγ–Venus (29Masuho Ostrovskaya O. Kramer G.M. Jones C.D. Xie Distinct discrimination actions protein-coupled receptors.Sci. 8: ra123Crossref (158) 30Muntean Association plasma membrane potentiating catalytic (RGS) subfamily.J. Chem. 2016; 291: 7195-7204Abstract (10) Approximately exhibited increased application, 1D). Among several previously identified (KCTD2, KCTD12). On other hand, KCTD16, bind generated only mild experiments. elicited higher BRET, indicate prior stimulation. addition, revealed had known To gain insight patterns, phylogenetic tree aligned heatmap fold change 1E). analysis consistency subgroups. One exception observation lack KCTD9, differing robust group KCTD17). Given small netBRET wanted ensure approach did limit detection interactions. next IP examine experiment, myc tag carboxy ORF tandem Gβγ–Venus. Cells lysed followed pulldown GFP antibody. first tested whether promoting would enhance therefore lysis/IP presence absence AlF4- 2A). purpose, cotransfected (highest netBRET) KCTD20 (no netBRET). Probing total lysate anti-myc antibody corresponding estimated weight 2B). Treatment band samples; however, detected regardless treatment. result fortifies KCTD2/KCTD20, suggests may window co-IP, demonstrates treatment will false positives (at least KCTD20). applied lysates myc-tag near predicted 2C). Stunningly, samples but (KCTD9 Although quantitative, results trend categories stronger 3, 5, 7, 8, 10, 15, 16, 17, 18, BTBD10, SHKBP1) weaker (KCTD1, 4, 6, 11, 12, 19, KCNRG, TNFAIP1) examined possibility nonspecific repeating Venus-transfected three weak binders (KCTD4, TNFAIP1), strong (BTBD10 KCTD18), nonbinder (KCTD20). levels board, no IP, 2D). collectively show while cells, IP. vary strength, suggest they do appear positives. sought understand broad Of noninteractors, shares isolated similarity KCTD9 belongs subfamily includes conservation clades (Figs. 1C S1). reasoned differences acids distinct signatures paucity profiles, started reducing architecture simplest shared domains: (i) varying length N terminus, (ii) BTB (iii) 3A). Comparison domains ∼50% identity KCTD17, greater than 75% 3B). Similarly, exhibit high termini (>60%), low (∼20%) hypothesized conferred terminus. made chimeras myc-tagged replacing repeated chimera able 3C). if impede KCTD9. Interestingly, retained ability experiment 3D), interplay elements within bioinformatics components region charged polar conserved 3E). mutant where swapped residues ones Likewise, replaced counterpart (although seemingly lesser wildtype), charged/polar 3F). These clade order determined placement influenced mCherry 4A). Placement position equivalent profile 4B). Next, asked alone mapped generating N-terminal tags 5A). (mCh-FL), deleted (mCh-ΔN), expressed (mCh-BTB), (mCh-C-term). Starting dispensable 5B). coexpression separate vectors also unable provide same 5C), likely owing degree KCTD2. showed Gβγ, 5D). Intriguingly, contains coiled-coil (CC) 5E). fusion constructs mapping involvement 5F). Unfortunately, mCherry-CC express 5G). (95–314) studies Gβγ; domain.Figure 5KCTD17 A, scheme (FL = full length, ΔN= deletion only, C-Term only). B, (IP) anti-GFP probing mCherry-KCTD2 anti-mCherry Experiments (30 μM) lysis buffer. Representative blot C, mCherry-KCTD5 D, mCherry-KCTD17 E, alignment animo 17. Purple highlights unique F, indicated G, HEK293, line; KCTD, domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) consequences context signaling. real-time dynamics imaging TEpacVV FRET-based biosensor (31Klarenbeek Goedhart Hink Gadella T.W. Jalink mTurquoise-based sensor FLIM ratiometric read-out improved dynamic range.PLoS 6e19170Crossref (157) 32Muntean Zucca MacMullen C.M. Dao M.T. Johnston Iwamoto al.Interrogating landscape neuromodulatory intact neurons circuits.Cell Rep. 22: 255-268Abstract (37) inputs (AC)–mediated production, AC (AC5) prolonged Gi/o involves (33Muntean Masuho Sutton L.P. al.Galphao determinant pathophysiology movement disorders.Cell 34108718Abstract (36) 34Watts V.J. Molecular heterologous adenylate cyclase.J. Exp. 302: 1-7Crossref optimized robustly interrogate influence cAMP. overexpressed D2R, AC5, 6A). Stimulation endogenous Gαs-coupled β2-adrenergic μM isoproterenol increase 6B). overexpression (pertussis toxin S1 subunit; PTX) scavenger (GRK3ct) 6C). conditions, alter isoproterenol-induced responses D E). induced AC5 stimulating 100 h, resulted subsequent application 6F). PTX blocked sensitized amplitude non-dopamine treated GRK3ct significantly smaller PTX-treated because ongoing inhibition D2R→Gαi Overall, readout assessed impact overexpression. agreement studies, 6G). slightly inhibited sensitization, potential non-Gβγ effect 6H). containing decreased consistent obtained model sequesters AC5-mediated reconstituted w

Language: Английский

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Investigation of crystal structures, spectral (FT-IR and NMR) analysis, DFT, and molecular docking studies of novel piperazine derivatives as antineurotic drugs

Journal of Molecular Structure, Journal Year: 2023, Volume and Issue: 1278, P. 134937 - 134937

Published: Jan. 10, 2023

Language: Английский

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Synaptic Effects Induced by Alcohol

Current topics in behavioral neurosciences, Journal Year: 2023, Volume and Issue: unknown

Published: Jan. 1, 2023

Language: Английский

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Amyloid-β oligomers trigger sex-dependent inhibition of GIRK channel activity in hippocampal neurons in mice

Science Signaling, Journal Year: 2024, Volume and Issue: 17(856)

Published: Oct. 1, 2024

Alzheimer's disease (AD) is a progressive neurodegenerative characterized by amyloid plaques and cognitive decline, the latter of which thought to be driven soluble oligomeric amyloid-β (oAβ). The dysregulation G protein-gated inwardly rectifying K

Language: Английский

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Continuous cell type diversification throughout the embryonic and postnatal mouse visual cortex development

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 6, 2024

The mammalian cortex is composed of a highly diverse set cell types and develops through series temporally regulated events that build out the type circuit foundation for cortical function. mechanisms underlying development different remain elusive. Single-cell transcriptomics provides capacity to systematically study across entire temporal range development. Here, we present comprehensive high-resolution transcriptomic epigenomic atlas developing mouse visual cortex. was built from single-cell RNA-sequencing dataset 568,674 high-quality transcriptomes single-nucleus Multiome 194,545 nuclei providing both chromatin accessibility profiles, densely sampled throughout embryonic postnatal developmental stages E11.5 P56. We computationally reconstructed trajectory map all excitatory, inhibitory, non-neuronal in cortex, identifying branching points marking emergence new at specific ages defining molecular signatures cellular diversification. In addition neurogenesis, gliogenesis early postmitotic maturation stage which gives rise classes nearly subclasses, find increasingly refined emerge differentiation process, including late many during eye-opening (P11-P14) onset critical period (P21), suggesting continuous diversification Throughout development, cooperative dynamic changes gene expression types, peaks potentially regulating genes transcription factors peaks. Furthermore, single can be by multiple associated with and/or stages. Collectively, our most detailed directly individual reveals logic refinement identities

Language: Английский

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Ethosuximide: Subunit‐ and Gβγ‐dependent blocker and reporter of allosteric changes in GIRK channels

British Journal of Pharmacology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 15, 2025

Background and Purpose The antiepileptic drug ethosuximide (ETX) suppresses epileptiform activity in a mouse model of GNB1 syndrome, caused by mutations Gβ 1 protein, likely through the inhibition G‐protein gated K + (GIRK) channels. Here, we investigated mechanism ETX (block) different GIRKs. Experimental Approach We studied GIRK channels expressed Xenopus oocytes with or without their physiological activator, G protein subunit dimer Gβγ. binding site mode action were analysed using molecular dynamic (MD) simulations kinetic modelling, predictions tested mutagenesis functional testing. Key Results show that is subunit‐selective, allosteric blocker potency block increased Gβγ, parallel channel activation. MD locate GIRK2 to region associated phosphatidylinositol‐4,5‐bisphosphate (PIP 2 ) regulation, suggest acts closing helix bundle crossing (HBC) gate altering channel's interaction PIP . apparent affinity highly sensitive changes gating subunits. Conclusion Implications GIRKs allosteric, subunit‐specific, enhanced Gβγ an intricate network interactions within molecule. Our findings pose as potential therapeutic target for potent tool probing gating‐related conformational GIRK.

Language: Английский

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Activation of G protein gated inwardly rectifying potassium (GIRK) channels in keratinocytes mediates peripheral kappa opioid receptor-mediated antinociception.

Neuropharmacology, Journal Year: 2025, Volume and Issue: unknown, P. 110326 - 110326

Published: Jan. 1, 2025

Language: Английский

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Unraveling the transcriptome of pyramidal neurons from human hippocampus under aging, amnestic mild-cognitive impairment, and sex-interactions

Exploration of Medicine, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

Aim: Amnestic mild cognitive impairment (aMCI) is a transitional stage toward Alzheimer’s disease (AD). For late-onset AD (95% of cases), aging the main risk factor. Systematizing transcriptome hippocampal neurons under native conditions this essential, as information scarce and hippocampus highly vulnerable cerebral region. Methods: Public microarray data corresponding to homogenates human Healthy-Younger, Healthy-Elder, Elder-with-MCI individuals were re-analyzed herein. Through an optimized computational pipeline, those genes having splice forms that belong neuronal type identified. The differential arising from each group then characterized by contrasting ontologies (functions, cellular components, pathways). Additionally, re-arranged factorially determine first- second-order sex interactions. Results: Around 76% relevant changes actually occurred during healthy-aging process, being further balanced or not MCI. “Cognition”, “behavior”, “glutamatergic synaptic transmission”, “lipid rafts”, “catecholamines” decreased across three groups analyzed, whereas “associative/visual learning”, “gliogenesis”, “neuro-inflammation”, “corticosteroids”, “p75NTR”, “ER-stress” “autophagy” peaked in Elders. On contrary, “Learning/memory”, “GAP junctions”, “GABAergic “GNDF” showed minimum “transcriptional regulators” (MeCP2, NPAS4, DREAM), “BNDF/NGFR”, “Ca2+ transport”, “CRHR1” “CXCL12” minimal From Elder MCI, “MAPKs”, “MEF2C”, “RGS7”, “CCKBR1”, “ErbB4”, “ERK5”, “Ca2+-Na+-K+ channels” (RYR2, SCNA1/A8, KCNQ2/Q3, KCNN3, KCNIP2) appeared downregulated. “Long-term depression” (LTD) increased sharply Most findings detected contrasted against 250 reports. Conclusions: multiple alterations basic mechanisms—mainly CA3 dendrites mossy fibers could be compatible with hyper-excitability, diminished transmission, theta/gamma/SWR rhythms. Many functionalities conditioned sex-interactions. Particularly, women “pure” sex-effects interactions “cross-over” effects. Due its consequences on higher-order functions, all these predictions should confirmed experimentally.

Language: Английский

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Epilepsy in a mouse model of GNB1 encephalopathy arises from altered potassium (GIRK) channel signaling and is alleviated by a GIRK inhibitor

Frontiers in Cellular Neuroscience, Journal Year: 2023, Volume and Issue: 17

Published: May 18, 2023

mutations in

Language: Английский

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