Cov tshuaj tua kab ua lub luag haujlwm tseem ceeb hauv kev daws teeb meem kev tsis txaus noj thoob ntiaj teb thiab kev tawm tsam cov kab mob uas kis los ntawm tib neeg. Txawm li cas los xij, qhov teeb meem loj hlob ntawm kev tiv thaiv tshuaj tua kab xav tau kev tshawb pom cov tshuaj tshiab uas tsom mus rau cov hom phiaj tsis tau siv. Cov kab transient receptor potential (TRPV) channels—Nanzhong (Nan) thiab inactive (Iav)—tuaj yeem tsim cov heterologous channels (Nan-Iav) thiab localize rau mechanosensory organisms uas mediate geotropism, hearing, thiab proprioception hauv kab. Qee cov tshuaj tua kab, xws li aphidopyrrolidone (AP), tsom rau Nan-Iav los ntawm cov txheej txheem tsis paub. AP muaj txiaj ntsig zoo tiv thaiv cov kab tho-nqus (hemipterans), tiv thaiv kev noj los ntawm kev cuam tshuam kev ua haujlwm ntawm cov filaments. AP tsuas yog tuaj yeem khi rau Nan, tab sis tsuas yog Nan-Iav tuaj yeem cuam tshuam nrog agonists, suav nrog endogenous nicotinamide (NAM), yog li ua rau pom cov channel ua haujlwm. Txawm hais tias Nan-Iav lub peev xwm ua lub hom phiaj tua kab, me ntsis paub txog nws cov channel sib dhos, cov chaw khi cai, thiab Ca2+-dependent kev tswj hwm, cuam tshuam kev tsim cov tshuaj tua kab ntxiv. Hauv txoj kev tshawb fawb no, cryo-electron microscopy tau siv los txiav txim siab qhov qauv ntawm Nan-Iav hauv Hemiptera kab hauv lub xeev calmodulin-ligand-free, nrog rau nrog AP thiab NAM ntawm ciam teb ntawm ankyrin rov ua cytoplasmic domain (ARD). Qhov xav tsis thoob, peb pom tias Nan protein nws tus kheej tuaj yeem tsim ib lub pentamer, uas tau ruaj khov los ntawm AP-mediated ARD kev sib cuam tshuam. Txoj kev tshawb fawb no qhia txog kev sib cuam tshuam molecular ntawm cov tshuaj tua kab thiab agonists thiab Nan-Iav, qhia txog qhov tseem ceeb ntawm ARD hauv kev ua haujlwm channel thiab kev sib dhos, thiab tshawb nrhiav cov txheej txheem ntawm Ca2+ kev tswj hwm.
Vim muaj kev hloov pauv ntawm huab cua thoob ntiaj teb uas hnyav zuj zus tuaj, kev ruaj ntseg zaub mov thoob ntiaj teb uas tsis zoo zuj zus yog ib qho ntawm cov teeb meem loj tshaj plaws ntawm lub xyoo pua 21st, nrog rau cov txiaj ntsig zoo rau lub zej zog.1,2Daim ntawv qhia txog Lub Xeev Kev Ruaj Ntseg Khoom Noj thiab Kev Noj Haus hauv Ntiaj Teb Xyoo 2023 (SOFI) ntawm Lub Koom Haum Ntiaj Teb Kev Noj Qab Haus Huv kwv yees tias kwv yees li ntawm 2.33 billion tus tib neeg thoob ntiaj teb raug kev txom nyem los ntawm kev tsis muaj zaub mov txaus, uas yog ib qho teeb meem uas tau muaj ntev los lawm.3,4Tu siab kawg li, kwv yees li ntawm 20% txog 30% lossis ntau dua ntawm cov qoob loo poob txhua xyoo rau cov kab tsuag thiab cov kab mob, thiab kev sov ntawm lub ntiaj teb xav tias yuav ua rau cov kab tsuag tsis kam thiab cov qoob loo tsis muaj zog.4,5,6,7,8Kev tsim cov tshuaj tua kab yog qhov tseem ceeb tsis yog tsuas yog los tiv thaiv cov qoob loo ntawm cov kab tsuag thiab txo qhov kev sib kis ntawm cov kab mob uas kis tau los ntawm cov kab mob xwb, tab sis kuj tseem yuav tsum tau tawm tsam cov kab mob uas kis los ntawm cov kab mob xws li mob dengue, malaria, thiab mob Chagas, uas tiv taus cov tshuaj tua kab ntau zuj zus.5,9,10,11
Ntawm cov hom phiaj tseem ceeb ntawm cov tshuaj tua kab neurotoxic, heterotetrameric TRPV channel Nanchung (Nan)-Inactive (Iav) sawv cev rau ib chav kawm ntawm cov hom phiaj tua kab uas pom tsuas yog nyob rau hauv kaum xyoo dhau los, suav nrog cov tshuaj tua kab muaj nyob hauv khw xws li imidacloprid thiab pyraclostrobin.12, 13, 14Cov tshuaj tua kab semisynthetic aphidopyrrolifen (AP) yog ib yam khoom tshiab uas tau tsim thiab muag uas nws cov khoom tseem ceeb yog cov tshuaj tua kab Inscalis®, uas khi AP ntawm qib kev ua haujlwm subnanomolar.15AP muaj tshuaj lom tsawg heev rau cov kab uas ua rau cov kab tsuag puas, cov kab uas pab tau, thiab lwm yam kab uas tsis yog hom phiaj, thiab thaum siv raws li cov lus qhia ntawm daim ntawv lo, nws tuaj yeem txo qhov kev tiv taus lwm yam tshuaj tua kab.16, 17, 18Nan thiab Iav muaj ntau yam nyob thoob plaws cov kab, tsuas yog sib koom ua ke hauv cov chordal stretch receptor neurons ntawm lub antennae thiab ceg, thiab tseem ceeb rau kev hnov, kev pom lub ntiajteb txawj nqus, thiab proprioception.13,16,19,20,21,22AP, imidacloprid, thiab pyraclostrobin ua kom lub Nan-Iav complex ua haujlwm los ntawm ib txoj hauv kev tshwj xeeb, thaum kawg inhibiting proprioceptive signal transduction.13, 16, 23Hauv cov kab uas nqus cov kab (hemipterans) xws li cov aphids thiab cov whiteflies, qhov tsis muaj peev xwm noj zaub mov ua rau lawv tsis muaj peev xwm noj mov, thiab thaum kawg ua rau tuag.13,24Qhov txaus siab yog, AP qhia tau tias muaj kev sib raug zoo rau Nan-Iav complex thiab kev sib raug zoo tsawg rau Nan ib leeg. Kev khi ntawm AP rau Nan-Iav ua rau muaj hluav taws xob, tab sis kev khi rau Nan ib leeg tsis ua rau muaj kev ua haujlwm ntawm cov channel. Iav nws tus kheej tsis khi rau AP hlo li.16Qhov no qhia tau hais tias Nan thiab Iav yuav khi ua ke los tsim cov channel sib txawv Nan-Iav (piv txwv li, nrog cov piv txwv sib txawv stoichiometric lossis cov kev npaj sib txawv hauv tib qho piv txwv stoichiometric) lossis tias AP yuav khi rau ntau qhov chaw. Ntxiv mus, cov tshuaj nicotinamide (NAM) khi rau Drosophila Nan-Iav nrog micromolar affinity, ua rau pom cov teebmeem zoo ib yam li cov aphids (AP) hauv vitro.16,25thiab inhibit aphid reproduction thiab pub mov, thaum kawg ua rau lawv tuag25,26. Cov ntaub ntawv no tsa ntau cov lus nug. Piv txwv li, nws tseem tsis meej tias Nan-Iav heterodimer tsim li cas, cov chaw khi twg siv los hloov cov molecules me me, thiab cov molecules me me no tswj cov channel ua haujlwm li cas los ntawm kev tswj hwm proprioception. Ntxiv mus, cov laj thawj vim li cas Nan nws tus kheej tsis ua haujlwm thiab muaj qhov sib raug zoo tsawg rau AP, thaum Nan-Iav heterodimer ua haujlwm thiab khi AP nrog qhov sib raug zoo siab dua, tseem tsis meej. Thaum kawg, me ntsis paub txog Ca2 +-dependent kev tswj hwm ntawm Nan-Iav ua haujlwm thiab nws koom ua ke li cas rau hauv cov txheej txheem neuronal signaling.. 13,21
Hauv txoj kev tshawb fawb no, kev sib txuas ua ke ntawm cryo-electron microscopy, electrophysiology, thiab radioligand binding techniques, peb tau piav qhia txog kev sib sau ua ke ntawm Nan-Iav thiab cov txheej txheem ntawm nws txoj kev sib txuas rau cov tshuaj me me. Ntxiv mus, peb tau kuaj pom cov calmodulin (CaM) uas khi rau Iav thiab AP-stabilized Nan pentamers. Cov txiaj ntsig no muab cov kev nkag siab tseem ceeb rau kev tswj hwm cov calcium ions hauv cov channel, kev sib dhos channel, thiab cov yam ntxwv txiav txim siab ligand binding affinity. Qhov tseem ceeb tshaj, peb tau lees paub tias ARD ua lub luag haujlwm tseem ceeb hauv cov txheej txheem no. Peb txoj kev tshawb fawb txog cov kab uas khi rau cov tshuaj tua kab ua liaj ua teb.27, 28, 29qhib kev cia siab rau kev txhim kho kev lag luam tshuaj tua kab, txhim kho qhov ua tau zoo thiab qhov tshwj xeeb ntawm cov tshuaj tua kab, thiab ua kom muaj peev xwm siv cov tshuaj TRPV-targeted rau lwm hom tsiaj los daws teeb meem kev ruaj ntseg zaub mov thoob ntiaj teb thiab kev kis tus kab mob los ntawm cov kab mob vector.
Peb kuj pom tias Nan-Iav raug tswj hwm los ntawm Ca2+, thiab cov txheej txheem ntawm kev tswj hwm yog los ntawm CaM uas khi ua ke. Qhov tseem ceeb, qhov kev tswj hwm Ca2+ no ntawm Nav los ntawm CaM txawv ntawm cov txheej txheem ntawm kev tswj hwm ntawm lwm cov ion channels (piv txwv li, voltage-gated Na+ channels thiab TRPV5/6 channels)52,53,54,55,56,57Hauv Nav1.2 channel, C-terminal domain ntawm CaM helically associate nrog C-terminal domain (CTD), thiab Ca2+ induces binding ntawm nws N-terminal domain rau distal portion ntawm CTD.56Hauv TRPV5/6 channel, C-terminal domain ntawm CaM khi rau CTH, thiab Ca2+ ua rau nws N-terminal domain nthuav dav mus rau hauv qhov pore, yog li thaiv cation permeability.53,54. Peb muab ib qho qauv rau Ca2+-tswj kev ua haujlwm ntawm Nan-Iav-CaM (Daim duab 4h). Hauv tus qauv no, N-terminal domain ntawm CaM txuas nrog C-terminal domain (CTH) ntawm Iav. Hauv lub xeev so (qis [Ca2+] concentration), C-terminal domain ntawm CaM cuam tshuam nrog Nan, ua kom ARD conformation ruaj khov thiab yog li ntawd txhawb kev qhib channel. Kev khi ntawm agonist/insecticide rau channel ua rau pore qhib, ua rau Ca2+ influx. Ca2+ ces khi rau CaM, ua rau kev sib cais ntawm C-terminal domain los ntawm ARD ntawm Nan. Vim tias kev thaiv CaM binding yeej tshem tawm qhov inhibitory effect ntawm Ca2+, qhov kev sib cais no modulates ARD mobility, yog li ua rau Ca2+-dependent inhibition lossis desensitization. Kev rov qab sai ntawm cov channel currents tom qab calcium ion elution (Daim duab 4g) qhia tias lub tshuab no pab txhawb kev teb sai rau Ca2+-mediated neuronal signals. Tsis tas li ntawd xwb, thaj tsam C-terminal ntawm Iav, uas tseem tsis tau nkag siab zoo, tau tshaj tawm tias ua lub luag haujlwm lwm yam hauv kev tsom mus rau cov channel thiab kev cai lij choj tam sim no.21
Thaum kawg, peb txoj kev tshawb fawb nthuav tawm cov qauv siab daws teeb meem ntawm cov tshuaj tua kab-tshuaj tua kab TRP channel complex ntawm kev ua liaj ua teb tseem ceeb - qhov kev tshawb pom yav dhau los peb tsis paub. Qhov tseem ceeb, peb tau piav qhia txog cov qauv thiab kev ua haujlwm ntawm cov kab channel hauv tib neeg lub hlwb (HEK293S GnTi–) es tsis yog hauv cov kab hlwb. Thaum muaj kev tiv thaiv kab tsuag ntau ntxiv thiab kev nyuaj siab rau kev ruaj ntseg zaub mov thiab cov kab mob, peb txoj haujlwm muab cov ntaub ntawv tseem ceeb uas yuav pab txhawb kev tsim cov tshuaj tua kab tshiab rau kev noj qab haus huv tib neeg thiab kev ruaj ntseg zaub mov thoob ntiaj teb. Cov kev tshawb fawb tau qhia tias cov tshuaj tua kab xws li AP muaj txiaj ntsig zoo rau qee cov kab tsuag thaum siv raws li cov lus qhia ntawm daim ntawv lo thiab muaj kev lom tsawg rau cov kab mob pollinators, qhia txog lawv txoj kev nyab xeeb ib puag ncig.13,16Ntxiv mus, kev sim qee cov AP derivatives ntawm cov yoov tshaj cum tau qhia tias lawv thaum kawg poob lawv lub peev xwm ya. Kev nkag siab txog yuav ua li cas cov tshuaj modulating no khi rau Nan-Iav yuav pab txhawb kev hloov kho ntawm cov tshuaj uas twb muaj lawm lossis kev tsim cov tshuaj tshiab kom ua haujlwm tau zoo dua thiabmeejkev tswj kab tsuag. Peb txoj kev tshawb fawb qhia tau hais tias Nan-Iav ARD interface tsis yog qhov tseem ceeb rau kev tswj hwm cov haujlwm ntawm cov tshuaj endogenous, cov tshuaj tua kab, thiab Ca2+-CaM, tab sis kuj rau kev sib dhos channel. Peb xav tias kev cuam tshuam kev sib dhos heterodimer nrog cov molecule me me yuav yog txoj hauv kev tshwj xeeb thiab muaj kev cia siab rau kev tsim cov ion channel inhibitors.
Ntawm yim lub noob caj noob ces orthologous, cov noob caj noob ces puv ntoob ntawm cov kab laug sab xim av (Halyomorpha halys) Nanchung thiab Inactive tau raug xaiv, qhia txog kev ruaj khov zoo heev hauv cov tshuaj ntxuav tes. Cov noob caj noob ces synthesized tau codon-optimized rau tib neeg kev qhia tawm thiab cloned rau hauv pBacMam pCMV-DEST vector (Life Technologies) siv XhoI thiab EcoRI restriction sites. Qhov no ua kom ntseeg tau tias cov clones tau nyob hauv thav duab nrog C-terminal GFP-FLAG-10xHis thiab mCherry-FLAG-10xHis tags, uas tau cleaved los ntawm HRC-3C protease (PPX), tso cai rau kev ywj pheejkev qhia tawmCov primers siv los clone Nanchung thiab Inactive rau hauv pBacMam vector yog raws li nram no:
Cov duab thaij me me ntawm cov khoom me me tau txais los ntawm Titan Krios G2 transmission electron microscope (FEI) uas muaj lub koob yees duab K3 thiab Gatan BioQuantum energy filter. Lub microscope tau ua haujlwm ntawm 300 keV, nrog rau lub zog teeb tsa ntawm 20 eV, qhov loj ntawm cov qauv pixel ntawm 1.08 Å/pixel (nominal magnification ntawm 81,000x), thiab qhov defocus gradient xws li ntawm -0.8 txog -2.2 μm. Kev kaw video tau ua tiav ntawm 40 thav duab ib ob siv Latitude S microscope (Gatan) nrog tus nqi koob tshuaj nominal ntawm 25 e-px−1 s−1, lub sijhawm raug ntawm 2.4 s, thiab tag nrho cov koob tshuaj ntawm kwv yees li 60 e-Å−2.
Kev kho qhov kev txav mus los ntawm beam thiab kev ntsuas qhov hnyav ntawm cov koob tshuaj tau ua tiav rau ntawm zaj duab xis siv MotionCor2 hauv RELION 4.061. Kev kwv yees qhov sib piv ntawm kev hloov pauv ntawm cov haujlwm (CTF) tau ua tiav hauv cryoSPARC siv txoj kev kwv yees CTF raws li thaj chaw62. Cov duab thaij duab nrog qhov kev daws teeb meem CTF haum ≥4 Å tau raug tshem tawm ntawm kev tshuaj xyuas tom qab. Feem ntau, ib pawg ntawm 500–1000 photomicrographs tau siv rau kev xaiv cov ntsiab lus hauv cryoSPARC, ua raws li ntau qhov kev faib tawm 2D tom qab lim kom tau txais cov duab siv rau kev xaiv cov khoom me me raws li qauv. Cov khoom me me tau raug rho tawm siv 64-pixel bounding boxes thiab 4-fold binning. Ntau qhov kev faib tawm 2D tau ua tiav los tshem tawm cov pawg khoom me me uas tsis xav tau. Tus qauv 3D thawj zaug tau rov tsim dua siv ab initio reconstruction thiab kho kom zoo siv kev kho kom zoo tsis sib xws hauv cryoSPARC. Kev faib tawm 3D tau ua tiav hauv cryoSPARC lossis RELION raws li ARD heterogeneity. Tsis muaj qhov sib txawv tseem ceeb ntawm cov cheeb tsam membrane tau pom. Cov khoom me me tau kho kom zoo siv cov txheej txheem C1 thiab C2; Cov khoom me me uas muaj qhov daws teeb meem C2 siab dua tau suav tias yog qhov sib xws nrog C2 thiab xa mus rau RELION rau Bayesian refinement. Cov khoom me me tom qab ntawd tau xa rov qab mus rau cryoSPARC rau qhov kawg tsis sib xws thiab kev kho kom zoo hauv zos. Qhov kev daws teeb meem kawg thiab suav cov khoom me me tau qhia hauv Rooj 1.
Thaum ua cov Nan+AP pentamers, peb tau tshawb nrhiav ntau txoj hauv kev los txhim kho qhov kev daws teeb meem ntawm cov cheeb tsam membrane (tshwj xeeb tshaj yog thaj chaw pore), xws li kev rho tawm teeb liab thiab TMD masking. Txawm li cas los xij, cov kev sim no tsis ua tiav vim muaj kev tsis sib haum xeeb hauv thaj chaw pore thiab qhov sib txawv ntawm TMD. Qhov kev daws teeb meem kawg tau suav los ntawm kev siv lub ntsej muag uas tsim los ntawm txoj kev ua haujlwm tsis sib xws hauv cryoSPARC, feem ntau yog tsom mus rau thaj chaw ARD. Qhov no tau ua tiav qhov kev daws teeb meem siab dua li ntawm cov cheeb tsam membrane (tshwj xeeb tshaj yog thaj chaw VSLD).
Cov qauv de novo thawj zaug ntawm cov qauv apo ntawm Nanchung thiab Inactive kab laum tau tsim thawj zaug siv Coot63, thiab cov qauv ntawm Nan thiab Iav kab laum tau tsim los ntawm kev siv AlphaFold264 los txheeb xyuas cov cheeb tsam tsis muaj kev ntseeg siab. Calmodulin qauv yog raws li kev sib haum xeeb ntawm Ca2 + -binding thiab Ca2 + -free qauv hauv PDB accessions 4JPZ56 thiab 1CFD65, raws li. Cov qauv tau txhim kho siv kev txhim kho spherical kom ntseeg tau tias muaj stereochemistry raug thiab geometry zoo. Phosphatidylcholine, phosphatidylethanolamine, thiab phosphatidylserine tau raug qauv ua cov lipid densities zoo, thiab NAM thiab AP ligands tau muab tso rau hauv cov densities sib xws hauv cov kev sib tshuam nruj. Cov ntaub ntawv txwv tau tsim los ntawm SMILES hlua ntawm isoforms siv eLBOW hauv PHENIX66. Thaum kawg, cov qauv tau txhim kho hauv qhov chaw tiag tiag hauv PHENIX siv kev tshawb nrhiav hauv zos thiab kev txo qis thoob ntiaj teb nrog cov qauv theem nrab. Lub MolProbity server tau siv rau kev txhim kho qauv thiab kev tshuaj xyuas cov qauv, thiab cov duab kos tau ua tiav siv PyMOL thiab UCSF Chimera X. 67,68,69 Kev tshuaj xyuas qhov qhib tau ua tiav siv HOLE server,70 thiab kev kos duab kev txuag kab ke tau ua tiav siv Consurf server.71
Kev tshuaj xyuas cov ntaub ntawv tau ua tiav los ntawm kev siv Igor Pro 6.2, Excel Office 365, thiab GraphPad Prism 7.0. Tag nrho cov ntaub ntawv ntau npaum li cas tau nthuav tawm ua qhov nruab nrab ± qhov yuam kev txheem (SEM). Kev xeem t-xeem ntawm tus tub ntxhais kawm (ob-tailed, unpaired) tau siv los sib piv ob pawg. Kev tshuaj xyuas ib txoj kev ntawm qhov sib txawv (ANOVA) ua raws li Dunnett qhov kev xeem post hoc tau siv los sib piv ntau pawg. *P< 0.05, **P< 0.01, thiab ***P< 0.001 tau suav tias yog qhov tseem ceeb ntawm kev suav lej nyob ntawm qhov faib cov ntaub ntawv. Kd, Ki tus nqi, thiab lawv cov asymmetric 95% kev ntseeg siab tau suav los ntawm kev siv GraphPad Prism 10.
Yog xav paub ntxiv txog txoj kev kawm, thov saib Nature Portfolio Report Summary txuas rau hauv tsab xov xwm no.
Tus qauv pib tau tsim los ntawm kev siv cov qauv calmodulin los ntawm PDB 4JPZ thiab 1CFD databases. Cov kev sib koom ua ke tau muab tso rau hauv Protein Data Bank (PDB) raws li cov lej nkag 9NVN (Nan-Iav-CaM tsis muaj ligand), 9NVO (Nan-Iav-CaM khi rau nicotinamide), 9NVP (Nan-Iav-CaM khi rau nicotinamide thiab EDTA), 9NVQ (Nan-Iav-CaM khi rau aphenidolpyrrolline thiab calcium), 9NVR (Nan-Iav-CaM khi rau aphenidolpyrrolline thiab EDTA), thiab 9NVS (Nan pentamer khi rau aphenidolpyrrolline). Cov duab cryo-electron microscopy sib xws tau muab tso rau hauv Electron Microscopy Database (EMDB) raws li cov lej nkag hauv qab no: EMD-49844 (Nan-Iav-CaM tsis muaj ligand), EMD-49845 (Nan-Iav-CaM complex nrog nicotinamide), EMD-49846 (Nan-Iav-CaM complex nrog nicotinamide thiab EDTA), EMD-49847 (Nan-Iav-CaM complex nrog aphidopyrrolline thiab calcium), EMD-49848 (Nan-Iav-CaM complex nrog aphidopyrrolline thiab EDTA), thiab EMD-49849 (Nan pentamer complex nrog aphidopyrrolline). Cov ntaub ntawv raw rau kev tshuaj xyuas kev ua haujlwm tau nthuav tawm hauv daim ntawv no.
Lub sijhawm tshaj tawm: Lub Ib Hlis-28-2026