Mishonga yekuuraya zvipembenene inoita basa rakakosha mukugadzirisa kushomeka kwechikafu pasi rose uye kurwisa zvirwere zvevanhu zvinotapukirwa nezvipembenene. Zvisinei, dambudziko riri kukura rekusagona kurwisa zvipembenene rinoda kuti pave nekukurumidzira kuwanikwa kwemakemikari matsva anotarisa zvinangwa zvisingashandiswi zvakakwana. Nzira dzezvipembenene dzinokurumidza kushandisa zvipembenene (TRPV) - Nanzhong (Nan) uye dzisingashande (Iav) - dzinogona kugadzira nzira dzakasiyana-siyana (Nan-Iav) uye dzinoita kuti zvipembenene zvinzwe zviri mumasero emuviri anokonzera geotropism, kunzwa, uye proprioception muzvipembenene. Mimwe mishonga yekuuraya zvipembenene, yakaita seaphidopyrrolidone (AP), inotarisa Nan-Iav kuburikidza nenzira dzisingazivikanwe. AP inoshanda kurwisa zvipembenene zvinobaya-baya (hemipterans), ichidzivirira kudya nekuvhiringidza mashandiro efilaments. AP inogona kusunga Nan chete, asi Nan-Iav chete ndiyo inogona kusangana ne agonists, kusanganisira endogenous nicotinamide (NAM), nokudaro ichiratidza mashandiro enzira. Pasinei nekugona kweNan-Iav senzira yekuuraya zvipembenene, zvishoma zvinozivikanwa nezvekuungana kwayo, nzvimbo dzinobatanidza zvipembenene, uye Ca2+-dependent regulation, zvichitadzisa kukura kwezvipembenene. Muchidzidzo ichi, cryo-electron microscopy yakashandiswa kuona chimiro cheNan-Iav muzvipembenene zveHemiptera muchimiro che calmodulin-ligand-free, pamwe neAP neNAM pamuganhu we ankyrin repeat cytoplasmic domain (ARD). Zvinoshamisa kuti takaona kuti protein yeNan pachayo inogona kuumba pentamer, iyo inogadzikiswa nekudyidzana kwe ARD kunosanganisirwa ne AP. Chidzidzo ichi chinoratidza kudyidzana kwemamorekuru pakati pemishonga inouraya zvipembenene nema agonists neNan-Iav, zvichiratidza kukosha kweARD mukushanda kwe channel uye kuunganidza, uye kuongorora mashandiro eCa2+ regulation.
Zvichienderana nekuchinja kuri kuramba kuchiitika kwemamiriro ekunze pasi rose, kudzikira kwekuchengetedzwa kwechikafu pasi rose ndeimwe yematambudziko makuru ezana remakore rechi21, nemhedzisiro yakaipa munharaunda.1,2Mushumo weWorld Health Organization weState of Food Security and Nutrition in the World 2023 (SOFI) unofungidzira kuti vanhu vanosvika mabhiriyoni 2.33 pasi rose vanotambura nekushaya chikafu kwakaringana kusvika kwakanyanya, dambudziko ravepo kwenguva refu.3,4Zvinosuwisa kuti, goho rezvirimwa rinofungidzirwa kuva pakati pe20% kusvika 30% kana kupfuura rinorasika gore rega rega nekuda kwezvipembenene nezvirwere, uye kudziya kwepasi rose kunotarisirwa kuwedzera kusimba kwezvipembenene uye kushaya simba kwezvirimwa.4, 5, 6, 7, 8Kugadzirwa kwemishonga yekuuraya zvipembenene kwakakosha kwete chete kudzivirira zvirimwa kubva kuzvipembenene uye kuderedza kupararira kwezvirwere zvinotapukira, asiwo kurwisa zvirwere zvinotapukira kuvanhu zvakaita sedengue fever, malaria, uye chirwere cheChagas, izvo zviri kuramba zvichirwisa mishonga yekuuraya zvipembenene.5,9,10,11
Pakati pezvinangwa zvikuru zvemishonga inouraya zvipembenene ine chepfu muuropi, heterotetrameric TRPV channel Nanchung (Nan)-Inactive (Iav) inomiririra mhando yezvinangwa zvezvipembenene zvakawanikwa mumakore gumi apfuura chete, kusanganisira mishonga inouraya zvipembenene inowanikwa kutengeswa yakadai seimidacloprid nepyraclostrobin.12, 13, 14Mushonga wezvipembenene unonzi aphidopyrrolifen (AP) unoshandiswa muzvikamu zvakasiyana-siyana, wakagadzirwa munguva pfupi yapfuura uye wakatengeswa, une chikamu chikuru chemushonga wezvipembenene unonzi Inscalis®, uyo unobatanidza AP padanho rekuita zvinhu zvishoma nezvishoma.15AP inoratidza chepfu shoma kune zvinoyamwisa, zvipembenene zvinobatsira, nezvimwe zvipenyu zvisingatarisirwi, uye kana ikashandiswa zvichienderana nemirayiridzo yakanyorwa, inogona kuderedza kumanikidzwa kwemishonga yekuuraya zvipembenene kune mimwe mishonga yekuuraya zvipembenene.16, 17, 18Nan neIav zvakapararira zvakanyanya mumarudzi ezvipembenene, zvinoburitswa pamwe chete muchordal stretch receptor neurons dzeantennae nemakumbo, uye zvakakosha pakunzwa, kunzwisisa simba rinokwevera pasi, uye kuziva chimiro chemuviri.13,16,19,20,21,22AP, imidacloprid, uye pyraclostrobin zvinokurudzira Nan-Iav complex kuburikidza nenzira yakasiyana, zvichizopedzisira zvadzivisa proprioceptive signal transduction.13,16,23Muzvipembenene zvinoyamwa (hemipterans) zvakaita seaphids newhiteflies, kurasikirwa neproprioception kunokanganisa kugona kwadzo kudya, zvichizoguma zvaita kuti dzife.13,24Zvinofadza kuti, AP inoratidza hukama hwakanyanya neNan-Iav complex uye hukama hushoma neNan chete. Kubatana kweAP neNan-Iav kunokonzera magetsi, asi kubatana neNan chete hakukurudziri mashandiro emugero. Iav pachayo haibatanidzi neAP zvachose.16Izvi zvinoratidza kuti Nan neIav zvinogona kubatana kuti zvigadzire maNan-Iav channel complexes akasiyana (semuenzaniso, ne stoichiometric ratios dzakasiyana kana marongerwo akasiyana mukati me stoichiometric ratio imwechete) kana kuti AP inogona kubatana kune nzvimbo dzakawanda. Uyezve, natural agonist nicotinamide (NAM) inobatanidza Drosophila Nan-Iav ne micromolar affinity, ichiratidza mhedzisiro yakafanana neye aphids (AP) in vitro.16,25uye zvinodzivisa kuberekana kwezvipenyu zvema aphid uye kudya, zvichizopedzisira zvaita kuti zvife.25,26. Data iri rinomutsa mibvunzo yakawanda. Semuenzaniso, hazvisati zvajeka kuti Nan-Iav heterodimer inoumbwa sei, kuti ndeapi ma "binding sites" anoshandiswa kugadzirisa mamorekuru madiki, uye kuti mamorekuru madiki aya anodzora sei mashandiro e "channel" nekudzvinyirira proprioception. Uyezve, zvikonzero nei Nan pachayo isingashande uye ine "low affinity" yeAP, nepo Nan-Iav heterodimer ichishanda uye inobatanidza AP ne "high affinity", hazvisati zvajeka. Chekupedzisira, zvishoma zvinozivikanwa nezveCa2+-dependent regulation yeNan-Iav function uye kuti inosanganiswa sei mu neuronal signaling processes.. 13,21
Muchidzidzo ichi, tichisanganisa cryo-electron microscopy, electrophysiology, uye nzira dzekubatanidza ma radioligand, takajekesa kuungana kweNan-Iav uye mashandiro ekubatana kwayo ne small molecule regulators. Uyezve, takaona constitutively bound calmodulin (CaM) kuIav neAP-stabilized Nan pentamers. Mhedzisiro iyi inopa ruzivo rwakakosha mukudzora ma calcium ions muma channels, channel assembly, uye zvinhu zvinosarudza ligand binding affinity. Chinonyanya kukosha, takasimbisa kuti ARD ine basa guru mumaitiro aya. Chidzidzo chedu che channels dzakazara dzakasungirirwa kumishonga yekurima yakakodzera.27, 28, 29Inovhura mikana yekukura kweindasitiri yemishonga yezvipembenene, ichivandudza kushanda uye kujeka kwemishonga yezvipembenene, uye ichigonesa kushandiswa kwemishonga inotarirwa neTRPV kune mamwe marudzi emhuka kugadzirisa kuchengetedzwa kwechikafu pasi rose uye kupararira kwezvirwere zvinotapukira.
Takawanawo kuti Nan-Iav inodzorwa neCa2+, uye mashandiro ekutonga anotungamirirwa neCaM yakasungwa. Zvakakosha, kudzorwa uku kweNav neCaM kunoenderana neCa2+ kwakasiyana zvakanyanya nemaitiro ekutonga mamwe maion channels (semuenzaniso, mavoltage-gated Na+ channels uye TRPV5/6 channels)52,53,54,55,56,57MuNav1.2 channel, C-terminal domain yeCaM inobatana neC-terminal domain (CTD), uye Ca2+ inobatanidza N-terminal domain yayo nechikamu cheCTD chiri kure.56Munzira yeTRPV5/6, nzvimbo yeC-terminal yeCaM inobatanidzwa neCTH, uye Ca2+ inokurudzira kuwedzera kwenzvimbo yayo yeN-terminal muburi, nokudaro ichivhara cation permeability.53,54. Tinokurudzira modhi yebasa rinodzorwa neCa2+ reNan-Iav-CaM (Mufananidzo 4h). Mumuenzaniso uyu, nzvimbo yeN-terminal yeCaM inobatanidzwa neC-terminal domain (CTH) yeIav. Mumamiriro ekuzorora (kuwanda kwakaderera kweCa2+), nzvimbo yeC-terminal yeCaM inosangana neNan, ichidzikamisa ARD conformation uye nokudaro ichikurudzira kuvhurwa kwenzira. Kusunga mushonga weagonist/zvipembenene kune nzira kunokonzera kuvhurwa kwepore, zvichikonzera kubuda kweCa2+. Ca2+ inobva yabatana neCaM, zvichikonzera kuparadzaniswa kwenzvimbo yeC-terminal kubva kuARD yeNan. Nekuti kuvharira kubatana kweCaM kunobvisa simba rekudzivirira reCa2+, kuparadzaniswa uku kunogadzirisa kufamba kweARD, nokudaro zvichikonzera kudziviswa kana kusanzwa kweCa2+. Kudzoka nekukurumidza kwenzira dzemagetsi mushure mekubvisa calcium ion (Mufananidzo 4g) kunoratidza kuti nzira iyi inobatsira kupindura nekukurumidza kuzviratidzo zveneuronal zveCa2+. Uyezve, nzvimbo yeC-terminal yeIav, iyo isinganyatsonzwisiswi, inonzi ine mamwe mabasa mukutsvaga nzira dzekufambisa zvinhu pamwe nemutemo wemazuva ano.21
Chekupedzisira, chidzidzo chedu chinoratidza chimiro chepamusoro-soro cheTRP channel complex yezvipembenene-zvipembenene inokosha pakurima—kuwana kwatakanga tisingazive kare. Zvakakosha kuti takatsanangura chimiro nebasa remugero wezvipembenene mumasero evanhu (HEK293S GnTi–) kwete mumasero ezvipembenene. Tichitarisana nekukura kwekusagona kuuraya zvipembenene uye kumanikidzwa kuri kuramba kuripo pakuchengetedzeka kwechikafu nezvirwere, basa redu rinopa ruzivo rwakakosha ruchaita kuti pave nekugadzirwa kwemishonga mitsva yezvipembenene kuitira kuti hutano hwevanhu uye kuchengetedzeka kwechikafu pasi rose. Zvidzidzo zvakaratidza kuti mishonga yezvipembenene yakaita seAP inoshanda kurwisa zvimwe zvipembenene kana ikashandiswa zvichienderana nemirayiridzo yakanyorwa uye ine chepfu shoma kune zvinomera zvinobatsira, zvichiratidza kuchengetedzeka kwayo munzvimbo.13,16Uyezve, kuyedzwa kwemamwe maAP derivatives paumhutu kwakaratidza kuti pakupedzisira anorasikirwa nekukwanisa kwawo kubhururuka. Kunzwisisa kuti mamodulating compounds aya anobatana sei neNan-Iav kuchaita kuti kuchinjwe kwemamodules aripo kana kugadzirwa kwemamodules matsva kuti ashande zvakanaka uye abudirire.chaiyokudzora zvipembenene. Chidzidzo chedu chinoratidza kuti Nan-Iav ARD interface yakakosha kwete chete pakudzora mashandiro emakomichi e endogenous, mishonga yezvipembenene, uye Ca2+-CaM, asiwo pakuungana kwenzira. Tinokurudzira kuti kukanganisa kuungana kweheterodimer nemamorekuru madiki kungave nzira yakasiyana uye inovimbisa yekugadzira inhibitor dzeion channel.
Pamajini masere ane orthologous, majini akazara ebrown beetle (Halyomorpha halys) Nanchung neInactive akasarudzwa, zvichiratidza kugadzikana kwakanaka mu detergents. Majini akagadzirwa akagadziriswa codon kuti akwanise kutaura nevanhu uye akaiswa mu pBacMam pCMV-DEST vector (Life Technologies) achishandisa nzvimbo dzeXhoI neEcoRI restriction. Izvi zvakaita kuti ma clones ave muchimiro chine C-terminal GFP-FLAG-10xHis uye mCherry-FLAG-10xHis tags, ayo anochekwa ne HRC-3C protease (PPX), zvichibvumira munhu wese.kutauraMa primer akashandiswa kukopa Nanchung neInactive mu pBacMam vector aive akadai:
Mifananidzo ye microscopic yezvikamu zvakasiyana-siyana yakawanikwa paTitan Krios G2 transmission electron microscope (FEI) ine kamera yeK3 uye Gatan BioQuantum energy filter. Maikorosikopu iyi yakashandiswa pa300 keV, ine simba re20 eV, saizi yesampuro yepikisiki ye1.08 Å/pixel (nominal magnification ye81,000x), uye defocus gradient inotangira pa -0.8 kusvika -2.2 μm. Kurekodha vhidhiyo kwakaitwa pamafuremu makumi mana pasekondi uchishandisa Latitude S microscope (Gatan) ine nominal dose rate ye25 e–px−1 s−1, nguva yekuratidzwa ye2.4 s, uye total dose yeinenge 60 e–Å−2.
Kugadziriswa kwemafambiro anokonzerwa nemwenje uye kuyerwa kwemuyero wedosi kwakaitwa pafirimu uchishandisa MotionCor2 muRELION 4.061. Kuongororwa kweparameter yeContrast transfer function (CTF) kwakaitwa mucryoSPARC uchishandisa nzira yeCTF estimation yakavakirwa papatch62. Maphotomicrograph ane resolution yeCTF fitting ≥4 Å akabviswa muongororo yakatevera. Kazhinji, subset ye500–1000 photomicrographs yakashandiswa pakusarudza mapoinzi mucryoSPARC, ichiteverwa nerounds dzinoverengeka dze2D classification mushure mekusefa kuti pave nemufananidzo wakajeka wekusarudza template-based particle. Particles dzakazobviswa uchishandisa mabhokisi e64-pixel bounding uye 4-fold binning. Rounds dzinoverengeka dze2D classification dzakaitwa kubvisa macategories eparticles asingadiwe. Modhi yekutanga ye3D yakagadzirwazve uchishandisa ab initio reconstruction uye yakanatswa uchishandisa nonuniform finance mucryoSPARC. Kupatsanurwa kwe3D kwakaitwa mucryoSPARC kana RELION zvichibva paARD heterogeneity. Hapana heterogeneity yakakosha ye membrane domains yakaonekwa. Zvidimbu zvakanatswa uchishandisa nzira dzeC1 neC2; zvidimbu zvine resolution yepamusoro yeC2 zvakaonekwa sezvine symmetric maererano neC2 uye zvakapinzwa muRELION kuti zvinatswe muBayesian. Zvidimbu zvakazodzoserwa ku cryoSPARC kuti zvinatswe zvekupedzisira zvisina kufanana uye zvemunharaunda. Resolution yekupedzisira uye nhamba dzezvidimbu zvinoratidzwa muTafura 1.
Pakugadzira maNan+AP pentamers, takatsvaga nzira dzakasiyana-siyana dzekuvandudza kugadziriswa kwema membrane domains (kunyanya nzvimbo yepore), dzakadai sekubvisa ma signal uye TMD masking. Zvisinei, kuedza uku hakuna kubudirira nekuda kwekukanganisika kwakanyanya munzvimbo yepore uye kusiyana kwese kweTMD. Resolution yekupedzisira yakaverengerwa uchishandisa mask inogadzirwa otomatiki nenzira isina kufanana mu cryoSPARC, ichinyanya kunanga nzvimbo yeARD. Izvi zvakawana resolution yakakwira kupfuura iyo yema membrane domains (kunyanya nzvimbo yeVSLD).
Mamodheru ekutanga matsva emhando dze apo dzeNanchung neInactive bugs akatanga kugadzirwa achishandisa Coot63, uye mamodheru eNan neIav bugs akagadzirwa achishandisa AlphaFold264 kuti aone nzvimbo dzisina chivimbo. Calmodulin modeling yakavakirwa pa rigid-body fit dzemamodheru eCa2+-binding neCa2+-free muPDB accessions 4JPZ56 ne1CFD65, zvichiteerana. Mamodheru aya akagadziriswa achishandisa spherical refinement kuti ave nechokwadi chekuti stereochemistry yakarurama uye geometry yakanaka. Phosphatidylcholine, phosphatidylethanolamine, uye phosphatidylserine zvakazogadzirwa se well-defined lipid densities, uye NAM neAP ligands zvakaiswa mu densities dzinoenderana mu tight junctions. Constraint files dzakagadzirwa kubva kuSMILES string ye isoforms uchishandisa eLBOW muPHENIX66. Pakupedzisira, mamodheru akagadziriswa munzvimbo chaiyo muPHENIX uchishandisa local grid search uye global minimization ne secondary structure constraints. Sevha yeMolProbity yakashandiswa pakunatsiridza modhi uye kuongorora chimiro, uye mifananidzo yakaitwa uchishandisa PyMOL neUCSF Chimera X. 67,68,69 Kuongorora kweAperture kwakaitwa uchishandisa HOLE server,70 uye sequence conservation mapping yakaitwa uchishandisa Consurf server.71
Kuongororwa kwenhamba kwakaitwa uchishandisa Igor Pro 6.2, Excel Office 365, uye GraphPad Prism 7.0. Data rese rehuwandu rinopihwa se mean ± standard error (SEM). T-test yemudzidzi (two-tailed, unpaired) yakashandiswa kuenzanisa mapoka maviri. One-way analysis of variance (ANOVA) ichiteverwa ne post hoc test yaDunnett yakashandiswa kuenzanisa mapoka akawanda. *P< 0.05, **P< 0.01, uye ***P<0.001 zvakaonekwa sezvine kukosha zvichienderana nekugoverwa kwedata. Kd, Ki values, uye 95% confidence intervals dzavo dzakaverengerwa uchishandisa GraphPad Prism 10.
Kuti uwane rumwe ruzivo nezve nzira yekudzidza, ndapota ona Nature Portfolio Report Summary yakabatana nechinyorwa chino.
Modhi yekutanga yakagadzirwa uchishandisa mamodheru e calmodulin kubva kumadhatabhesi ePDB 4JPZ ne 1CFD. Ma coordinates akaiswa muProtein Data Bank (PDB) pasi penhamba dze accession 9NVN (Nan-Iav-CaM isina ligand), 9NVO (Nan-Iav-CaM yakasungwa ku nicotinamide), 9NVP (Nan-Iav-CaM yakasungwa ku nicotinamide ne EDTA), 9NVQ (Nan-Iav-CaM yakasungwa ku aphenidolpyrrolline ne calcium), 9NVR (Nan-Iav-CaM yakasungwa ku aphenidolpyrrolline ne EDTA), uye 9NVS (Nan pentamer yakasungwa ku aphenidolpyrrolline). Mifananidzo ye cryo-electron microscopy inoenderana inoiswa muElectron Microscopy Database (EMDB) pasi penhamba dzinotevera dzekupinda: EMD-49844 (Nan-Iav-CaM isina ligand), EMD-49845 (Nan-Iav-CaM complex ine nicotinamide), EMD-49846 (Nan-Iav-CaM complex ine nicotinamide neEDTA), EMD-49847 (Nan-Iav-CaM complex ine aphidopyrrolline necalcium), EMD-49848 (Nan-Iav-CaM complex ine aphidopyrrolline neEDTA), uye EMD-49849 (Nan pentamer complex ine aphidopyrrolline). Data raw re functional analysis rinopihwa mupepa rino.
Nguva yekutumira: Ndira-28-2026