Izibulala-zinambuzane zidlala indima ebalulekile ekubhekaneni nokuntuleka kokudla emhlabeni wonke kanye nokulwa nezifo zabantu ezithwalwa yizinambuzane. Kodwa-ke, inkinga ekhulayo yokumelana nezibulala-zinambuzane idinga ngokushesha ukutholakala kwamakhemikhali amasha ahlose izinhloso ezingasetshenziswa kahle. Iziteshi ze-Insect transient receptor potential (TRPV)—i-Nanzhong (Nan) kanye ne-inactive (Iav)—zingakha iziteshi ezingafani (i-Nan-Iav) futhi zifinyelele ezithweni ze-mechanosensory ezilawula i-geotropism, ukuzwa, kanye nokutholwa kwezinambuzane. Ezinye izibulala-zinambuzane, njenge-aphidopyrrolidone (AP), zihlose i-Nan-Iav ngezindlela ezingaziwa. I-AP iyasebenza ngokumelene nezinambuzane ezibhoboza-ezimunca (ama-hemipteran), ivimbela ukudla ngokuphazamisa umsebenzi we-filaments. I-AP ingabopha kuphela i-Nan-Iav, kodwa i-Nan-Iav kuphela engasebenzisana nama-agonists, kufaka phakathi i-nicotinamide e-endogenous (NAM), ngaleyo ndlela ibonise umsebenzi wesiteshi. Naphezu kwamandla e-Nan-Iav njengenhloso yezinambuzane, kuncane okwaziwayo mayelana nokuhlanganiswa kwesiteshi sayo, izindawo zokubopha ezilawulayo, kanye nomthethonqubo oncike ku-Ca2+, okuvimbela ukuthuthukiswa okuqhubekayo kwezinambuzane. Kulolu cwaningo, i-cryo-electron microscopy yasetshenziswa ukunquma isakhiwo se-Nan-Iav ezinambuzaneni ze-Hemiptera esimweni sokungabi ne-calmodulin-ligand, kanye ne-AP ne-NAM emngceleni we-ankyrin repeat cytoplasmic domain (ARD). Ngokumangazayo, sithole ukuthi iphrotheni ye-Nan ngokwayo ingakha i-pentamer, eqiniswa ukusebenzisana kwe-ARD okwenziwa yi-AP. Lolu cwaningo lwembula ukusebenzisana kwama-molecule phakathi kwama-insecticide nama-agonist kanye ne-Nan-Iav, okugqamisa ukubaluleka kwe-ARD emsebenzini wesiteshi kanye nokuhlanganiswa, kanye nokuhlola indlela yokulawulwa kwe-Ca2+.
Naphezu kwesimo sokushintsha kwesimo sezulu esibi kakhulu emhlabeni, ukuwohloka kokuphepha kokudla emhlabeni kungenye yezinselelo ezinkulu zekhulu lama-21, okunemiphumela emibi emphakathini.1,2Umbiko we-World Health Organization's State of Food Security and Nutrition in the World 2023 (SOFI) ulinganisela ukuthi cishe abantu abayizigidi eziyizinkulungwane ezingu-2.33 emhlabeni wonke bahlushwa ukungavikeleki kokudla okumaphakathi kuya kokubi, okuyinkinga ende.3,4Ngeshwa, kulinganiselwa ukuthi u-20% kuya ku-30% noma ngaphezulu wesivuno sezitshalo ulahleka minyaka yonke ngenxa yezinambuzane namagciwane, futhi ukufudumala komhlaba kulindeleke ukuthi kukhulise ukumelana nezinambuzane kanye nobuthakathaka bezitshalo.4,5,6,7,8Ukuthuthukiswa kwezibulala-zinambuzane kubalulekile hhayi nje ekuvikeleni izitshalo ezinambuzaneni nasekunciphiseni ukusabalala kwamagciwane athwalwa yizinambuzane, kodwa futhi nasekulweni nezifo zabantu ezithwalwa yizinambuzane ezifana nomkhuhlane we-dengue, umalaleveva, kanye nesifo se-Chagas, ezimelana kakhulu nezibulala-zinambuzane.5,9,10,11
Phakathi kwezinhloso ezinkulu zemithi yokubulala izinambuzane enobuthi be-neuro, isiteshi se-TRPV se-heterotetrameric i-Nanchung (Nan)-Inactive (Iav) simelela uhlobo lwezinhloso zemithi yokubulala izinambuzane ezitholakale eminyakeni eyishumi edlule, okuhlanganisa imithi yokubulala izinambuzane etholakala kwezentengiselwano njenge-imidacloprid kanye ne-pyraclostrobin.12,13,14I-aphidopyrrolifen (AP) yezinambuzane ezihlanganisiwe ezihlanganisiwe iwumkhiqizo osanda kuthuthukiswa futhi othengiswa ngawo, ingxenye yawo eyinhloko iyi-inscalis® yezinambuzane ezisebenzayo, ebopha i-AP ezingeni lomsebenzi we-subnanomolar.15I-AP ikhombisa ubuthi obuphansi kakhulu ezithutheni zempova, izinambuzane ezizuzisayo, nezinye izinto eziphilayo ezingahlosiwe, futhi uma isetshenziswa ngokwemiyalelo yelebula, inganciphisa ingcindezi yokumelana nezinye izibulala-zinambuzane.16,17,18I-Nan ne-Iav zisakazeke kabanzi kuzo zonke izinhlobo zezinambuzane, zivezwa ndawonye kuphela kuma-neuron e-chordal stretch receptor ezimpondweni nasezithweni, futhi zibalulekile ekuzweni, ekuboneni amandla adonsela phansi, kanye nokuqonda ubunikazi.13,16,19,20,21,22I-AP, i-imidacloprid, kanye ne-pyraclostrobin zivuselela i-Nan-Iav complex ngendlela ehlukile, ekugcineni zivimbela ukudluliselwa kwesignali yobunikazi.13,16,23Ezinambuzaneni ezimunca izimbobo (ama-hemipteran) njenge-aphid kanye nezimpukane ezimhlophe, ukulahlekelwa ukuqonda okuqondile kuphazamisa ikhono lazo lokudla, okuholela ekufeni.13,24Ngokuthakazelisayo, i-AP ibonisa ukusondelana okuphezulu kwe-Nan-Iav complex kanye nokusondelana okuphansi kwe-Nan kuphela. Ukubopha kwe-AP ku-Nan-Iav kubangela ugesi, kodwa ukubopha ku-Nan kuphela akukhuthazi umsebenzi wesiteshi. I-Iav ngokwayo ayibopheli ku-AP nhlobo.16Lokhu kusikisela ukuthi i-Nan ne-Iav zingabophana ukuze zakhe ama-complexes ahlukene e-Nan-Iav channel (isb., ngezilinganiso ezahlukene ze-stoichiometric noma amalungiselelo ahlukene ngaphakathi kwesilinganiso esifanayo se-stoichiometric) noma ukuthi i-AP ingabophana ezindaweni eziningi. Ngaphezu kwalokho, i-agonist yemvelo i-nicotinamide (i-NAM) ibophana ne-Drosophila Nan-Iav nge-micromolar affinity, ikhombisa imiphumela efana neye-aphid (i-AP) in vitro.16,25kanye nokuvimbela ukuzala nokufuya ama-aphid, okuholela ekufeni kwawo ekugcineni25,26. Le datha iphakamisa imibuzo eminingi. Isibonelo, akukacaci ukuthi i-heterodimer ye-Nan-Iav yakhiwa kanjani, yiziphi izindawo zokubopha ezisetshenziswa ukuguqula ama-molecule amancane, nokuthi la ma-molecule amancane alawula kanjani umsebenzi wesiteshi ngokucindezela i-proprioception. Ngaphezu kwalokho, izizathu zokuthi kungani i-Nan ngokwayo ingasebenzi futhi ine-affinity ephansi ye-AP, kuyilapho i-heterodimer ye-Nan-Iav isebenza futhi ibopha i-AP nge-affinity ephezulu, azikacaci. Okokugcina, kuncane okwaziwayo ngokulawulwa okuncike ku-Ca2+ komsebenzi we-Nan-Iav nokuthi uhlanganiswa kanjani nezinqubo zokubonisa izimpawu ze-neuronal.. 13,21
Kulesi sifundo, esihlanganisa i-cryo-electron microscopy, i-electrophysiology, kanye namasu okubopha i-radioligand, sichaze ukuhlanganiswa kwe-Nan-Iav kanye nendlela yokubopha kwayo kubalawuli bama-molecule amancane. Ngaphezu kwalokho, sithole i-calmodulin (CaM) eboshiwe ngokuqhubekayo ku-Iav kanye nama-pentamers e-Nan azinzile e-AP. Le miphumela inikeza ukuqonda okubalulekile ngokulawulwa kwama-ion e-calcium eziteshini, ukuhlanganiswa kweziteshi, kanye nezici ezinquma ukuhlangana kwe-ligand. Okubaluleke kakhulu, siqinisekisile ukuthi i-ARD idlala indima ebalulekile kulezi zinqubo. Ucwaningo lwethu lweziteshi zezinambuzane eziphelele eziboshelwe ezibulala-zinambuzane zezolimo ezifanele.27, 28, 29kuvula amathuba okuthuthukiswa kwemboni yezibulala-zinambuzane, kuthuthukiswe ukusebenza kahle kanye nokucaciswa kwezibulala-zinambuzane, futhi kuvumele ukusetshenziswa kwamakhemikhali aqondiswe ku-TRPV kwezinye izinhlobo ukuze kubhekwe ukuphepha kokudla emhlabeni jikelele kanye nokusabalala kwezifo ezithwalwa yizinambuzane.
Sithole nokuthi i-Nan-Iav ilawulwa yi-Ca2+, futhi indlela yokulawula iqondiswa yi-CaM eboshwe ngokuqinile. Okubalulekile, lokhu kulawulwa okuncike ku-Ca2+ kwe-Nav yi-CaM kuhluke kakhulu ezindleleni zokulawula ezinye iziteshi ze-ion (isb., iziteshi ze-Na+ ezivalwe nge-voltage kanye neziteshi ze-TRPV5/6)52,53,54,55,56,57Kusiteshi se-Nav1.2, isizinda se-C-terminal se-CaM sihlotshaniswa nesizinda se-C-terminal (CTD), kanti i-Ca2+ ibangela ukubopha kwesizinda sayo se-N-terminal engxenyeni ekude ye-CTD.56. Kusiteshi se-TRPV5/6, isizinda se-C-terminal se-CaM sibopha ku-CTH, kanti i-Ca2+ ibangela ukwanda kwesizinda sayo se-N-terminal singene embotsheni, ngaleyo ndlela ivimbele ukungena kwe-cation.53,54. Siphakamisa imodeli yomsebenzi olawulwa yi-Ca2+ we-Nan-Iav-CaM (Isithombe 4h). Kulo modeli, isizinda se-N-terminal se-CaM sibopha ngokuqinile kwisizinda se-C-terminal (CTH) se-Iav. Esimweni sokuphumula (ukuhlushwa okuphansi kwe-[Ca2+]), isizinda se-C-terminal se-CaM sisebenzisana ne-Nan, siqinise ukwakheka kwe-ARD futhi ngaleyo ndlela sikhuthaze ukuvulwa kwesiteshi. Ukubopha i-agonist/izinambuzane esiteshini kubangela ukuvulwa kwe-pore, okuholela ekungeneni kwe-Ca2+. I-Ca2+ bese ibopha ku-CaM, okubangela ukuhlukaniswa kwesizinda se-C-terminal ku-ARD ye-Nan. Ngoba ukuvimba ukubopha kwe-CaM empeleni kuqeda umphumela wokuvimbela we-Ca2+, lokhu kuhlukaniswa kuguqula ukuhamba kwe-ARD, ngaleyo ndlela kubangele ukuvinjelwa noma ukuncishiswa kwe-Ca2+ okuncike ku-ca. Ukubuyiselwa okusheshayo kwemigudu yesiteshi ngemva kokususwa kwe-calcium ion (Isithombe 4g) kusikisela ukuthi le ndlela yenza kube lula ukuphendula ngokushesha kumasignali e-neuronal aphethwe yi-Ca2+. Ngaphezu kwalokho, isifunda se-C-terminal sase-Iav, esingakaqondakali kahle, kubikwe ukuthi sidlala ezinye izindima ekuqondisweni kweziteshi kanye nomthethonqubo wamanje.21
Okokugcina, ucwaningo lwethu luveza isakhiwo esinombono ophezulu se-TRP channel complex ye-insecticide-insecticide ebaluleke kakhulu kwezolimo—okutholwe esasingakwazi ngaphambili. Okuphawulekayo ukuthi sichaze isakhiwo kanye nomsebenzi we-insect channel emaseli abantu (HEK293S GnTi–) kunasemaseli ezinambuzane. Njengoba sibhekene nokwanda kokumelana ne-insecticide kanye nengcindezi eqhubekayo ekuphepheni kokudla kanye namagciwane, umsebenzi wethu unikeza ulwazi olubalulekile oluzokwenza kube lula ukuthuthukiswa kwe-insecticide entsha ukuze kuzuze impilo yabantu kanye nokuphepha kokudla emhlabeni jikelele. Izifundo zibonise ukuthi i-insecticide efana ne-AP iyasebenza ngokumelene nezinye izinambuzane uma isetshenziswa ngokwemiyalelo yelebula futhi inobuthi obuphansi kakhulu kubathuthi bempova abazuzisayo, okubonisa ukuphepha kwayo kwemvelo.13,16Ngaphezu kwalokho, ukuhlolwa kwezinye izinhlobo ze-AP ezitholakala komiyane kubonise ukuthi ekugcineni balahlekelwa ikhono labo lokundiza. Ukuqonda ukuthi lezi zinhlanganisela eziguqulayo zibopha kanjani ku-Nan-Iav kuzokwenza kube lula ukuguqulwa kwezinhlanganisela ezikhona noma ukuthuthukiswa kwezinhlanganisela ezintsha ukuze zisebenze kahle futhi zisebenze kangcono.okuqondileukulawulwa kwezinambuzane. Ucwaningo lwethu lubonisa ukuthi i-interface ye-Nan-Iav ARD ibalulekile hhayi nje ekulawuleni umsebenzi wamakhemikhali angokwemvelo, izibulala-zinambuzane, kanye ne-Ca2+-CaM, kodwa futhi nasekuhlanganisweni kwesiteshi. Siphakamisa ukuthi ukuphazamisa ukuhlanganiswa kwe-heterodimer ngama-molecule amancane kungaba yindlela eyingqayizivele nethembisayo yokuthuthukisa izithibi zesiteshi se-ion.
Kuma-gene ayisishiyagalombili ane-orthologous, ama-gene aphelele e-brown beetle (Halyomorpha halys) Nanchung kanye ne-Inactive akhethiwe, abonisa ukuzinza okuhle kakhulu kuma-detergent. Ama-gene ahlanganisiwe alungiselelwe i-codon ukuze aveze ukubonakaliswa komuntu futhi ahlanganiswa ku-pBacMam pCMV-DEST vector (Life Technologies) kusetshenziswa izindawo zokuvimbela i-XhoI kanye ne-EcoRI. Lokhu kuqinisekisile ukuthi ama-clone ahambisana namathegi e-C-terminal GFP-FLAG-10xHis kanye ne-mCherry-FLAG-10xHis, ahlukaniswa yi-HRC-3C protease (PPX), okuvumela ukuzimela.inkulumoAma-primer asetshenziswa ukuhlanganisa i-Nanchung kanye ne-Inactive ku-vector ye-pBacMam ayelandelayo:
Izithombe ezincane kakhulu zezinhlayiya ngazinye zitholwe ku-Titan Krios G2 transmission electron microscope (FEI) efakwe ikhamera ye-K3 kanye nesihlungi samandla se-Gatan BioQuantum. I-microscope yasebenza ku-300 keV, ngokusetha kwamandla okungu-20 eV, usayizi wephikseli yesampula ongu-1.08 Å/pixel (ukukhulisa okujwayelekile okungu-81,000x), kanye ne-gradient ye-defocus esukela ku--0.8 kuya ku--2.2 μm. Ukuqoshwa kwevidiyo kwenziwe kumafreyimu angu-40 ngomzuzwana kusetshenziswa i-Latitude S microscope (Gatan) enesilinganiso somthamo esijwayelekile esingu-25 e–px−1 s−1, isikhathi sokuvezwa esingu-2.4 s, kanye nomthamo ophelele ongaba ngu-60 e–Å−2.
Ukulungiswa kokunyakaza okubangelwa yi-beam kanye nokulinganisa umthamo kwenziwe efilimini kusetshenziswa i-MotionCor2 ku-RELION 4.061. Ukulinganiswa kwepharamitha yomsebenzi wokudlulisa umehluko (CTF) kwenziwe ku-cryoSPARC kusetshenziswa indlela yokulinganisa i-CTF esekelwe ku-patch62. Ama-photomicrograph anesisombululo sokulinganisa i-CTF ≥4 Å akhishwe ekuhlaziyweni okulandelayo. Ngokuvamile, isethi encane yama-photomicrograph angu-500–1000 yasetshenziswa ekukhetheni amaphuzu ku-cryoSPARC, kulandelwa imijikelezo eminingana yokuhlukaniswa kwe-2D ngemuva kokuhlunga ukuze kutholakale isithombe esicacile sokubhekisela sokukhethwa kwezinhlayiya ezisekelwe kuthempulethi. Izinhlayiya zabe sezikhishwa kusetshenziswa amabhokisi okubopha angama-pixel angu-64 kanye nokugoba okuphindwe kane. Imijikelezo eminingana yokuhlukaniswa kwe-2D yenziwa ukuze kususwe izigaba zezinhlayiya ezingafuneki. Imodeli yokuqala ye-3D yakhiwa kabusha kusetshenziswa ukwakhiwa kabusha kwe-ab initio futhi yahlungwa kusetshenziswa ukuhlunga okungavamile ku-cryoSPARC. Ukuhlukaniswa kwe-3D kwenziwa ku-cryoSPARC noma ku-RELION ngokusekelwe kukungafani kwe-ARD. Akukho ukungafani okuphawulekayo kwezizinda ze-membrane okwabonwa. Izinhlayiya zahlungwa kusetshenziswa izindlela ze-C1 kanye ne-C2; izinhlayiya ezinesinqumo esiphezulu se-C2 zabhekwa njengezilinganayo maqondana ne-C2 futhi zangeniswa ku-RELION ukuze kuhlungwe i-Bayesian. Izinhlayiya zabe sezidluliselwa emuva ku-cryoSPARC ukuze kuhlungwe okokugcina okungalingani kanye nendawo. Isixazululo sokugcina kanye nokubalwa kwezinhlayiya kuboniswe kuThebula 1.
Lapho sicubungula ama-pentamer e-Nan+AP, sihlole izindlela ezahlukahlukene zokuthuthukisa ukuxazululwa kwezizinda ze-membrane (ikakhulukazi isifunda se-pore), njengokususa isignali kanye nokufihla i-TMD. Kodwa-ke, le mizamo ayiphumelelanga ngenxa yokuphazamiseka okungenzeka kube kubi kakhulu esifundeni se-pore kanye nokungafani okuphelele kwe-TMD. Isixazululo sokugcina sibalwa kusetshenziswa imaski ekhiqizwa ngokuzenzakalelayo yindlela yokucubungula engalingani ku-cryoSPARC, ehlose kakhulu isifunda se-ARD. Lokhu kufinyelele ukuxazululwa okuphezulu kakhulu kunezizinda ze-membrane (ikakhulukazi isifunda se-VSLD).
Amamodeli okuqala amasha ezinhlobo ze-apo ze-Nanchung kanye ne-Inactive bugs akhiqizwa okokuqala kusetshenziswa i-Coot63, kanti amamodeli e-Nan kanye ne-Iav bugs akhiqizwa kusetshenziswa i-AlphaFold264 ukuze kutholakale izifunda ezingazethembi. Ukumodela kwe-Calmodulin kwakusekelwe ekulinganeni komzimba okuqinile kwamamodeli e-Ca2+-binding kanye ne-Ca2+-free kuma-PDB accessions 4JPZ56 kanye ne-1CFD65, ngokulandelana. Amamodeli acwengisiswe kusetshenziswa ukulungiswa kwe-spherical ukuqinisekisa i-stereochemistry efanele kanye ne-geometry enhle. I-Phosphatidylcholine, i-phosphatidylethanolamine, kanye ne-phosphatidylserine yabe isilinganiswa njengobuningi be-lipid obuchazwe kahle, kanti ama-NAM kanye nama-AP ligands abekwa kubuningi obuhambisanayo kuma-tight junctions. Amafayela omkhawulo akhiqizwa kusukela ku-SMILES string yama-isoforms kusetshenziswa i-eLBOW ku-PHENIX66. Ekugcineni, amamodeli acwengisiswe endaweni yangempela ku-PHENIX kusetshenziswa usesho lwegridi yendawo kanye nokunciphisa umhlaba wonke ngemikhawulo yesakhiwo yesibili. Iseva ye-MolProbity isetshenziswe ekuhlaziyweni kwemodeli kanye nokuhlaziywa kwesakhiwo, futhi imifanekiso yenziwe kusetshenziswa i-PyMOL kanye ne-UCSF Chimera X. 67,68,69 Ukuhlaziywa kwe-Aperture kwenziwa kusetshenziswa iseva ye-HOLE,70 kanye nemephu yokulondolozwa kwe-sequence yenziwa kusetshenziswa iseva ye-Consurf.71
Ukuhlaziywa kwezibalo kwenziwe kusetshenziswa i-Igor Pro 6.2, i-Excel Office 365, kanye ne-GraphPad Prism 7.0. Yonke idatha yokulinganisa yethulwa njengephutha elijwayelekile ± (SEM). Ukuhlolwa kwe-t komfundi (okunemisila emibili, okungabhangqiwe) kusetshenziswe ukuqhathanisa amaqembu amabili. Ukuhlaziywa kwe-One-way of variance (ANOVA) okulandelwe ukuhlolwa kwe-post hoc kukaDunnett kusetshenziswe ukuqhathanisa amaqembu amaningi. *P< 0.05, **P< 0.01, kanye no-**POkungaphansi kuka-0.001 kubhekwe njengokubalulekile ngokwezibalo kuye ngokusatshalaliswa kwedatha. Amanani e-Kd, i-Ki, kanye nezikhawu zawo zokuzethemba ezingama-95% ezingalingani kubalwe kusetshenziswa i-GraphPad Prism 10.
Ukuze uthole imininingwane eyengeziwe ngendlela yokufunda, sicela ubheke Isifinyezo Sombiko Wephothifoliyo Yemvelo esixhunywe kulesi sihloko.
Imodeli yokuqala yakhiwe kusetshenziswa amamodeli e-calmodulin avela kudathabheyisi ye-PDB 4JPZ kanye ne-1CFD. Ama-coordinates afakwe ku-Protein Data Bank (PDB) ngaphansi kwezinombolo zokungena ezingu-9NVN (Nan-Iav-CaM ngaphandle kwe-ligand), i-9NVO (Nan-Iav-CaM eboshelwe ku-nicotinamide), i-9NVP (Nan-Iav-CaM eboshelwe ku-nicotinamide kanye ne-EDTA), i-9NVQ (Nan-Iav-CaM eboshelwe ku-aphenidolpyrrolline kanye ne-calcium), i-9NVR (Nan-Iav-CaM eboshelwe ku-aphenidolpyrrolline kanye ne-EDTA), kanye ne-9NVS (Nan pentamer eboshelwe ku-aphenidolpyrrolline). Izithombe ze-cryo-electron microscopy ezihambisanayo zifakwa ku-Electron Microscopy Database (EMDB) ngaphansi kwezinombolo ezilandelayo zokungena: EMD-49844 (i-Nan-Iav-CaM ngaphandle kwe-ligand), EMD-49845 (i-Nan-Iav-CaM complex ene-nicotinamide), EMD-49846 (i-Nan-Iav-CaM complex ene-nicotinamide ne-EDTA), EMD-49847 (i-Nan-Iav-CaM complex ene-aphidopyrrolline ne-calcium), EMD-49848 (i-Nan-Iav-CaM complex ene-aphidopyrrolline ne-EDTA), kanye ne-EMD-49849 (i-Nan pentamer complex ene-aphidopyrrolline). Idatha eluhlaza yokuhlaziywa kokusebenza ivezwe kuleli phepha.
Isikhathi sokuthunyelwe: Jan-28-2026