Izibulali-zinambuzane zidlala indima ebalulekileyo ekusombululeni ukunqongophala kokutya kwihlabathi liphela nasekulweni nezifo zabantu ezithwalwa yivektha. Nangona kunjalo, ingxaki ekhulayo yokuchasana nezibulali-zinambuzane ifuna ngokungxamisekileyo ukufunyanwa kweekhompawundi ezintsha ezijolise kwiithagethi ezingasetyenziswanga kakuhle. Iziteshi ze-Insect transient receptor potential (TRPV)—iNanzhong (Nan) kunye ne-inactive (Iav)—zinokwenza iziteshi ze-heterologous (Nan-Iav) kwaye zifikelele kwiindawo ezisebenza nge-mechanosensory organs ezilawula i-geotropism, ukuva, kunye nokuqonda izinambuzane kwizinambuzane. Ezinye izibulali-zinambuzane, ezifana ne-aphidopyrrolidone (AP), zijolise kwiNan-Iav ngeendlela ezingaziwayo. I-AP iyasebenza ngokuchasene nezinambuzane ezihlabayo (i-hemipterans), ithintela ukondla ngokuphazamisa umsebenzi weefilaments. I-AP inokubopha kuphela kwiNan, kodwa yiNan-Iav kuphela enokusebenzisana ne-agonists, kubandakanya i-endogenous nicotinamide (NAM), ngaloo ndlela ibonisa umsebenzi wesiteshi. Nangona amandla eNan-Iav njengethagethi yezinambuzane, kuncinci okwaziwayo malunga nokuhlanganiswa kwesiteshi sayo, iindawo zokubopha ezilawulayo, kunye nolawulo oluxhomekeke kwiCa2+, okuthintela uphuhliso oluqhubekayo lwezinambuzane. Kolu phononongo, i-cryo-electron microscopy isetyenzisiwe ukufumanisa ulwakhiwo lweNan-Iav kwizinambuzane zeHemiptera kwimeko yokungabi na-calmodulin-ligand, kunye ne-AP kunye ne-NAM kumda we-ankyrin repeat cytoplasmic domain (ARD). Okumangalisayo kukuba, sifumanise ukuba iproteni yeNan ngokwayo inokwenza i-pentamer, eqiniswa kukusebenzisana kwe-ARD okubangelwa yi-AP. Olu phononongo lubonisa ukusebenzisana kweemolekyuli phakathi kwezinambuzane kunye ne-agonists kunye neNan-Iav, igxininisa ukubaluleka kwe-ARD kumsebenzi wesiteshi kunye nokuhlanganiswa, kwaye ihlola indlela yokusebenza komgaqo weCa2+.
Ngenxa yotshintsho olukhulu lwemozulu kwihlabathi liphela, ukungabikho kokutya okufunekayo kukhuseleko lwehlabathi yenye yezona ngxaki ziphambili zenkulungwane yama-21, nto leyo enemiphumo emibi kuluntu.1,2Ingxelo ye-World Health Organisation's State of Food Security and Nutrition in the World 2023 (SOFI) iqikelela ukuba malunga ne-2.33 yeebhiliyoni zabantu kwihlabathi liphela banengxaki yokungondleki kakuhle kokutya okuphakathi ukuya kokukhulu, ingxaki ekudala ikho.3,4Ngelishwa, kuqikelelwa ukuba i-20% ukuya kwi-30% okanye ngaphezulu yesivuno sesivuno siyalahleka minyaka le ngenxa yezinambuzane kunye neentsholongwane, kwaye ukufudumala kwehlabathi kulindeleke ukuba kubangele ukwanda kokumelana nezinambuzane kunye nokuba sesichengeni kwezityalo.4,5,6,7,8Uphuhliso lwezibulali-zinambuzane lubalulekile kungekuphela nje ekukhuseleni izityalo kwizinambuzane nokunciphisa ukusasazeka kwezifo ezithwalwa zizilwanyana, kodwa nasekulweni nezifo zabantu ezithwalwa zizilwanyana ezifana nomkhuhlane wedengue, imalariya, kunye nesifo seChagas, eziye zakwazi ukumelana nezibulali-zinambuzane.5,9,10,11
Phakathi kwezona zinto ziphambili ezijoliswe kuzo izibulali-zinambuzane ezinetyhefu kwimithambo-luvo, i-heterotetrameric TRPV channel Nanchung (Nan)-Inactive (Iav) imele udidi lweethagethi zezibulali-zinambuzane ezifunyenwe kuphela kwiminyaka elishumi edlulileyo, kuquka nezibulali-zinambuzane ezithengiswayo ezifana ne-imidacloprid kunye ne-pyraclostrobin.12,13,14I-aphidopyrrolifen (AP) yezinambuzane ezingasebenziyo ngokwe-semisynthetic yimveliso esandul’ ukuphuhliswa nethengiswayo enenxalenye ephambili yezinambuzane ezisebenzayo i-Inscalis®, ebopha i-AP kwinqanaba lomsebenzi we-subnanomolar.15I-AP ibonakalisa ubuthi obuphantsi kakhulu kwizinambuzane ezithumela impova, izinambuzane eziluncedo, kunye nezinye izinto eziphilayo ezingajoliswanga kuzo, kwaye xa isetyenziswa ngokwemiyalelo yeleyibhile, inokunciphisa uxinzelelo lokuxhathisa ezinye izibulali-zinambuzane.16,17,18UNan kunye no-Iav zisasazeke kakhulu kwiintlobo zezinambuzane, zibonakaliswa kuphela kwi-chordal stretch receptor neurons ze-antennae kunye nemilenze, kwaye zibalulekile ekuveni, ukuqonda amandla adonsela phantsi komhlaba, kunye nokuqonda iproprioception.13,16,19,20,21,22I-AP, imidacloprid, kunye ne-pyraclostrobin zivuselela i-Nan-Iav complex ngendlela eyahlukileyo, ekugqibeleni zithintela ukudluliselwa kwesignali ye-proprioceptive.13,16,23Kwizinambuzane ezifunxayo (ii-hemipterans) ezifana nee-aphids kunye neempukane ezimhlophe, ukulahleka kweproprioception kuphazamisa amandla azo okutya, ekugqibeleni kukhokelela ekufeni.13,24Okunomdla kukuba, i-AP ibonisa ulwalamano oluphezulu kwi-Nan-Iav complex kunye nolwalamano oluphantsi kwi-Nan yodwa. Ukubopha i-AP kwi-Nan-Iav kubangela umsinga wombane, kodwa ukubopha kwi-Nan yodwa akukhuthazi umsebenzi wesiteshi. I-Iav ngokwayo ayibopheleli kwi-AP kwaphela.16Oku kuthetha ukuba iNan kunye neIav zinokubophelelana ukuze zenze ii-complexes ezahlukeneyo zeNan-Iav channel (umz., ngee-ratios ezahlukeneyo ze-stoichiometric okanye amalungiselelo ahlukeneyo ngaphakathi kwe-stoichiometric ratio efanayo) okanye i-AP inokubophelelana kwiindawo ezininzi. Ngaphezu koko, i-agonist yendalo i-nicotinamide (NAM) ibophelela kwi-Drosophila Nan-Iav nge-micromolar affinity, ebonisa iziphumo ezifana nezo ze-aphids (AP) in vitro.16,25kunye nokuthintela ukuzala nokutyisa ii-aphid, ekugqibeleni okukhokelela ekufeni kwazo25,26. Le datha iphakamisa imibuzo emininzi. Umzekelo, akukacaci ukuba yenziwa njani i-heterodimer ye-Nan-Iav, zeziphi iindawo zokubopha ezisetyenziselwa ukuguqula iimolekyuli ezincinci, kunye nendlela ezi molekyuli zincinci ezilawula ngayo umsebenzi wesiteshi ngokuthintela i-proprioception. Ngaphezu koko, izizathu zokuba i-Nan ngokwayo ingasebenzi kwaye ine-affinity ephantsi ye-AP, ngelixa i-heterodimer ye-Nan-Iav isebenza kwaye ibopha i-AP nge-affinity ephezulu, azikacaci. Okokugqibela, kuncinci okwaziwayo malunga nolawulo oluxhomekeke kwi-Ca2+ lomsebenzi we-Nan-Iav kunye nendlela edityaniswe ngayo kwiinkqubo zokubonisana ze-neuronal.. 13,21
Kolu phononongo, sidibanisa i-cryo-electron microscopy, i-electrophysiology, kunye neendlela zokubopha i-radioligand, sicacise ukuhlanganiswa kwe-Nan-Iav kunye nendlela yokubopha kwayo kwii-molecule regulators ezincinci. Ngaphezu koko, sifumene i-calmodulin (CaM) ebotshelelwe ngokudibeneyo kwi-Iav kunye nee-pentamers ze-Nan ezizinzileyo ze-AP. Ezi ziphumo zibonelela ngengqiqo ebalulekileyo malunga nokulawulwa kwee-calcium ions kwiitshaneli, ukuhlanganiswa kwetshaneli, kunye nezinto ezimisela ukuhambelana kwe-ligand. Okubaluleke ngakumbi, siqinisekisile ukuba i-ARD idlala indima ephambili kwezi nkqubo. Uphononongo lwethu lweendlela ezipheleleyo zezinambuzane ezibotshelelwe kwii-pesticides zezolimo ezifanelekileyo27, 28, 29ivula amathuba ophuhliso lweshishini lee-pesticide, iphucula ukusebenza kakuhle kunye nokuchaneka kwee-pesticide, kwaye ivumela ukusetyenziswa kwee-compounds ezijoliswe kwi-TRPV kwezinye iintlobo zezilwanyana ukujongana nokhuseleko lokutya lwehlabathi kunye nokusasazeka kwezifo ezithwalwa yizilwanyana.
Sikwafumanise ukuba iNan-Iav ilawulwa yiCa2+, kwaye indlela yokulawula ilawulwa yiCaM ebotshelelwe ngokwendalo. Okubalulekileyo kukuba, le ndlela yokulawula iNav yiCaM exhomekeke kwiCa2+ yahlukile kakhulu kwindlela yokulawula ezinye iitshaneli ze-ion (umz., iitshaneli zeNa+ ezigalelwe yi-voltage kunye neetshaneli zeTRPV5/6)52,53,54,55,56,57Kwitshaneli ye-Nav1.2, idomeyini ye-C-terminal ye-CaM ngokwe-heli inxulumana nedomeyini ye-C-terminal (CTD), kwaye i-Ca2+ ibangela ukubopha idomeyini yayo ye-N-terminal kwinxalenye ekude ye-CTD.56Kwitshaneli ye-TRPV5/6, idomeyini ye-C-terminal ye-CaM ibopha kwi-CTH, kwaye i-Ca2+ ibangela ukwandiswa okuphezulu kwedomeyini yayo ye-N-terminal ukuya kwi-pore, ngaloo ndlela ithintela ukungena kwe-cation.53,54. Sicebisa imodeli yomsebenzi olawulwa yiCa2+ weNan-Iav-CaM (Umzobo 4h). Kule modeli, i-N-terminal domain yeCaM ibopha rhoqo kwi-C-terminal domain (CTH) yeIav. Kwimeko yokuphumla (uxinzelelo oluphantsi lwe[Ca2+]), i-C-terminal domain yeCaM isebenzisana neNan, izinzisa ukwakheka kwe-ARD kwaye ngaloo ndlela ikhuthaza ukuvulwa kwetshaneli. Ukubopha i-agonist/i-insecticide kwitshaneli kubangela ukuvulwa kwe-pore, okukhokelela ekungeneni kweCa2+. I-Ca2+ emva koko ibopha kwiCaM, okubangela ukwahlukana kwe-C-terminal domain kwi-ARD yeNan. Ngenxa yokuba ukuvala ukubopha kweCaM ngokusisiseko kuphelisa isiphumo sokuthintela seCa2+, oku kwahlukana kuguqula ukuhamba kwe-ARD, ngaloo ndlela kubangele ukuthintela okanye ukungazinzisi okuxhomekeke kwiCa2+. Ukubuyiselwa ngokukhawuleza kwe-channel currents emva kokususwa kwe-calcium ion (Umzobo 4g) kubonisa ukuba le ndlela inceda ukuphendula ngokukhawuleza kwimiqondiso ye-neuronal elawulwa yiCa2+. Ngaphezu koko, ummandla we-C-terminal wase-Iav, ongaqondwa kakuhle, uxelwe ukuba udlala ezinye iindima ekujoliseni iziteshi kunye nomthetho wangoku.21
Okokugqibela, uphando lwethu lubonisa isakhiwo esigqibeleleyo se-TRP channel complex ye-insecticide-insecticide ebalulekileyo kwezolimo—ukufunyanwa esasingakwazi ngaphambili. Okuphawulekayo kukuba, sichaze isakhiwo kunye nomsebenzi we-insect channel kwiiseli zabantu (HEK293S GnTi–) endaweni yeeseli zezinambuzane. Xa sijongene nokwanda kokumelana ne-insecticide kunye noxinzelelo oluqhubekayo kukhuseleko lokutya kunye ne-pathogens, umsebenzi wethu ubonelela ngolwazi olubalulekileyo oluya kwenza kube lula ukuphuhliswa kwe-insecticide ezintsha ukuze kuzuze impilo yabantu kunye nokhuseleko lokutya lwehlabathi. Izifundo zibonise ukuba i-insecticide ezifana ne-AP ziyasebenza ngokuchasene nezinye izinambuzane xa zisetyenziswa ngokwemiyalelo yeleyibhile kwaye zinetyhefu ephantsi kakhulu kwi-pollinators eziluncedo, nto leyo ebonisa ukhuseleko lwazo kwindalo esingqongileyo.13,16Ngaphezu koko, uvavanyo lwezinye ii-AP derivatives kwiingcongconi lubonise ukuba ekugqibeleni ziyalahlekelwa kubuchule bazo bokubhabha. Ukuqonda indlela ezi compounds zokuguqula ezidibana ngayo neNan-Iav kuya kwenza kube lula ukuguqulwa kwee compounds ezikhoyo okanye ukuphuhliswa kwee compounds ezintsha ukuze zisebenze ngakumbi kwaye zisebenze ngakumbi.ngqoulawulo lwezinambuzane. Uphononongo lwethu lubonisa ukuba ujongano lwe-Nan-Iav ARD lubalulekile kungekuphela nje ekulawuleni umsebenzi weekhompawundi zendalo, izibulali-zinambuzane, kunye ne-Ca2+-CaM, kodwa nakwindibano yesiteshi. Sicebisa ukuba ukuphazamisa ukuhlanganiswa kwe-heterodimer ngeemolekyuli ezincinci kunokuba yindlela eyahlukileyo nethembisayo yokuphuhlisa izithinteli zesiteshi se-ion.
Kwizakhi zofuzo ezisibhozo ze-orthologous, kukhethwe izakhi zofuzo ezipheleleyo ze-brown beetle (Halyomorpha halys) Nanchung kunye ne-Inactive, ezibonisa uzinzo oluhle kakhulu kwiisepha. Izakhi zofuzo ezidityanisiweyo zenziwe ngcono nge-codon ukuze zikwazi ukubonakalisa ubuntu kwaye zadityaniswa kwi-pBacMam pCMV-DEST vector (Life Technologies) kusetyenziswa iindawo zokuthintela i-XhoI kunye ne-EcoRI. Oku kuqinisekisile ukuba ii-clones zihambelana neethegi ze-C-terminal GFP-FLAG-10xHis kunye ne-mCherry-FLAG-10xHis, eziqhekeka yi-HRC-3C protease (PPX), ezivumela ukuzimela.intethoIiprimer ezisetyenzisiweyo ukuhlanganisa iNanchung kunye neInactive kwi-pBacMam vector zezi zilandelayo:
Imifanekiso ye-microscopic yamasuntswana ngamanye ifunyenwe kwi-Titan Krios G2 transmission electron microscope (FEI) exhotyiswe ngekhamera ye-K3 kunye nesihluzo samandla seGatan BioQuantum. I-microscope isebenze kwi-300 keV, kunye nokuseta amandla kwe-20 eV, ubungakanani be-pixel yesampulu ye-1.08 Å/pixel (ukwandiswa okuqhelekileyo kwe-81,000x), kunye ne-defocus gradient ukusuka kwi--0.8 ukuya kwi--2.2 μm. Ukurekhodwa kwevidiyo kwenziwe kwiifreyimu ezingama-40 ngomzuzwana kusetyenziswa i-Latitude S microscope (Gatan) enesantya sedosi esiqhelekileyo se-25 e–px−1 s−1, ixesha lokuvezwa kwe-2.4 s, kunye nedosi iyonke ye-60 e–Å−2.
Ukulungiswa kwentshukumo ebangelwa yimitha kunye nobunzima bedosi kwenziwe kwifilimu kusetyenziswa iMotionCor2 kwiRELION 4.061. Uqikelelo lweparameter yomsebenzi wokudlulisa umahluko (CTF) lwenziwe kwi-cryoSPARC kusetyenziswa indlela yokulinganisa i-CTF esekelwe kwi-patch62. Iifotomicrographs ezinesisombululo sokufakela i-CTF ≥4 Å azizange zikhutshwe kuhlalutyo olulandelayo. Ngokwesiqhelo, icandelo elincinci leefotomicrographs ezingama-500–1000 lasetyenziswa ekukhetheni amanqaku kwi-cryoSPARC, kulandele imijikelo eliqela yokuhlelwa kwe-2D emva kokucoca ukuze kufunyanwe umfanekiso ocacileyo wokukhethwa kwe-particle esekwe kwitemplate. Amasuntswana emva koko akhutshwa kusetyenziswa iibhokisi ezibophelelayo ze-64-pixel kunye ne-binning ephindwe kane. Imijikelo eliqela yokuhlelwa kwe-2D yenziwe ukususa iindidi zeesuntswana ezingafunekiyo. Imodeli yokuqala ye-3D yakhiwa ngokutsha kusetyenziswa ukwakhiwa kwakhona kwe-ab initio kwaye yacocwa kusetyenziswa ukuphuculwa okungekuko kwi-cryoSPARC. Ukuhlelwa kwe-3D kwenziwe kwi-cryoSPARC okanye kwi-RELION ngokusekelwe kukungafani kwe-ARD. Akukho kungafani okubalulekileyo kweendawo ze-membrane eziboniweyo. Amasuntswana acociwe kusetyenziswa iindlela ze-C1 kunye ne-C2; amasuntswana anesisombululo esiphezulu se-C2 athathwa njengalinganayo ngokubhekiselele kwi-C2 aze angeniswa kwi-RELION ukuze kucocwe i-Bayesian. Amasuntswana aphinde adluliselwe kwi-cryoSPARC ukuze kucocwe okokugqibela okungalinganiyo kunye nokwasekuhlaleni. Isisombululo sokugqibela kunye nokubalwa kwamasuntswana kuboniswe kwiTheyibhile 1.
Xa sicubungula ii-pentamers zeNan+AP, sihlolisise iindlela ezahlukeneyo zokuphucula isisombululo seendawo ze-membrane (ingakumbi indawo ye-pore), ezifana nokukhupha isignali kunye nokufihla i-TMD. Nangona kunjalo, le mizamo ayiphumelelanga ngenxa yokuphazamiseka okunokwenzeka kakhulu kwindawo ye-pore kunye nokungafani ngokubanzi kwe-TMD. Isisombululo sokugqibela sibalwe kusetyenziswa imaski eveliswa ngokuzenzekelayo yindlela yokucubungula engalinganiyo kwi-cryoSPARC, ikakhulu ijolise kummandla we-ARD. Oku kufezekise isisombululo esiphezulu kakhulu kuneso seendawo ze-membrane (ingakumbi indawo ye-VSLD).
Iimodeli zokuqala ezintsha ze-apoforms ze-Nanchung kunye ne-Inactive bugs zaqala ukuveliswa kusetyenziswa i-Coot63, kwaye iimodeli ze-Nan kunye ne-Iav bugs zadalwa kusetyenziswa i-AlphaFold264 ukuchonga iindawo ezingaqinisekiyo. Imodeli ye-Calmodulin yayisekelwe kwi-rigid-body fit yeemodeli ze-Ca2+-binding kunye ne-Ca2+-free kwi-PDB accessions 4JPZ56 kunye ne-1CFD65, ngokwahlukeneyo. Iimodeli zacocwa kusetyenziswa i-spherical refinement ukuqinisekisa i-stereochemistry echanekileyo kunye ne-geometry elungileyo. I-Phosphatidylcholine, i-phosphatidylethanolamine, kunye ne-phosphatidylserine emva koko zalinganiswa njenge-lipid densities ezichazwe kakuhle, kwaye ii-NAM kunye ne-AP ligands zabekwa kwi-densities ehambelanayo kwi-tight junctions. Iifayile ze-Constraint zenziwe kwi-SMILES string ye-isoforms kusetyenziswa i-eLBOW kwi-PHENIX66. Ekugqibeleni, iimodeli zacocwa kwindawo yokwenyani kwi-PHENIX kusetyenziswa i-local grid search kunye ne-global minimization kunye ne-secondary structure limits. Iseva yeMolProbity isetyenziselwe ukuhlaziya imodeli kunye nohlalutyo lwesakhiwo, kwaye imifanekiso yenziwe kusetyenziswa iPyMOL kunye ne-UCSF Chimera X. 67,68,69 Uhlalutyo lwe-Aperture lwenziwe kusetyenziswa iseva ye-HOLE,70 kwaye imephu yolondolozo lwe-sequence yenziwe kusetyenziswa iseva yeConsurf.71
Uhlalutyo lwezibalo lwenziwe kusetyenziswa i-Igor Pro 6.2, i-Excel Office 365, kunye ne-GraphPad Prism 7.0. Yonke idatha yobungakanani iboniswa njengempazamo ephakathi ± standard (SEM). Uvavanyo lwe-t lomfundi (olunemisila emibini, olungabhangqwanga) lusetyenzisiwe ukuthelekisa amaqela amabini. Uhlalutyo lwe-One-way of variance (ANOVA) olulandelwe luvavanyo lwe-post hoc lukaDunnett lusetyenzisiwe ukuthelekisa amaqela amaninzi. *P< 0.05, **P< 0.01, kunye ***PI-<0.001 zithathwe njengezibalulekileyo ngokwezibalo ngokuxhomekeke kulwabiwo lwedatha. Amaxabiso e-Kd, e-Ki, kunye ne-95% confidence intervals zazo ezingafaniyo zibalwa kusetyenziswa i-GraphPad Prism 10.
Ukuze ufumane iinkcukacha ezithe vetshe malunga nendlela yokufunda, nceda ujonge isishwankathelo seNgxelo yePotfoliyo yeNdalo edityaniswe kweli nqaku.
Imodeli yokuqala yakhiwe kusetyenziswa iimodeli ze-calmodulin ezivela kwiidathabheyisi ze-PDB 4JPZ kunye ne-1CFD. Ezi coordinates zifakwe kwiProtein Data Bank (PDB) phantsi kwamanani okungena kwi-9NVN (i-Nan-Iav-CaM ngaphandle kwe-ligand), i-9NVO (i-Nan-Iav-CaM ibotshelelwe kwi-nicotinamide), i-9NVP (i-Nan-Iav-CaM ibotshelelwe kwi-nicotinamide kunye ne-EDTA), i-9NVQ (i-Nan-Iav-CaM ibotshelelwe kwi-aphenidolpyrrolline kunye ne-calcium), i-9NVR (i-Nan-Iav-CaM ibotshelelwe kwi-aphenidolpyrrolline kunye ne-EDTA), kunye ne-9NVS (i-Nan pentamer ibotshelelwe kwi-aphenidolpyrrolline). Imifanekiso ye-cryo-electron microscopy ehambelanayo ibekwe kwi-Electron Microscopy Database (EMDB) phantsi kwala manani alandelayo okufikelela: EMD-49844 (i-Nan-Iav-CaM engenalo i-ligand), EMD-49845 (i-Nan-Iav-CaM complex ene-nicotinamide), EMD-49846 (i-Nan-Iav-CaM complex ene-nicotinamide kunye ne-EDTA), EMD-49847 (i-Nan-Iav-CaM complex ene-aphidopyrrolline kunye ne-calcium), EMD-49848 (i-Nan-Iav-CaM complex ene-aphidopyrrolline kunye ne-EDTA), kunye ne-EMD-49849 (i-Nan pentamer complex ene-aphidopyrrolline). Idatha eluhlaza yohlalutyo olusebenzayo iboniswe kweli phepha.
Ixesha leposi: Jan-28-2026