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Isiteshi sephrothoni esine-voltage ye-Hv1 siyinkimbinkimbi ye-dimeric ehlanganisa izizinda ezimbili ezizwa amandla kagesi (i-VSD), ngasinye sazo esiqukethe indlela ye-proton enesango.I-Dimerization ilawulwa isizinda sekhoyili ekhoyili ye-cytoplasmic. isimo esivulekile kuwo womabili ama-VSD kwaziwa ukuthi senzeka ngokubambisana; nokho, kuncane okwaziwayo mayelana nezinqubo eziyisisekelo.Izixhumanisi ze-Intersubunit zidlala indima ebalulekile ezinqubweni ze-allosteric; kodwa-ke, lokho kuxhumana akutholakalanga eziteshini ezivulekile ze-Hv1.Lapha sibonisa ukuthi okuphuma ku-2-guanidinothiazole vimba ama-VSD amabili e-Hv1 ngendlela yokubambisana futhi asebenzise enye yalezi zinhlanganisela njenge-probe yokuhlanganisa i-allosteric phakathi kwama-subunits avulekile.Sithole ukuthi i-extracellular Ukuphela kocezu lokuqala lwe-transmembrane lwe-VSD lwakha isixhumi esibonakalayo se-intersubunit esilamula ukuhlangana phakathi kwamasayithi abophayo, kuyilapho isizinda sekhoyili ehlanganisiwe asihileleki ngokuqondile kule nqubo.Sithole nobufakazi obuqinile bokuthi isihlungi sesiteshi esikhethayo se-proton silawula ukusebenzisana okubophezela okuvimbelayo. .
Iziteshi ze-proton ezinamasango kagesi zidlala indima ebalulekile ezinhlobonhlobo zezinto eziphilayo, kusukela ku-phytoplankton kuya kubantu1.Emangqamuzana amaningi, lezi ziteshi zilamula ukuphuma kwe-proton kusuka kulwelwesi lwe-proton futhi zilawula umsebenzi we-NADPH oxidase.Ukuphela kwesiteshi seproton esine-voltage-gated kubantu. i-Hv1, ewumkhiqizo we-HVCN1 gene2,3.Hv1 (aka VSOP) iboniswe ukuthi idlala indima ekwandeni kwe-B cell4, ukukhiqizwa kwezinhlobo ze-oksijini ezisebenzayo yi-innate immune system5,6,7,8, i-sperm cell. i-motility9 kanye nokulawulwa kwe-pH ye-airway surface fluid10. Lesi siteshi sihilelekile eziningana Ezicindezelekile kakhulu ezinhlotsheni zomdlavuza ezifana ne-B-cell malignancies4,11 kanye nomdlavuza webele kanye nomdlavuza we-colorectal12,13.Umsebenzi oweqile we-Hv1 watholakala ukwandisa amandla e-metastatic amangqamuzana omdlavuza 11, 12 .Engqondweni, i-Hv1 ivezwa nge-microglia, futhi umsebenzi wayo uboniswe ukwandisa ukulimala kobuchopho kumamodeli we-ischemic stroke.
Iphrotheni ye-Hv1 iqukethe isizinda se-voltage-sensing (VSD), esinezingxenye ezine ze-transmembrane ezibizwa nge-S1 kuya ku-S414. I-VSD ifana nezizinda ezihambisanayo zamashaneli e-voltage-gated Na+, K+ kanye ne-Ca2+ kanye nama-phosphatase azwela amandla kagesi, njenge-CiVSP evela I-Ciona gutis15.Kulawa amanye amaprotheni, i-C-terminus ye-S4 inamathiselwe kumojula ye-effector, isizinda se-pore noma i-enzyme.Ku-Hv1, i-S4 ixhunywe kusizinda sekhoyili ekhoyili (CCD) etholakala ohlangothini lwe-cytoplasmic lolwelwesi. .Isiteshi siyinkimbinkimbi ye-dimeric ehlanganisa ama-VSD amabili, ngalinye liqukethe i-gated proton permeation pathway16,17,18.Lawa mayunithi amabili e-Hv1 atholwe evuleke ngokubambisana19,20,21,22, okuphakamisa ukuthi ukuhlangana kwe-allosteric kanye nokusebenzisana kwe-inter-subunit kuyadlala indima ebalulekile enqubweni yokungena.I-interface phakathi kwama-subunits ngaphakathi kwesizinda sekhoyili ehlanganisiwe ichazwe kahle ngoba izakhiwo zekristalu zezizinda ezimbili ezihlukene ziyatholakala22,23.Ngakolunye uhlangothi, ukuxhumana phakathi kwe-VSD ngaphakathi kwe-membrane ayikho. kuqondwe kahle.Isakhiwo sekristalu sephrotheni ye-chimeric ye-Hv1-CiVSP ayinikezi ulwazi mayelana nalesi sixhumi esibonakalayo, njengoba inhlangano ye-trimeric yenxanxathela yesiteshi esicwebezelayo ingase ibe umphumela wokushintshwa kwe-CCD yomdabu ye-Hv1 yi-yeast leucine uziphu GCN424.
Ucwaningo lwakamuva lwenhlangano ye-subunit yesiteshi se-Hv1 luphethe ngokuthi ama-helice amabili e-S4 ashintshela ku-CCD ngaphandle kokuphazamiseka okukhulu kwesakhiwo sesibili, okuholela kuma-helice amade aqala kusukela kulwelwesi kanye nephrojekthi ku-cytoplasm. lolu cwaningo luphakamisa ukuthi ama-Hv1 VSD axhumane eceleni kwengxenye ye-S4. Nokho, ezinye izifundo ziphakamise ezinye izindlela zokusebenzelana phakathi kwama-VSD. Lokhu kuxhumana kuhlanganisa izingxenye ze-S1 17, 21, 26 kanye neziphetho zangaphandle ze-S2 ingxenye 21.Isizathu esingaba isizathu sokungqubuzana imiphumela yalezi zifundo iwukuthi ukuhlangana kwe-allosteric phakathi kwe-VSD kwahlolisiswa maqondana nenqubo yokungena, okuncike ezifundeni ezivaliwe nezivulekile, futhi ukuxhumana phakathi kwama-VSD kungahluka ezinguqukweni ezihlukene zesimo ekuhambisaneni.
Lapha, sithole ukuthi i-2-guanidinothiazole ivimbela iziteshi ze-Hv1 ngokubophezela ngokuhlanganyela kuma-VSD amabili avuliwe, futhi yasebenzisa enye yezinhlanganisela, i-2-guanidinobenzothiazole (GBTA), ukuhlola ukusebenzisana phakathi kwamayunithi amancane endaweni evulekile. esibonakalayo.Sithole ukuthi ijika elibophezelayo le-GBTA lingachazwa kahle ngemodeli yobuningi lapho ukubophezela kwe-inhibitor kuyunithi eyodwa kuphumela ekwenyukeni kokuhlangana okubophezelayo kwe-subunit eseduze.Sithole futhi ukuthi izinsalela ze-D112, izihlungi zokukhetha ze isiteshi 27, 28 kanye nengxenye yesayithi elibophezelayo eliphuma ku-guanidine 29 lilawula i-GBTA ebophezelayo ukusebenzisana. lamula ukuhlangana kwe-allosteric phakathi kwezindawo ezibophezela i-GBTA.Ngokuphambene, sithola ukuthi ucezu lwe-S1 luyingxenye yesixhumi esibonakalayo phakathi kwamayunithi amancane futhi siphakamisa ukuhlelwa kwama-VSD aseduze nokuphela kwe-extracellular ye-S4 helix kude nendawo ye-dimer ukuze kuvunyelwe. ucezu lwe-S1 luzoba sesimweni esivulekile.
Ama-molecule amancane e-Hv1 inhibitors awusizo njengezidakamizwa ezilwa nomdlavuza kanye nama-neuroprotective agents.Nokho, kuze kube manje, izinhlanganisela ezimbalwa zikwazile ukuvimbela isiteshi30,31,32,33.Phakathi kwazo, i-2-guanidinobenzimidazole (2GBI, inhlanganisela [1] kuFig. . I-1a) kanye nokuphuma kwayo kutholakale ukuthi kuvimbela i-VSD29,32 ukungena kwama-proton ngokusebenzisa isiteshi.Ukubophezela kwamakhemikhali anjalo kucatshangwa ukuthi kwenzeka ngokuzimela kuma-subunits amabili avulekile.2-guanidinobenzothiazole (GBTA, i-compound [2] ku-Figure 1a) kwaba ngaphambili kuboniswe ukuvimbela i-Hv1 cishe ngokuphumelelayo njenge-2GBI lapho ihlolwa ekuhlanganiseni kwe-200 μM (Umfanekiso 1b). umbhalo).Sinqume amajika empendulo yokugxilisa ingqondo kokuphuma kokunye kwe-thiazole (i-GBTA nezinhlanganisela [3], [6] kanye ne- [11], i-Fig. 1c) futhi sathola ukuthi ayeqina kakhulu kunalawo we-2GBI. Ama-coefficients we-Hill (h) kokuphuma kwe-thiazole kusuka ku-1.109 ± 0.040 kuya ku-1.306 ± 0.033 (Fig. 1c kanye ne-Supplementary Fig. 1). Ngokuphambene, i-Coefficient ye-Hill ye-2GBI yayingu-0.975 ± 0.024 29, Fig. 2a kanye ne-Supplementary Fig. 1).I-coefficient ye-Hill ngenhla kokuthi 1 ibonisa ukusebenzisana okubophezelayo.Ngoba iyunithi ngayinye ye-Hv1 ine-inhibitor yayo- indawo ebophayo,29,32 sicabange ukuthi ukubophezelwa kokuphuma kwe-thiazole kuyunithi eyodwa kungase kuthuthukise ukubopha kwe-molecule yesibili ye-inhibitor engxenyeni eseduze.I-GBTA yayiyinhlanganisela yokuhlola ene-coefficient ye-Hill ephakeme kakhulu.Ngakho-ke, sikhethe le nhlanganisela ukuthi funda ngokuqhubekayo indlela yokubopha i-synergy futhi yasebenzisa i-2GBI njengokulawula okungekuhle kwereferensi.
(a) Izinhlanganisela zokuhlola: [1] Inkomba ye-Hv1 inhibitor 2-guanidino-benzimidazole (2GBI).[2] I-2-guanidino-benzothiazole (GBTA), [3] (5-trifluoromethyl-1,3-benzothiazol-2-yl)guanidine, [4] naphtho[1,2-d][1, 3] Thiazol-2-yl -guanidine, [5](4-methyl-1,3-thiazol-2-yl)guanidine, [6](5-bromo-4-methyl-1,3-thiazol- 2-yl)guanidine, [7] famotidine, [8] 2-guanidino-5-methyl-1,3-thiazole-4-carboxylic acid ethyl ester, [9] 2-guanidino-4-methyl Ethyl-1,3-thiazole-5-carboxylate, [10] ](2-guanidino-4-methyl-1,3-thiazol-5-yl)ethyl acetate, [11]1-[4-(4 -Chlorophenyl)-1,3-thiazol-2-yl]guanidine, [ 12]1-[4-(3,4-dimethoxyphenyl)-1,3-thiazol-2-yl]guanidine .(b) Ukuvinjwa komsebenzi we-Hv1 womuntu nge-guanidinothiazoles ekhonjiwe kanye nenhlanganisela yereferensi engu-2GBI (amabha eluhlaza okwesibhakabhaka) .I-Hv1 proton currents ikalwe ngama-plaque angaphakathi-ngaphandle e-Xenopus oocyte ukuphendula ukuchithwa kwepolar kusuka kumandla okubamba angu- -80 mV kuya ku-+120 mV.I-inhibitor ngayinye yengezwe kubhavu ekuhlanganiseni okungu-200 μM.pHi = pHo = 6.0 .Idatha isho ±SEM (n≥4).(c) Ukuvinjelwa okuncike ekugxilweni kwe-Hv1 yomuntu ngezinhlanganisela [2], [3], [6] kanye ne- [11].Iphuzu ngalinye limelela isilinganiso sokuvinjelwa ± SD sika-3 kuya ku-15 izilinganiso. Ulayini uwukulingana kwe-Hill okusetshenziselwa ukuthola amanani abonakalayo e-Kd abikwe Kuthebula Lokwengeza 1.Ama-coefficients e-Hill anqunywe kusukela ekulinganeni okubikwe kokuthi I-Supplementary Fig. 1: h(1) = 0.975 ± 0.024 h(2) = 1.306 ± 0.033, h(3) = 1.25 ± 0.07, h(6) = 1.109 ± 0.040, h (11) = 1.179 ± 0.036 (bona Izindlela).
(a,b) Ihlanganisa i-2GBI ne-GBTA evinjiwe i-dimeric ne-monomeric Hv1 ngendlela encike ekugxiliseni. Iphuzu ngalinye limelela isilinganiso sokuvinjwa esingu-± SD sezilinganiso ezingu-3 kuya kwezingu-8 futhi ijika lilingana ne-Hill.Ama-coefficients e-Hill (h) aboniswe ama-histograms afakiwe anqunywa kusukela ekulinganeni okubikwe ku-Supplementary Figs 3 kanye no-4.Impendulo yokugxilisa ingqondo ye-GBTA eboniswe ku-(a) iyafana naleyo ekuFig. 1c.Bheka Ithebula Lokwengeza 1 ukuze uthole amanani abonakalayo e-Kd.(c) Ukumodela ukubophezela ngokubambisana kwe-GBTA ku-dimeric Hv1. Ulayini omnyama oqinile umele ukulingana kwedatha yokuhlola ngesibalo (6), esichaza imodeli ebophezelayo eboniswe kokuthi (d). -i-dissociation equilibrium curves yemicimbi ebophezelayo yokuqala neyesibili, ngokulandelanayo (I-Sub 1: OO + B ⇄ BO*, Kd1 = 290 ± 70 μM; I-Sub 2: BO* + B ⇄ B*O*, Kd2 = 29.3 ± 2.5 μM ).(d) Umdwebo oyisikimu wendlela ehlongozwayo yebhulokhi ye-Hv1. Esimeni se-GBTA, ukubophezela kuyunithi eyodwa evulekile kukhulisa ukuhambisana kweyunithi evulekile eseduze (Kd2 Iziteshi ze-Hv1 zingenziwa zibe monomers ngokushintsha izizinda zazo ze-N- ne-C-terminal cytoplasmic ngezingxenye ezihambisanayo ze-Ciona Intestinalis voltage-sensitive phosphatase phosphatase CiVSP (Hv1NCCiVSP)18,34. ziqhathanise naleyo ye-dimeric channel (wild-type) inhibition (Fig. 2a,b).Sithole ukuthi umehluko ekubambisaneni okubophezelayo phakathi kwezinhlanganisela ezimbili waqedwa ku-monomeric Hv1, okusekela incazelo yokuthi ukubophezela kwe-GBTA kuyunithi eyodwa kwandisa ukuhambisana kwelinye iyunithi encane ye-inhibitor.Sicabange ukuthi ukubophezela kwe-GBTA kuyunithi eyodwa ye-Hv1 kungase kuholele ekuhleleni kabusha isayithi elibophezelayo (inducing fit35), okungase kuqalise ukuhlelwa kabusha kwesizinda esingenalutho esibophezelayo seyunithi eseduze, okuholela ekwenyukeni kokubophezela. ubuhlobo.
Ukuze sichaze ngokwesilinganiso ukubophezela ngokubambisana kwe-GBTA esiteshini, sisebenzise imodeli lapho noma iyiphi iyunithi ingabopha i-molecule yokuqala ye-inhibitor elandela ukusabela kwe-bimolecular nge-dissociation engaguquki i-Kd1 (Fig. 2c, Sub 1, OO+ B ⇆ BO* ). Ukubophezela kubangela ukuthi isiteshi sithathe isimo lapho amayunithi angenalutho asele ebophezela ku-inhibitor kulandela ukusabela okuyingqayizivele kwe-bimolecular nge-dissociation constant Kd2, lapho i-Kd2 Ulayini omnyama oqinile kuMfanekiso 2c ulingana nemodeli yesibalo kwijika lempendulo yokugxilisa ingqondo yokuhlola, enikeza i-Kd1 engu-~290 μM kanye ne-Kd2 engu-~29 μM (isigaba sezindlela, isibalo (6)). ukubophezela kwe-2GBI, lapho i-Kd2 ≈ Kd1 = Kd (Fig. 2d).Ku-monomeric Hv1, kunesayithi eyodwa kuphela ebophayo kanye ne-Kdm eyodwa (O° + B ⇆ B°).Endabeni ye-GBTA, i-Kdm icishe ibe ngu-54 μM, efana kakhulu ne-Kd1 kune-Kd2 (Fig. 2d). Ngamanye amazwi, isayithi elibophezelayo le-monomer likulungiselelo (O°) elifana kakhulu nesimo sokuhlangana okuphezulu (BO*) kune-low- isimo se-affinity (OO) se-dimer, cishe kungenxa yokuqedwa kwesixhumi esibonakalayo phakathi kwamayunithi amancane.
Njengoba kuboniswe ngaphambilini ku-2GBI32, sithole ukuthi i-GBTA icindezele amaza e-Hv1 ngokuvimba isiteshi lapho sivuliwe kunokuthi isenze kube nzima ukusivula (I-Supplementary Fig. 2a,b,d).2GBI iyaziwa nangokuthi ifake imisinga yomsila ebola kancane. ekuphenduleni i-membrane repolarization, kodwa lesi senzakalo asizange sibonwe ku-GBTA (I-Supplementary Fig. 2c) .Ebukhoneni be-Hv1 ukungasebenzi phambi kwe-2GBI, amasango ku-subunit ngayinye awakwazi ukuvala kuze kube yilapho i-blocker ishiya indawo yokubopha (i-“ foot gate” mechanism), kanye ne-2GBI ikhulula kancane kunamasango avalwayo.Uma iyunithi eyodwa ye-Hv1 ivuliwe futhi ivaliwe, kuyilapho iyunithi eseduze ihlala ivinjiwe, ukukhulula ama-molecule e-2GBI asele kuba kancane (i-blocker capture).Izinhlobo zamashaneli eziphila isikhathi eside iyunithi eyodwa kuphela evinjiwe ephethe amaphrothoni okwesikhashana ngaphambi kokuvala futhi yenza umnikelo obalulekile kumanje obola kancane womsila.
Ukuthola ukuthi ukubola kwemisinga yomsila we-Hv1 akuzange kwehliswe kakhulu phambi kwe-GBTA (I-Supplementary Fig. 2c) ihambisana nendlela yokubopha i-synergistic yalesi sivimbeli (I-Fig. 2d). Uma i-molecule ye-GBTA ingaboshiwe kusuka kuyunithi encane ye-Hv1 , ukuhlobana kwesivimbeli neyunithi eseduze kwehla cishe ngokuphindwe ka-10, kuvuna inqubo engabopheli.Lokhu kusho ukuthi i-molecule yesibili ye-GBTA inethuba eliphakeme kakhulu lokuqaqa ngaphambi kokuba ibambeke esiteshini.Izinhlobo zeziteshi eziphila isikhathi eside ukukhiqiza iyunithi eyodwa kuphela yokuvimbela kulindeleke ukuthi ibe kuningi kakhulu ebukhoneni be-GBTA kunaphambi kwe-2GBI, okuholela ekuboleni kwamanje komsila ngokushesha.
Ukuze i-GBTA ibophezele ngokubambisana nawo womabili amayunithi amancane e-Hv1, amasayithi abophezelayo kufanele ahlanganiswe ngokwezibalo. inguquko esuka ekudambiseni okuphansi ukuya ekubopheni okuhambisanayo okuphezulu.Sicabange ukuthi uma izinsalela ezithile ngaphakathi kwesayithi elibophezelayo zibe nomthelela enqubweni yokubambisana, ukuguqulwa kwazo kufanele kuguqule i-coefficient ye-Hill yejika lokuphendula lokugxilisa ingqondo le-GBTA evimbela i-Hv1.Residues D112, F150 , i-S181 ne-R211 ngaphambilini ziboniswe njengengxenye yendawo ebophezelayo ye-2GBI29 futhi sacabanga ukuthi zizobandakanyeka ngokufanayo ekubopheni i-GBTA (Fig. 3A).Silinganise amajika empendulo yokugxilisa evimbelayo we-GBTA kumashaneli aguqukayo i-D112E, F150A, S181A , kanye no-R211S (Umfanekiso 3) futhi baqhathanisa ama-coefficients abo e-Hill nalawo ohlobo olusendle lwe-Hv1, kusetshenziswa i-2GBI njengereferensi.Insalela engu-V109 isebusweni obufanayo bengxenye ye-S1 njenge-D112 futhi inokujika okukodwa kwe-helical okukodwa ngaphakathi kweseli. Kusukela I-V109 ayizange ibambe iqhaza ekubopheni i-2GBI29, sasebenzisa i-V109A mutant njengokulawula (I-Fig. 3b).
(a) Izinsalela ze-Hv1 eziphakanyisiwe ezihilelekile ekubopheni kokuphuma kokuphuma ku-guanidine.Amajika aluhlaza okwesibhakabhaka ahaqekile azungeza amaketango aseceleni ayehlongozwe ngaphambili asebenzisana nezingxenye ezihlukene ze-compound 2GBI29.(bf) Ukuvinjelwa okuncike ekugxiliseni kokuguquguquka okubonisiwe kwe-Hv1 ngenhlanganisela 2GBI (cyan) ne-GBTA ( okubomvu okumnyama).Iphuzu ngalinye limelela isilinganiso sokuvinjwa kwezilinganiso ezingu-3 kuya kwezingu-12 ± SD V109 njengesilawuli esinegethivu.Amajika ayi-Hill fits asetshenziselwa ukuthola amanani abonakalayo e-Kd (bona Ithebula Le-Supplementary 1). Ama-coefficients we-Hill (h) aboniswe ku- ama-histogram afakiwe anqunywa ngokulingana okubikwe ku-Supplementary Figs 3 kanye no-4. Amanani wereferensi h we-Hv1 WT aboniswa njengemigqa edayishiwe.Izinkanyezi zibonisa umehluko ophawulekayo ngokwezibalo phakathi kwamavesi e-mutant ne-WT (p Sithole ukuthi ukuguqulwa kwe-D112E kwehlise kakhulu i-coefficient ye-Hill (p Ukuthola ukuthi i-coefficient ye-Hill yokubophezela kwe-GBTA iphakeme kuno-1 ku-Hv1 dimer futhi iba ~1 kusiteshi se-monomeric (I-Fig. 2b) ihambisana nokuba khona kokusebenzisana kwe-allosteric phakathi kwamasayithi abophayo kumayunithi amabili amancane. icala, i-Coefficient ye-Hill ye-GBTA ebophezela ku-dimer lapho isivimbeli esibophele kuyunithi eyodwa kufanele ibe ~1.Sihlole lesi sibikezelo ku-Hv1 F150A-WT exhunywe i-dimer (Fig. 4a), lapho ukuhambisana kwe-F150A i-subunit ye-GBTA yayingaphezu kwama-oda angu-2 obukhulu obuphezulu kunaleyo ye-WT subunit (Figs. 1c kanye ne-3d). Sabe sesilinganisa amajika okuphendula okugxilile ekuvinjweni kweyunithi encane ye-WT ekugxilweni okuyisisekelo kwe-2 μM GBTA. , isivimbeli kulindeleke ukuthi sibophe cishe u-99% weyunithi encane ye-F150A kanye no- (a) Umdwebo oyisikimu we-F150A-WT ye-dimer esetshenziselwa ukukhiqiza i-hemichannel evimbayo ({F150A}b-WT/BAO*).Amadayimane amhlophe abonisa indawo yokuguqulwa.Ezifundeni ze-BAO* kanye ne-BA*B*, ukuhlobana kweyunithi encane ye-F150A kulindeleke ukuthi ibe phezulu kunaleyo yeyunithi encane ye-WT.(b) Ama-proton currents asuka eziteshini ze-F150A-WT akalwa ngokusabela kushintsho lwamandla olwelwesi oluvela ku-−80 mV ukuya ku-+120 mV.pHi = pHo = 6.0 .Iminonjana empunga imelela imisinga elinganiswa lapho ingekho inhibitor.I-trace (control) emnyama imelela isilinganiso samanje esikalwa ngemva kokwengezwa kwe-2 μM GBTA. Kulokhu kugxiliswa, iyunithi encane ye-WT ayizange ivinjwe kakhulu, kuyilapho iyunithi encane ye-F150A iboshelwe cishe ngokuphelele ku-GBTA. ({F150A}b-WT).Ukwanda okwengeziwe kokugxilisa ingqondo kwe-GBTA kubangele ukuvinjelwa kweyunithi encane ye-WT (imikhondo ewolintshi nebomvu).(c) Ukuvinjelwa okuncike ekugxilweni kwe-{F150A}b-WT dimer (i-inhibitor eboshwe ngaphambili I-subunit ye-F150A).Iphuzu ngalinye limelela isilinganiso sokuvinjwa okungu-± SD kwezingu-3 kuya kwezingu-7. Amajika ayewukulingana kwe-Hill okusetshenziselwa ukuthola amanani asobala e-Kd abikwe kuThebula Lokwengeza 1. Ama-coefficients we-Hill aboniswe kuma-histogram afakwe ngaphakathi anqunywa ekulinganeni okubikwe kokuthi I-Supplementary Figs 3 kanye no-4. Amanani angu-h we-Hv1 WT angu- kuboniswa njengemigqa enedeshi.Ama-asterisk abonisa umehluko obalulekile ngokwezibalo uma kuqhathaniswa ne-WT dimer Hv1 (p Njengoba ukuvinjelwa kwe-GBTA kwe-Hv1 kukalwa esiteshini esivulekile, sacabanga ukuthi ukusebenzisana okubophezelayo kwe-GBTA kungasetshenziswa ukutadisha indlela yokuhlangana kwe-intersubunit endaweni evulekile. 22 kanye nokuhlangana kwe-intersubunit okuhilelekile ekungeneni kwesango kuhlongozwe ukuthi kulamule isizinda se-cytoplasmic coiled-coil (CCD)22,25.Ngakho-ke, sibuze ukuthi ingabe i-CCD nayo iyabandakanyeka yini ekuhlanganisweni kwezindawo ezibophezela i-GBTA endaweni evulekile.I-Triglycine ukuguqulwa kwesixhumi esibonakalayo phakathi kwesiqephu se-transmembrane ye-S4 kanye nesizinda sekhoyili ekhoyiliyo kubonisiwe ocwaningweni lwangaphambili ukuze kunqanyulwe lesi sizinda kusuka kuso sonke isiteshi ngenkathi kugcinwa ubuqotho be-dimerization.Ngakho-ke, sihlole umphumela wokuguqulwa kwe-V220G, K221G kanye ne-T222G (Fig . 5a, i-GGG mutant) ku-GBTA ebophezela ukusebenzisana.Sithole ukuncipha okungabalulekile ngokwezibalo (p > 0.05) ku-coefficient ye-Hill ye-GBTA ebophezela eziteshini ze-GGG uma kuqhathaniswa nohlobo lwasendle (Fig. 5c), okuphakamisa ukuthi naphezu kwendima yayo ekugcineni i-GBTA amayunithi amancane amabili ndawonye abalulekile, kodwa i-CCD ayilamuli ngokuqondile ukuhlangana kwe-allosteric phakathi kwama-subunit phakathi kwamasayithi abophezelayo e-GBTA.
(a) Umdwebo oyisikimu we-Hv1 dimer onokuguquka kwe-triglycine ekugcineni kwangaphakathi kwe-S4 oklanyelwe ukuphazamisa ukuhlangana kwe-intersubunit okulamula isizinda sekhoyili ehlanganisiwe ye-cytoplasmic (imicibisholo eluhlaza okwesibhakabhaka).(b) Ukumelwa okuhleliwe kwama-dimers e-Hv1 nama-ligation dimers ane-cytoplasmic coiled-coil domain. ukuguqulwa okubonisiwe, okudizayinelwe ukuhlola izingcezu ze-S1 ezihilelekile ekuhlanganeni phakathi kwamayunithi amancane (imicibisholo eluhlaza okwesibhakabhaka).(c–h) 2GBI (cyan) kanye ne-GBTA (okubomvu okumnyama) zivimbela ukwakhiwa okubonisiwe ngendlela encike ekugxiliseni.Iphuzu ngalinye limelela ukuvimbela okumaphakathi. ± SD yezilinganiso ezi-3 kuye kweziyi-10. Ijika laliwukulingana kwe-Hill esetshenziselwa ukuthola amanani abonakalayo e-Kd (bona Ithebula Lokwengeza 1). Ama-coefficients we-Hill kuma-histogram ahlanganisiwe anqunywa njengoba kuchazwe esigabeni Sezindlela (bona ama-Supplementary Figs 3 no-4). Amanani ayireferensi angu-h I-Hv1 WT ikhonjiswa njengemigqa enedeshi.Izinkanyezi zibonisa umehluko obalulekile ngokwezibalo phakathi kwamavesi e-mutant ne-WT (p Njengoba sikhiphe i-CCD njengomlamuleli oqondile wokubophezela ngokubambisana kwe-GBTA, sibuze ukuthi ingabe ukusebenzelana phakathi kwe-VSD kuyabandakanyeka yini ekuhlanganiseni i-allosteric phakathi kwamasayithi abophayo. engxenyeni ye-transmembrane ye-S1.I-S1 iqukethe ezinye izinsalela ezimbili ezinecala elibi, i-E119 ne-D123, etholakala ohlangothini olulodwa lwe-helix njenge-D112, kodwa eduze nokuphela kwangaphandle kwe-24 yesiqephu.Imifanekiso yokuqala ye-molecular dynamics ibonise ukuthi lezi zinsalela zazixhunywe. kuya ku-D112 ngemigqa yamanzi36,37.Ngakho-ke, sihlole ukuthi i-E119 ne-D123 bayabandakanyeka yini ekuhlanganiseni i-allosteric phakathi kwezindawo ezibophezela i-GBTA (Fig. 5b).Njengokulawula okungekuhle, sihlole indawo egcinwe nge-positively charged K125, etholakala eduze ne-D123 kodwa ngakolunye uhlangothi lwe-S1 helix (Fig. 5b).
Silinganise ukuvinjelwa okuncike ekugxilweni kweziteshi ze-E119A, D123A kanye ne-K125A nge-2GBI ne-GBTA futhi sathola ukuthi ukuguqulwa kwe-E119A ne-K125A akuzange kushintshe kakhulu i-coefficient ye-Hill yanoma iyiphi i-inhibitor uma kuqhathaniswa nohlobo lwasendle (p > 0.05, Fig. 5d,i ). ). Ngakolunye uhlangothi, ukuguqulwa kwe-D123A kwehlise kakhulu i-Hill coefficient ye-GBTA (p 0.05/14).
Njengoba ukwehlisa inkokhiso endaweni engu-123 kuwo womabili amayunithi angaphansi kubangele ushintsho oluqinile ekusebenzisaneni okubophezelayo kwe-GBTA, kuyilapho ukubuyisela emuva inkokhiso kuwo womabili amayunithi kube nomphumela omncane, sanweba ukuhlaziya ukuze kufake iyunithi eyodwa kuphela neshaneli yokuguqula yenkokhelo. kukhiqizwe ama-dimer axhumene ne-Hv1 ngokushintshwa kwe-D123R kuyunithi ye-C-terminal (Fig. 5b) futhi yakala ukuvinjelwa kwempendulo yokugxilisa ingqondo nge-GBTA kanye ne-2GBI. yohlobo lwasendle i-Hv1 (p Siphinde sathola ukuthi ukuguqulwa kwe-D112E kuqede ukwenyuka kwe-GBTA-binding Hill coefficient ekhiqizwe ingemuva le-WT-D123R (Fig. 5b,h kanye ne-Supplementary Fig. 4). Fig. 5h kanye ne-Supplementary Fig. 3).Lokhu okutholakele kusikisela ukuthi ukuhlangana kwe-allosteric okuthuthukisiwe phakathi kwamayunithi amancane okubangelwa ukukhokhiswa okuphambene endaweni ye-123 akuhumushi ekuhlanganyeleni okubophayo okuqinile lapho isayithi elibophezelayo endaweni D112 liphazamiseka.
Sicabange ukuthi uma izinsalela ze-D123 kuma-subunit aseduze avulekile zisondele ngokwanele ukuthi zihlanganyele ngogesi, ukusebenzisana okunengekayo phakathi kwezindleko ezingezinhle kuzoguqulelwa ekubeni yinhlanganisela yezindleko ezingezinhle nezihle ngokufaka i-aspartic acid esikhundleni se-arginine. ukusebenzisana okukhangayo phakathi kwabo.I-subunit eyodwa (i-WT-D123R dimer).Ukusebenzelana okukhangayo kungaqinisa ukuxhumana phakathi kwemikhawulo yangaphandle yesiqephu se-S1, okuholela ekuhlanganiseni okuqinile kwe-allosteric phakathi kwama-subunits kanye nokwenyuka kokubambisana okubophezela kwe-GBTA.
Ukuze sisekele lo mbono, sibheke indlela yokuqinisa ukuxhumana phakathi kwamaphethelo angaphandle engxenye ye-S1, ehlukile ekushintsheni kwenkokhiso endaweni engu-123.Lee et al.Ukuguqulwa kwensalela ye-Hv1 I127 kuya ku-cysteine kwatholakala ngaphambilini. ukuphumela ekuxhumaneni okuzenzakalelayo kwe-intersubunit cross-linking17.Ngaphezu kwalokho, isakhiwo sekristalu se-Hv1-CiVSP chimeric VSD sibonise ukuthi i-I127 ihlukaniswe nokuphela kwangaphandle kwe-S1 helix yinsalela eyodwa kuphela24.Ngakho-ke sibuze ukuthi ngabe ukwakheka kwesibopho esihlangene phakathi ama-cysteine esikhundleni se-127, okulindeleke ukuthi aphumele ekusebenzisaneni okunamandla kwe-S1-S1, anomthelela ekubopheni i-GBTA ukubambisana okufana nalokho okubonwa ngokushintsha inkokhiso endaweni ye-123.
Silinganise ukuvinjelwa okuncike ekugxiliseni kwe-Hv1 I127C nge-GBTA kanye ne-2GBI lapho kukhona kanye nokungabikho kwe-10 mM β-mercaptoethanol (βME) (Fig. 6a). Ngesikhathi esifanayo njengoba i-inhibitor yengezwa kusixazululo se-intracellular, ukunciphisa i-ejenti yengezwa kusixazululo se-extracellular.I-dimer exhunyiwe ne-cysteine substitution endaweni eyodwa kuphela (WT-I127C) isetshenziswe njengesilawuli esingalungile (Fig. 6a).Asikwazanga ukukala noma yimuphi umphumela wokuxhumanisa okuzenzakalelayo kwe-intersubunit cross-linking. phakathi kwama-subunits e-I127C ekuvinjweni kwe-2GBI, ngenxa yokuthi i-Coefficient ye-Hill ye-I127C ebophezela ku-Hv1, ene-βME noma ngaphandle kwayo, yayihluke kakhulu kulokho okubophezela kuma-dimers esikhundleni se-monocysteine. I-coefficient ye-Hill ayikwazanga ukuhlukanisa i-WT-I127C (I-Fig. 6b kanye ne-Supplementary Fig. 3). Ngokuphambene kakhulu, silinganise umphumela omangalisayo wokuphambana okuzenzakalelayo kwe-intersubunit ku-GBTA inhibition.I-coefficient ye-Hill yokubophezela ku-Hv1 I127C ingekho i-βME (i-crosslink on) yayiphakeme kakhulu kunokulinganiswa phambi kwe-βME (i-crosslink off) noma isebenzisa i-WT-127C ukuxhumanisa i-dimer (uma kungekho crosslink) (Fig. 6c kanye ne-Supplementary Fig. 4), okubonisa ukuthi ukuhlanganisa i-allosteric phakathi kwezindawo ezibophayo kuthuthukiswa kakhulu ngokwakhiwa kwebhondi ye-disulfide phakathi kweziphetho zangaphandle zezingcezu ze-S1. Le miphumela isekela ukuhumusha kwethu ukuthi ukusebenzisana okukhangayo kwe-electrostatic kwe-aspartate ne-arginine endaweni 123 ku-WT-D123R dimer kuphumela ekwandeni kokuhlangana kwe-allosteric phakathi ama-subunits.
Ukuxhumanisa kombuso ovulekile kulamula ukusebenzisana okuqondile ngokomzimba phakathi kwamaphethelo angaphandle engxenye ye-S1 kumayunithi amabili.
(a) Ukumelwa okuhleliwe kwama-mutant Hv1 dimers nama-linker dimers, okudizayinelwe ukuphenya ukuthi i-intersubunit cysteine crosslinks ikuthinta kanjani ukusebenzisana okubophezelayo kwe-GBTA.(bd) Ukuvinjelwa okuncike ekugxilweni kwezakhiwo ezibonisiwe nge-2GBI (b,d) kanye ne-GBTA (c, e) uma kukhona noma ukungabikho kwe-10 mM βME kusixazululo esingaphandle kwe-extracellular.Iphuzu ngalinye limelela ukuvimbela okumaphakathi ± SD kwezilinganiso ezingu-3 kuya kwezingu-10. Ijika laliwukulingana kwe-Hill okusetshenziselwa ukuthola amanani e-Kd (bona Ithebula Le-Supplementary 1). Ama-coefficients angu-Hill kuma-histogram afakiwe anqunywa ngokulingana okubikwe ku-Supplementary Figs 3 kanye no-4.Asterisks ku-(c,e) abonisa umehluko obalulekile wezibalo. phakathi kwezimo ezihlukene zokuvimbela i-GBTA (p 0.05).
Siphinde sathola ukuthi uma ingekho i-βME, i-Hill coefficient ye-GBTA ebophezela ku-D112E I127C Hv1 dimer yayiphezulu kakhulu kunaleyo elinganiswa lapho kukhona ama-ejenti anciphisayo (Fig. 6e kanye ne-Supplementary Fig. 4), okusho ukuthi ukuguqulwa kwe-D112E ayizange ikuqedele ukukhuphuka kokubambisana okubophezelayo kwe-GBTA okubangelwa ukuxhumanisa kwe-cysteine 127.I-Coefficient ye-Hill ye-2GBI ebophezela ku-D112E, i-I127C Hv1 dimers nayo ayizange ithintwe kakhulu ukuguqulwa kwe-D112E (Umfanekiso 1).6d kanye Nokwengeza. Umfanekiso wesi-3).
Sekuhlanganiswe ndawonye, lokhu okutholakele kusikisela ukuthi ukubophezela kwe-GBTA kuthuthukiswa ukuxhumana phakathi kweziphetho zangaphandle zezingcezu ze-S1 kumayunithi aseduze we-Hv1 ngokusebenzisana okukhangayo kwe-electrostatic noma ngokwakhiwa kwezibopho eziqinile phakathi kwama-cysteines ashintshiwe Ukuhlangana kwe-allosteric phakathi kwamasayithi kuyakhuphuka futhi kubangele ukubambisana okubophezelayo. Nakuba umthelela wokusebenzelana okukhangayo kwe-electrostatic ekusebenzisaneni kungaqedwa ngokuguqulwa kwe-D112E, umphumela wamabhondi aqinile awukwazi.
Ekuhloleni kwethu isikhala samakhemikhali esitholakalayo sokubopha okuphuma kokuphuma kwe-guanidine eziteshini ze-Hv1, sithole ukuthi ama-2-guanidinothiazoles afana ne-GBTA ancike kakhulu ekugxiliseni kune-2-guanidinobenzimidazoles (Fig. 1c) .Ukuhlaziywa kwe-coefficient ye-Hill ye-GBTA ebophezela kukho kokubili i-dimeric kanye neziteshi ze-monomeric (Fig. 2a,b) kanye nesiteshi se-dimeric lapho iyunithi eyodwa yayiboshwe ngaphambili ku-inhibitor (Fig. 4) yasiholela ekuphetheni ngokuthi ukuvinjelwa kwe-Hv1 nge-GBTA Isenzo siyinqubo ye-synergistic, futhi izindawo ezibophezelayo zezinhlanganisela kumayunithi amabili amancane ahlanganiswe nge-allosterically.Ukutholakala ukuthi i-GBTA ibophezela esiteshini esivulekile, njengoba kuboniswe ngaphambilini ngenhlanganisela ehlobene engu-2GBI32, kuphakamisa ukuthi ukuhlanganisa i-allosteric kungahlolwa ngokuqondile esimweni esivulekile.Imodeli yethu yokuhlanganisa ngokubambisana ikwazile ukuchaza ngobuningi ukuvinjelwa kwe-Hv1 nge-GBTA (Fig. 2c) futhi ichaze imiphumela ehlukene ye-2GBI ne-GBTA ekuboleni kwemisinga yomsila wesiteshi ngemva kokwenziwa kabusha kwe-membrane, okusekela incazelo yethu yenqubo yokubopha.
I-coefficient ye-Hill ephezulu engafinyelelwa kuphrotheni ye-allosteric enezindawo ezimbili ezibophezelayo (njenge-Hv1) ngu-2.Silinganise i-GBTA ukuze sibophe i-Hv1 yohlobo lwasendle nge-coefficient engu-1.31, ekhuphuke yaba ngu-1.88 ku-Hv1 I127C. amandla amahhala e-synergistic, umehluko phakathi kwamandla amahhala abophayo wezindawo ezisondelene eziphansi neziphakeme kakhulu (bona Izindlela), kwakungu-1.3 kcal/imvukuzane esimweni se-Hv1 wild-type kanye no-2.7 kcal/mole esimweni se-Hv1 I127C.Isibopho we-oksijeni ku-hemoglobin iyisibonelo esidume kakhulu nesifundwe kahle senqubo yokusebenzisana38.Ku-hemoglobin yomuntu (i-tetramer enezindawo ezine ezihlanganisa i-allosterically), i-coefficient ye-Hill isukela ku-2.5-3.0, ngamavelu asukela ku-1.26 kuya ku-3.64 kcal/mol, kuye ngezimo zokuhlola38.Ngakho-ke, ngokwemibandela yamandla omhlaba, ukusebenzisana kwe-GBTA ebophezela ku-Hv1 akuhlukile kakhulu kulokho kwe-O2 ebophezela ku-hemoglobin lapho kucatshangelwa izinombolo ezihlukene zamayunithi amaprotheni ezinhlelweni zombili.
Kumodeli yethu ye-synergy, ukubophezela kwama-molecule e-GBTA kuyunithi eyodwa kuholela ekwandeni kokuhlangana okubophezelayo kweyunithi eseduze.Sibona ngeso lengqondo inqubo lapho ukuhlelwa kabusha kwendawo ebophezelayo okubangelwa umcimbi wokuqala obophayo (ukulingana okudalwe) kuholela ekutheni izinguquko ekusebenzelaneni phakathi kwamayunithi amancane.Ekuphenduleni lezi zinguquko, izingxenye ezincane eziseduze zishintsha amasayithi awo abophezelayo, okuholela ekubopheni kwe-GBTA okuqinile.Phakathi nale nqubo, i-S1 aspartate D112 inesibopho sokuhlela kabusha isayithi elibophezelayo elihlotshaniswa nokwenyuka kobudlelwane obubophezelayo.D112 ngaphambilini yaboniswa ukuthi iyingxenye ye-Hv1 proton permeation pathway futhi isebenza njengesihlungi sokukhetha27,28. Imiphumela yethu ibonisa ukuthi izihlungi zokukhetha kuma-subunits amabili e-Hv1 zihlanganiswe ngokwe-allosterically endaweni evulekile.Umfanekiso 7a ubonisa indawo ecishe ifane yesizinda sokubopha i-GBTA kanye nendawo yezinsalela i-D112, D123, K125 kanye ne-I127 kuhlelo lwe-Hv1 VSD esekelwe. esakhiweni sekristalu se-chimera ye-Hv1-CiVSP.Izikhombisi-ndlela eziphakanyisiwe zokuhlanganisa i-allosteric ezibandakanya ukuphela kwe-extracellular ye-S1 ziboniswa ngemicibisholo emnyama.
(a) I-Schematic ye-Hv1 VSD.Ngokusekelwe esakhiweni sekristalu se-chimera ye-Hv1-CiVSP, izingxenye ze-helical ziboniswa njenge-cylindrical.Kulokhu kulungiselelwa, isiphetho sangaphakathi se-S4 segment sihlangana ne-CCD (engaboniswa). Izindawo ezibikezelwe ze-GBTA eboshiwe ziboniswa njengama-ovals ampunga.Imicibisholo emnyama ibonisa izindlela ezihilelekile ekuhlanganeni kwe-allosteric phakathi kwamasayithi abophayo kumayunithi amabili aseduze.Izindawo zezinsalela ze-S1 eziphenyiwe zimakwe ngama-sphere anombala.(b) Umdwebo oyisikimu wamayunithi amancane e-Hv1 ahleliwe ekucushweni okubili okuhlukene kwe-dimer njengoba kubonakala ohlangothini olungaphandle kwendiza ye-membrane.Kuphaneli yesokunxele, isixhumi esibonakalayo se-dimer sakhiwe i-S4 helix.Ukuzungeza kwama-VSD amabili ama-degree angu-20 ngokuhambisana newashi azungeze i-axis perpendicular kuya endizeni yolwelwesi, kuphelezelwa ukuhlukaniswa kwemikhawulo yangaphandle yezindiza ezimbili ze-S4 (imicibisholo edayishiwe), kukhiqiza ilungiselelo eliboniswa kwesokudla.Kulokhu kumisa, izinsalela ze-D123 ne-I127 ezivela kumayunithi aseduze zivunyelwe ukusondela ndawonye.(c) Ukumelwa okuhleliwe kwe-CiVSP VSD ekucushweni kwe-dimer okutholakala ku-4G80 crystal structure.Isikhundla se-P140 ku-CIVSP sihambisana nesikhundla se-D123 ku-Hv1.
Imiphumela yethu ibonisa ukuthi ukusebenzisana kwe-electrostatic okunengekayo phakathi kwezinsalela ezingu-123 (123D/123D noma 123R/123R) kuhlotshaniswa nezinga “elivamile” lokusebenzelana okubophezela kwe-GBTA esimweni esivulekile futhi kushintsha ukusebenzisana kusuka ekunyanyeni kuya kokuthi Okukhangayo (123D/123R) , ukubambisana okwandisiwe (I-Fig. 5g) .Kulindeleke ukuthi ukususwa kokuxhumana okunyanyekayo nokushintshwa kwe-alanine kuzophinde kuholele ekwandiseni ukubambisana.Nokho, ukwehla kokubambisana kwe-123A / 123A dimer kwabonwa (Fig. 5e) .Okunye kungenzeka. incazelo iwukuthi umphumela ophazamisayo wokubeka izinsalela ze-hydrophobic endaweni ye-hydrophilic ungase udlule umphumela wokuzinzisa ngenxa yokuqedwa kokuxhumana okunyanyekayo phakathi kwezinsalela ze-D123, okuholela ekunciphiseni okuphelele kokubambisana okubophezelayo.Mony et al.39 muva nje kubika ukuthi ukufinyeleleka kwe-solvent at isikhundla se-D171 se-Ciona gutis Ci-Hv1 (ehambisana ne-D123 ku-Hv1 yomuntu) siyakhuphuka lapho sisebenza, sisekela umbono wokuthi i-D123 itholakala endaweni ene-hydrophilic endaweni evulekile.
Ukugaywa kwamashaneli e-Hv1 kuyaziwa ukuthi kwenzeka ngoshintsho oluningi19,20,26,39,40,41.Qiu et al26 bathole ukuthi lapho ulwelwesi lwe-depolarization, inzwa kagesi ye-Ci-Hv1 iba noshintsho lokuvumelana olushiya isiteshi sivuliwe kodwa sisavaliwe. , okulandelwa uguquko oluhlukile olubangela ukuba ama-proton avuleke ukuqhutshwa kuzo zombili izindlela ze-subunits.Ushintsho oluhambisanayo lwenzwa ye-voltage luqashwe yi-voltage-clamp fluorescence, futhi kwatholakala ukuthi uguquko lwesibili lwaluphazanyiswa ngokukhetha ukuguquka kwesimo se-D171. Ukuphazamiseka kwesignali ye-fluorescent kuhambisana nokuba khona kokusebenzisana kwe-electrostatic phakathi kwezinsalela ze-D171 zamayunithi aseduze ngesikhathi esithile eduze kwezixhumanisi zokusabela zoshintsho lokuvumelana. Le ncazelo ihambisana nokuthola kwethu ukuthi izinsalela ze-D123 zixhumana ngogesi endaweni evulekile. futhi ilamula ukuhlangana kwe-allosteric phakathi kwamasayithi abophezelayo e-GBTA.
U-Fujiwara et al25 uhlongoze ukuthi isixhumi esibonakalayo se-dimer sesizinda sekhoyili esikhoyili se-cytoplasmic sidlulele kulwelwesi ukuze siqukathe ama-S4 helices amabili (Fig. 7b, panel left). yonke i-VSD kanye nokuhlaziywa kokusebenza kwesifunda esixhuma i-S4 helix kanye neCCD. Uma ingekho i-transmembrane pH gradient, iziteshi ze-Hv1 zidinga ukuchithwa kwe-membrane okukhulu ukuze kuvuleke, futhi i-cysteine crosslinking kwenzeka ngaphansi kwezimo lapho isiteshi sivalwa kakhulu. Ngakho-ke, isikhombikubona se-S4-S4 esitholiwe cishe sibonisa ukucushwa kweyunithi ye-off-state.Ezinye izifundo zithole ubufakazi bokubandakanyeka kwe-S1 ne-S2 ekusebenzisaneni kwe-intersubunit ngesikhathi se-gating17,21,26 ephakamisa ukuthi iziteshi zingase zamukele ukucushwa kweyunithi ehlukile endaweni evulekile futhi. amazwe avaliwe, umbono ohambisana nokutholwe nguMony et al. I-S1 ihamba ngama-39 ngesikhathi sokungena.
Lapha, sibonisa ukuthi, esimweni esivulekile, iziphetho ze-extracellular ze-S1 helix zisondele ngokwanele ukusekela ukusebenzisana okuqondile kwe-electrostatic okulamula ukuhlangana kwe-allosteric phakathi kwama-subunits.Ekucushweni kwe-dimer nokusebenzisana okunwetshiwe kwe-S4-S4, ama-S1 helices akude kakhulu. ngaphandle komunye nomunye ukuze basebenzisane ngokuqondile.Nokho, ukuzungezisa okungu-20° okuhambisana newashi kwamayunithi amabili e-VSD azungeze i-axis perpendicular endizeni ye-membrane, kuhlanganiswe nokuhlukaniswa kwamaphethelo angaphandle ezindiza ezimbili ze-S4, kuveze ukucushwa kwe-S1-S1 okungaguquki. ngokuthola kwethu (Umdwebo 7b, iphaneli yesokudla).Sincoma lokhu kulungiselelwa kwesiteshi esimweni esivulekile.
Nakuba i-enzyme ye-CiVSP kucatshangwa ukuthi isebenza njenge-monomer, isakhiwo sekristalu se-VSD yayo ehlukanisiwe sithathwe esimweni se-dimer.Iziphetho zangaphandle zama-helice e-S1 kule dimer zisondelene ngendawo, futhi ukucushwa okuphelele kufana nalokho okuhlongozwayo. ye-Hv1 (Fig. 7c).Ku-dimer ye-CIVSP, izinsalela eziseduze kakhulu ezivela engxenyeni eseduze yayiyi-proline endaweni engu-140 (Fig. 7c). Kuyathakazelisa ukuthi i-P140 ku-CiVSP ihambisana nesikhundla D123 ku-Hv1.Ukufana ekucushweni kweyunithi phakathi kwe-Hv1 kanye ne-CiVSP dimers iphakamisa ukuthi ama-VSD alawa maprotheni anomkhuba wangaphakathi wokwenza i-interface lapho iziphetho ze-extracellular ze-S1 zixhumana khona.
Iqhaza elibalulekile le-Hv1 ekwenzeni kusebenze iseli yesidoda yenza lesi siteshi sibe yithagethi ekhangayo yomuthi wokulawula inzalo yabesilisa.Ngaphezu kwalokho, ukusebenzisa i-Hv1 ku-microglia kuboniswe kuthuthukisa ukululama ku-ischemic stroke.Umsebenzi othuthukisiwe we-Hv1 utholakale uhlotshaniswa nokwehla ukusinda ezigulini ezinomdlavuza webele 12 noma umdlavuza we-colorectal 13 futhi kucatshangwa ukuthi nomthelela emazingeni e-B-cell 11. Ngakho-ke, izidakamizwa ezincane ze-molecule eziqondise i-Hv1 zingasetshenziswa njengama-neuroprotective agents noma ama-anticancer therapeutics. i-subunit evulekile ye-Hv1, okuholela ekwenyukeni kobudlelwane obubophezelayo, kungase kuholele ekwakhiweni kwezidakamizwa ezinamandla kakhulu eziqondise iziteshi ze-Hv1.
I-mutagenesis eqondiswe kusayithi ye-Hv1 yomuntu yenziwa kusetshenziswa amasu ajwayelekile e-PCR.Ekwakheni kwe-Hv1NCCiVSP, izinsalela ezingu-1-96 kanye no-228-273 ze-Hv1 zathathelwa indawo izinsalela 1-113 kanye no-240-576 ze-CiVSP18.Ku-dimer exhunywe i-Hv1, i-C-terminus ye-subunit eyodwa ixhunywe ku-N-terminus ye-subunit yesibili ngokusebenzisa isixhumanisi se-GGSGGSGGSGSGGSGG. Ama-plasmids e-pGEMHE aqukethe izakhiwo ezihlukahlukene ahlanganiswe ne-Nhe1 noma i-Sph1 restriction enzymes (i-New England Biolabs) kanye nokuhlanganiswa kwe-RNA kwenziwa nge- I-T7 mMessage mMachine transcription kit (Ambion). Ezinsukwini ezingu-1-3 ngaphambi kwezilinganiso ze-electrophysiological, i-cRNA yajovwa kuma-Xenopus oocyte (50 nl ngeseli, 0.3-1.5 μg/μl). I-Oocyte yesigaba V kanye ne-VI evela ku-Xenopus laevis (NASCO) yatholwa kusukela ku-Ecocyte Bioscience.Ukulandela umjovo we-RNA, amaseli agcinwe ku-ND96 medium equkethe 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl, 1 mM MgCl, 10 mM HEPES, 5 mM pyruvate, 100 μg/ml 7° C7°C. .
2-guanidino-benzimidazole[1], 2-guanidino-benzothiazole[2], (4-methyl-1,3-thiazol-2-yl)guanidine [5], (5-bromo-4 -methyl-1,3 -thiazol-2-yl)guanidine) [6], ethyl 2-guanidino-5-methyl-1,3-thiazole-4-carboxylate [8], ethyl 2-guanidino-4- methyl-1,3-thiazole- I-5-carboxylate [9] kanye (2-guanidino-4-methyl-1,3-thiazol-5-yl)ethyl acetate [10] yayivela ku-Sigma-Aldrich.Famotidine [7] yayivela ku-MP Biomedicals.1-[4] -(4-Chlorophenyl)-1,3-thiazol-2-yl]guanidine[11] kanye ne-1-[4-(3,4-dimethoxyphenyl)-1,3- Thiazol-2-yl]guanidine [12] kusuka ku-Matrix Scientific.Lezi zinhlanganisela zingobumsulwa obuphakeme kakhulu obutholakala ngokwezentengiselwano.Zancibilika ku-DMSO eyomile ukuze kukhiqizwe isixazululo sesitoko esingu-100 mM, esabe sesihlanjululwa esixazululweni sokurekhoda ekugxiliseni okufiswayo kokugcina.Izingxube 3 kanye no-4 zahlanganiswa ngezansi ubumsulwa okungenani obungama-99%.
Ekumisweni kwe-2-amino-4-(trifluoromethyl)benzenethiol hydrochloride (1.02 g, 4.5 mmol) ku-25 mL we-aqueous hydrochloric acid (2.5 N) yengezwe i-dicyandiamide eqinile (380 mg, 4.5 mmol), kanye nomphumela wengxube ye-heterogeneous. i-refluxed ngokuvuselela ngamandla amahora angu-4. Ingxube yokusabela ipholile ekamelweni lokushisa futhi ingathathi hlangothi ngokungezwa kancane kancane kwe-10N potassium hydroxide.I-precipitate emhlophe eyakhiwe yahlungwa, igezwa ngamanzi abandayo (3 × 50 ml), yomiswa kuhhavini ( 65 °C) amahora ambalwa, bese kuphinda kufakwe ikristalu kusuka ku-ethyl acetate/petroleum ether ukuze kunikezwe okuqinile okumhlophe (500 mg, 48 %); mp 221–222 °C (ukukhanya 225–226 °C) 45; 1H NMR (500 MHz, DMSO-d6): δ [ppm] = 7.25 (ebanzi kakhulu s, 4 H), 7.40 (d, 1 H, J = 8.1 Hz), 7.73 (s, 1 H), 7.92 (d , 1 H, J = 8.1 Hz).13C NMR (200 MHz, DMSO-d6): δ = 114.2 (d, J = 3.5 Hz), 117.5 (d, J = 3.5 Hz), 121.7, 124.6 (q, J = 272 Hz), 126.1 (q, J = 272 Hz) = 31.6 Hz), 134.8, 152.1, 158.4, 175.5.HRMS (ESI): m/z inani elibaliwe.Ku-C9H8F3N4S (M + H)+041, itholiwe. : 261.0419.
I-Naphtho[1,2-d]thiazol-2-amine (300 mg, 1.5 mmol), ehlanganiswe njengoba kuchaziwe ngaphambili, yashiswa yaba ngu-200 °C endaweni yokugeza kawoyela epayipini elincane lokuhlola.300 mg (ukweqiwa okukhulu) i-cyanamide futhi I-1.0 ml conc.Esakhiweni esishisayo yengezwe i-hydrochloric acid ngokushesha futhi ingxube igcinwe kubhavu kawoyela cishe imizuzu emi-2, phakathi nalesi sikhathi amanzi amaningi ahwamuka.Ingxube yokusabela yabe isipholiswa ekamelweni lokushisa kanye nomphumela oqinile oqinile. yaphulwa yaba yizicucu ezincane futhi yagezwa ngamanzi ukuze inikeze okuqinile kwe-amorphous okuphuzi okuphaphathekile.(38 mg, 10%) mp 246-250 °C; 1H NMR (500 MHz, DMSO-d6, D2O): δ [ppm] = 7.59 (t, 1 H, J = 8.2 Hz), 7.66 (t, 1 H, J = 8.3 Hz), 7.77 (d, 1 H , J = 8.6 Hz), 7.89 (d, 1 H, J = 8.6 Hz), 8.02 (d, 1 H, J = 8.2 Hz), 8.35 (d , 1 H, J = 8.3 Hz).13C NMR (150 MHz, DMSO-d6): δ = 119.9, 122.7, 123.4, 123.6, 126.5, 127.1, 128.7, 132.1, 140.7, 169.1.HRMS (ESI): m/1 ivelu ye-C30: H + 12: m4 9 , kutholwe: 243.0704.
Ama-proton currents alinganiswa ngeziqephu zangaphakathi nezangaphandle zama-oocyte aveza ukwakheka okuhlukile kusetshenziswa i-Axopatch 200B amplifier elawulwa yi-Axon Digidata 1440A (Molecular Devices) ngesoftware ye-pClamp10.Izixazululo ze-Intracellular kanye nezangaphandle zinokwakheka okufanayo: 100 mM 2-(N-morpholino) ) i-ethanesulfonic acid (MES), 30 mM tetraethylammonium (TEA) mesylate, 5 mM TEA chloride, 5 mM ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), ilungiswe ukuze I-pH 6.0 nge-TEA hydroxide.Zonke izilinganiso zenziwe ku-22 ± 2 °C. I-pipette inokumelana nokufinyelela kwe-1.5–4 MΩ.Imikhondo yamanje yahlungwa ku-1 kHz, yathathwa isampula ku-5 kHz futhi yahlaziywa nge-Clampfit10.2 (Amadivayisi Emangqamuzana ) kanye ne-Origin8.1 (OriginLab).
Izixazululo eziqukethe ukugxila okuhlukahlukene kwe-Hv1 inhibitor futhi kwezinye izimo i-10 mM βME zangeniswa kubhavu amandla adonsela phansi ngokusebenzisa i-multifold exhunywe ohlelweni lwe-VC-6 perfusion valve (Warner Instr.), olwadluliselwa ngesofthiwe ye-pClamp TTL (Transistor- Amasiginali we-Transistor Logic).Ukuhlolwa kokugcwaliswa okusheshayo kwenziwa kusetshenziswa ipensela enamashubhu amaningi (i-AutoMate Sci.) enethiphu yokulethwa kwe-diameter engu-360 μm ebekwe ngaphambi kwe-patch pipette.Ukuvimbela isiteshi kunqunywa izilinganiso ze-isochronal amperometric ekupheleni kwe +120 mV depolarizing pulse.Izilinganiso ze-GV zenziwa njengoba kuchazwe ngaphambilini18,20.Imisinga yomsila yarekhodwa ku--40 mV ngemva kwezinyathelo ze-depolarization kuma-voltage ahlukene ukusuka ku--20 mV kuya ku-+120 mV ngaphandle kwalapho kushiwo ngenye indlela. esetshenziselwe ukulungisa ukubola kwamanje 18 .Igrafu ye-GV ilingana nesibalo se-Boltzmann:
Ama-dissociation constants (Kd) enhlanganisela ehlukene yamashaneli nama-inhibitors (Ithebula Lokwengeza 1) anqunywa ngokuhlanganisa ukuncika kokuncika kokuvinjelwa (kusho amanani angu-%inhib) nesibalo se-Hill:
lapho [I] iyinkomba ye-inhibitor I kanye no-h kuyi-coefficient ye-Hill.Ukuze ubale i-coefficient ye-Hill, isibalo (2) sihlelwa kabusha ngokuthi:
Isikhathi sokuthumela: Jun-07-2022
