Kubazwa intera ya intersubunit yumurongo wa Hv1 proton ifunguye hamwe na probe ihujwe hamwe

Urakoze gusura Kamere.com. Verisiyo ya mushakisha ukoresha ifite inkunga nkeya kuri CSS.Kuburambe bwiza, turagusaba ko wakoresha mushakisha ivuguruye (cyangwa kuzimya uburyo bwo guhuza muri Internet Explorer) .Muri hagati aho, kugirango ubyemeze gukomeza inkunga, tuzerekana urubuga rudafite stil na JavaScript. Umuyoboro wa Hv1 wongeyeho umuyoboro wa proton numuyoboro wa dimeric ugizwe na domaine ebyiri zumva voltage (VSDs), buriwese urimo inzira ya proton yinjira mumarembera. Kugabanuka bigenzurwa na cytoplasmeque coiled-coil.Inzibacyuho kuva leta ifunze ikajya muri coil. leta ifunguye muri VSDs izwiho kugaragara mubufatanye; icyakora, bike bizwi kubijyanye nuburyo bwibanze. Imigaragarire ya Intersubunit igira uruhare runini mubikorwa bya allosteric; icyakora, intera nkiyi ntiyamenyekanye mumiyoboro ya Hv1 ifunguye. Hano twerekana ko ibikomoka kuri 2-guanidinothiazole bibuza Hv1 VSD ebyiri muburyo bwa koperative kandi bigakoresha kimwe muri ibyo bikoresho nkubushakashatsi bwo guhuza allosteric hagati ya subunits zifunguye.Twabonye ko bidasanzwe. iherezo ryigice cya mbere cya transembrane ya VSD ikora interineti ihuza intersubunit ihuza guhuza hagati yimbuga zihuza, mugihe domaine ya coil-coil itagira uruhare rutaziguye muriki gikorwa.Twabonye kandi ibimenyetso bifatika byerekana ko umuyoboro wa proton-uhitamo akayunguruzo ugenzura ubufatanye-buhuza ubufatanye. . Imiyoboro ya poroteyine ya voltage ifite uruhare runini mubinyabuzima bitandukanye, kuva phytoplankton kugeza kubantu1.Mu selile nyinshi, iyi miyoboro ihuza proton efflux kuva muri proton membrane kandi ikagenga imikorere ya okiside ya NADPH. Umuyoboro uzwi gusa wa voltage-gated proton mubantu. ni Hv1, nigicuruzwa cya gene ya HVCN1,3. moteri9 na pH kugenga umuyaga wo hejuru wamazi10. Uyu muyoboro ugira uruhare runini muburyo bwa kanseri nka B-selile malignancies4,11 na kanseri yamabere na kanseri yibara 12.13.Ibikorwa bya Hv1 byagaragaye ko byongera ubushobozi bwimikorere ya selile kanseri 11, 12 .Mu bwonko, Hv1 iragaragazwa na microglia, kandi ibikorwa byayo byagaragaye ko byongera ubwonko mubwoko bwa stroke ischemic stroke. Poroteyine ya Hv1 ikubiyemo indangururamajwi (VSD), igizwe n'ibice bine bya transembrane bita S1 kugeza kuri S414. VSD isa na domaine ijyanye na voltage ya gati ya Na +, K + na Ca2 + hamwe na fosifata-yumvikanisha imbaraga, nka CiVSP kuva Ciona gutis15 .Umuyoboro ni complexe ya dimeric igizwe na VSD ebyiri, buri imwe irimo inzira ya proton yinjira mumarembo 16,17,18.Iyi subunitike ebyiri za Hv1 wasangaga zifungura ubufatanye19,20,21,22, byerekana ko guhuza allosteric no guhuza imikoranire hagati ya subunit ikina Uruhare rwingenzi mugikorwa cyo gutambuka. Imigaragarire hagati ya subunits murwego rwa coil coil irasobanuwe neza kuko imiterere ya kristu ya domaine ebyiri yitaruye irahari 22.23.Ku rundi ruhande, intera iri hagati ya VSDs muri membrane ntabwo ari byumvikane neza. Imiterere ya kristu ya proteine ​​ya Hv1-CiVSP ya chimeric ntabwo itanga amakuru kubyerekeranye niyi interface, kuko ishyirahamwe ryimyitozo ngororamubiri ryumuyoboro wa kristu rishobora guturuka ku gusimbuza kavukire ya Hv1 CCD n'umusemburo wa leucine zipper GCN424. Ubushakashatsi buherutse gukorwa ku ishyirahamwe rya subunit ryumuyoboro wa Hv1 bwanzuye ko S4 helices ebyiri zinjira muri CCD nta guhungabanya bikabije imiterere ya kabiri, bikavamo helices ndende zitangirira muri membrane n'umushinga muri cytoplazme.Bishingiye ku isesengura rya sisitemu, ubu bushakashatsi bwerekana ko Hv1 VSDs zandikirana mugice cya S4.Nyamara, ubundi bushakashatsi bwerekanye ubundi buryo bwo guhuza hagati ya VSD.Iyi ntera irimo S1 ibice 17, 21, 26 nimpera yinyuma yicyiciro cya S2 21.Impamvu ishobora gutera amakimbirane. ibisubizo by'ubu bushakashatsi ni uko guhuza allosteric hagati ya VSDs byasuzumwe bijyanye n'inzira yo gutambuka, biterwa na leta zifunze kandi zifunguye, kandi intera iri hagati ya VSDs irashobora gutandukana muguhinduka kwa leta zitandukanye muburyo bwo guhinduka. Hano, twasanze 2-guanidinothiazole ibuza imiyoboro ya Hv1 muguhuza hamwe na VSD ebyiri zifunguye, hanyuma tugakoresha kimwe mubice, 2-guanidinobenzothiazole (GBTA), kugirango tumenye imikoranire hagati ya subunits muri leta ifunguye. Imigaragarire.Twabonye ko umurongo wa GBTA uhuza umurongo ushobora gusobanurwa neza nicyitegererezo cyumubare aho guhuza inhibitor kuri subunit imwe bivamo kwiyongera muburyo bwo guhuza isano ya subunit yegeranye.Twabonye kandi ko ibisigisigi D112, guhitamo gushungura kuri umuyoboro wa 27, 28 hamwe nigice cya guanidine ikomoka kuri site 29 igenzura ubufatanye bwa GBTA.Twerekana ko guhuza amakoperative bikomezwa muri dimer ya Hv1, aho CCD itandukanya nigice cya S4, byerekana ko interineti ihuza intersubunit muri CCD idahuye neza. guhuza guhuza allosteric guhuza imbuga za GBTA-zihuza.Ibinyuranye, dusanga igice cya S1 kiri mubice byimbere hagati ya subunits hanyuma tugasaba gahunda ya VSD yegeranye hamwe nimpera zidasanzwe za S4 helix kure yikigo cya dimer kugirango yemere agace S1 kuba muri reta ifunguye. Ingirabuzimafatizo ntoya ya Hv1 ni ingirakamaro nk'imiti igabanya ubukana hamwe na neuroprotective agent.Nyamara, kugeza ubu, ibice bike byashoboye guhagarika umuyoboro30,31,32,33.Muri bo, 2-guanidinobenzimidazole (2GBI, compound [1] mu gishushanyo .. mbere yerekanwe kubuza Hv1 hafi nka 2GBI mugihe twageragejwe kuri 200 μM (Igicapo 1b) .Twasuzumye izindi nkomoko ya thiazole dusanga bamwe muribo babujije umuyoboro ufite imbaraga zisa cyangwa nyinshi kurenza GBTA (Ishusho 1 ninyongera) inyandiko) .Twagennye umurongo wo gusubiza umurongo wibikomoka kuri thiazole enye (GBTA hamwe n’ibicuruzwa [3], [6] na [11], igishusho 1c) dusanga byari bikomeye kuruta ibya 2GBI. ibikomoka kuri thiazole kuva kuri 1.109 ± 0.040 kugeza kuri 1.306 ± 0.033 (Ishusho. 1c hamwe n’inyongera Igishushanyo 1) .Ibinyuranye, coefficient de Hill ya 2GBI yari 0.975 ± 0.024 29, Igishushanyo cya 2a hamwe n’ishusho y'inyongera. 1 urubuga ruhuza, 29,32 twatekereje ko guhuza inkomoko ya thiazole kuri subunit imwe ishobora kongera guhuza molekile ya kabiri ya inhibitor kuri subunit yegeranye.GBTA yari ikizamini cyibizamini hamwe na coefficient yo hejuru ya Hill.Noneho rero, twahisemo iyi compound to ongera wige uburyo bwo guhuza imbaraga kandi ukoreshe 2GBI nkigenzura ribi. (a) Ibizamini byo gupima: [1] Reba Hv1 inhibitor 2-guanidino-benzimidazole (2GBI). 2-guanidino-benzothiazole (GBTA), [3] (5-trifluoromethyl-1,3-benzothiazol-2-yl) guanidine, [4] naphtho [1,2-d] -guanidine, [5] (4-methyl-1,3-thiazol-2-yl) guanidine, [6] (5-bromo-4-methyl-1,3-thiazol- 2-yl) guanidine, [7] umuryango wa famotidine, ] (2-guanidino-4-methyl-1,3-thiazol-5-yl) Ethyl acetate, [11] 1- [4- 12] 1- [4- (3,4-dimethoxyphenyl) -1,3-thiazol-2-yl] guanidine. .Hv1 ya proton yapimwe mubibaho byimbere bya oocytes ya Xenopus hasubijwe ko depolarisiyasi yavuye kuri mV 80 kugeza kuri mV120. Buri inhibitor yongerewe mubwogero bwa 200 μM.pHi = pHo = 6.0 .Data isobanura ± SEM (n≥4). kugeza ku bipimo 15. Umurongo ni Umusozi ukwiye gukoreshwa kugirango ubone indangagaciro za Kd zavuzwe mu mbonerahamwe yinyongera 1.Hefficient zose zagenwe uhereye ku bisobanuro byatanzwe mu gishushanyo cya 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 (reba Uburyo). . inset histogrammes zagenwe uhereye kumibare ivugwa mumashusho yinyongera ya 3 na 4.Igisubizo cyibisubizo bya GBTA cyerekanwe muri (a) ni kimwe no ku gishushanyo 1c. Reba Imbonerahamwe yinyongera ya 1 kubintu bigaragara Kd. (c) Kwerekana koperative ihuza GBTA na dimeric Hv1.Umurongo wumukara ukomeye ugereranya ibikwiranye namakuru yubushakashatsi ukoresheje ikigereranyo (6), gisobanura uburyo bwo guhuza bwerekanwe muri (d) .Imirongo yacagaguye yanditseho Sub 1 na Sub 2 yerekana ishyirahamwe bimolecular -gutandukanya kuringaniza umurongo wa mbere nuwa kabiri uhuza ibyabaye, (Sub 1: OO + B ⇄ BO *, Kd1 = 290 ± 70 μM; Sub 2: BO * + B ⇄ B * O *, Kd2 = 29.3 ± 2.5 μM ). ubururu muri leta yegeranye cyane.Ku GBTA ihuza monomeric Hv1, gutandukana guhoraho kubitekerezo: O ° + B ⇄ B ° iri hagati ya Kd1 na Kd2, ariko hafi yanyuma (imirongo yubururu-ubururu yerekana isano iri hagati). Imiyoboro ya Hv1 irashobora kwiharira mugusimbuza domaine ya N- na C-terminal cytoplasmic hamwe nibice bihuye na Ciona Intestinalis voltage-sensibilité ya fosifata CiVSP (Hv1NCCiVSP) 18,34.Twapimye uburyo bwo kwibanda ku kubuza monomeric Hv1 na GBTA na 2GBI na twabagereranije nu muyoboro wa dimeric (ubwoko bwishyamba) kubuza (Igishushanyo 2a, b) .Twabonye ko itandukaniro ryo guhuza ubufatanye hagati yibi bice byombi ryakuweho muri monomeric Hv1, dushyigikira ibisobanuro byerekana ko GBTA ihuza subunit imwe yongera u isano ya zindi subunit ya inhibitor.Twatekereje ko GBTA ihuza subunit imwe ya Hv1 ishobora gutuma hahindurwa gahunda yo guhuza urubuga (induc fit35), ibyo bikaba byavugurura gahunda yo guhuza ikibanza kidafite aho gihurira na subunit yegeranye, bigatuma guhuza byiyongera ubucuti. Kugirango dusobanure muburyo bwa koperative ihuza GBTA kumuyoboro, twakoresheje icyitegererezo aho subunit ishobora guhuza molekile ya mbere ya inhibitor nyuma ya bimolecular reaction hamwe no gutandukana guhoraho Kd1 (Ishusho 2c, Sub 1, OO + B ⇆ BO *). Guhambira bitera umuyoboro gufata leta aho subunits isigaye yubusa ihuza na inhibitor nyuma yimyitwarire idasanzwe ya bimolecular hamwe no gutandukana guhoraho Kd2, aho Kd2 Iyo molekile ya kabiri ya inhibitor imaze guhambirwa, subunits zombi zifite gutandukana guhoraho Kd2 (Igishusho 2d) .Kubuza umuyoboro wapimwe mubihe bitandukanijwe (+120 mV) .Ibinyabuzima byumuyoboro hamwe na subunit imwe ifunguye (reba inzibacyuho CC ⇄ OC ⇄ OO ku gishushanyo cya 2d) mbere wasangaga batanga umusanzu muke kuri Hv1 muri ibi bihe19,20 bityo ntibashyizwe muburyo bwo guhuza. Umurongo wumukara ukomeye mubishushanyo 2c nuburyo bukwiye bwo kugereranya icyitegererezo kugeragezwa kwibanda-gusubiza umurongo, gutanga Kd1 ya ~ 290 μM na Kd2 ya ~ 29 μM (Igice cyuburyo, kugereranya (6)). guhuza 2GBI, aho Kd2 ≈ Kd1 = Kd (Igishusho 2d) .Mu monomeric Hv1, hari urubuga rumwe rukomatanya na Kdm imwe (O ° + B ⇆ B °) .Mu bijyanye na GBTA, Kdm ni 54 μM, isa cyane na Kd1 kuruta Kd2 (Igishusho 2d). Muyandi magambo, urubuga ruhuza monomer ruri mu miterere (O °) rusa cyane na leta ihuza abantu benshi (BO *) kuruta hasi- affinity state (OO) ya dimer, birashoboka cyane bitewe no gukuraho intera hagati ya subunits. Nkuko byerekanwe mbere kuri 2GBI32, twasanze GBTA yahagaritse imigezi ya Hv1 ihagarika umuyoboro mugihe ifunguye aho kuyikomerera gukingura (Ishusho yinyongera. 2a, b, d) .2GBI nayo izwiho gutera imirizo yangirika buhoro buhoro. hasubijwe ko membrane repolarisation, ariko iki kintu nticyagaragaye muri GBTA (Igicapo cyinyongera. irembo ryikirenge "mehanisme", na 2GBI idindiza gahoro kurenza amarembo afunze.Niba subunit imwe ya Hv1 idafunzwe kandi igafungwa, mugihe subunit yegeranye ikomeza gufungwa, kudahuza molekile zisigaye 2GBI zigenda gahoro (gufata bloceri). imwe gusa yahagaritswe subunit yitwara proton mugihe gito mbere yo gufunga no gutanga umusanzu wingenzi kumurizo wangirika buhoro. Kubona ko kwangirika kwumurongo wumurizo wa Hv1 bitatinze cyane imbere ya GBTA (Umugereka winyongera. . kubyara umusaruro umwe gusa wo guhagarika subunit biteganijwe ko uzaba mwinshi imbere ya GBTA kuruta imbere ya 2GBI, bigatuma umurizo wangirika vuba. Kugirango GBTA ihuze ubufatanye na Hv1 subunits zombi, imbuga zihuza zigomba guhuzwa hamwe.Urubuga rwose ruhuza rugomba kuba rushoboye: 1) gukurura urunana rwibintu bivugana na subunits zegeranye zifatanije na inhibitor, na 2) guhuza abunzi inzibacyuho kuva mubuke-buke kugera kumurongo-mwinshi.Twatekereje ko niba ibisigara byihariye biri murwego rwo guhuza byagize uruhare mubikorwa bya koperative, ihinduka ryabo rigomba guhindura coefficente ya Hill yo guhuza ibitekerezo-byo gusubiza umurongo wa GBTA kubuza Hv1.Ibisigisigi D112, F150 . , na R211S (Igishusho 3) hanyuma ugereranya coefficient zabo zo ku Gisozi niz'ubwoko bwa Hv1 bwo mu gasozi, ukoresheje 2GBI nk'ibisobanuro. Igisigisigi V109 kiri ku isura imwe y'igice cya S1 na D112 kandi gifite impinduka imwe idasanzwe imbere muri selire.Since V109 ntabwo yagize uruhare muguhuza 2GBI29, twakoresheje mutant ya V109A nkigenzura (Ishusho. 3b). . umutuku wijimye) .Buri ngingo igereranya uburyo bwo kubuza ibipimo 3 kugeza kuri 12 ± SD V109 nkigenzura ribi. Imirongo ni imisozi ikoreshwa kugirango ibone indangagaciro za Kd (reba Imbonerahamwe yinyongera 1) .Ibice byo ku musozi (h) byerekanwe muri inset histogrammes zagenwe uhereye kumibare ivugwa mumashusho yinyongera ya 3 na 4.Icyerekezo h indangagaciro za Hv1 WT zerekanwa nkumurongo ucagaguye.Asterisks yerekana itandukaniro rishingiye ku mibare hagati yimiterere ya mutant na WT (p 0.05, Ishusho 5d, i) ). Ku rundi ruhande, ihinduka rya D123A ryagabanije cyane coefficente ya Hill ya GBTA (p 0.05/14). Kubera ko kutabogama kwishyurwa kumwanya wa 123 kuri subunits zombi byatumye habaho impinduka zikomeye mubufatanye bwa GBTA, mugihe guhindura amafaranga kuri subunits zombi byagize ingaruka nkeya gusa, twongereye isesengura kugirango dushyiremo subunit imwe gusa hamwe numuyoboro wa inversion.We yabyaye Hv1 ihuza ibipimo hamwe na D123R isimburwa muri C-terminal subunit (Igicapo 5b) hanyuma dupima uburyo bwo gukumira-gusubiza hamwe na GBTA na 2GBI. Twasanze ko coefficient de Hill ya GBTA ihuza imiyoboro ya WT-D123R yari hejuru cyane kurenza iyo y'ubwoko bw'ishyamba Hv1 (p 0.05). Twasanze kandi mugihe βME idahari, coefficient de Hill ya GBTA ihuza dimer ya D112E I127C Hv1 yari hejuru cyane ugereranije nuwapimwe imbere yo kugabanya imiti (Igicapo cya 6e nigishushanyo cya 4), bivuze ko ihinduka rya D112E. ntiyavanyeho ubwiyongere bwa koperative GBTA ihuza ubufatanye biturutse ku guhuza kwa sisitemu 127. Coefficient de Hill ya 2GBI ihuza D112E, ibipimo bya I127C Hv1 nayo ntiyigeze ihindurwa cyane na mutation ya D112E (Ishusho 1) .6d ninyongera. Igishushanyo 3). Ufatiye hamwe, ibi byagaragajwe byerekana ko guhuza GBTA byongerewe imbaraga n’imikoranire hagati yimpera zinyuma za S1 mubice byegeranye bya Hv1 binyuze mumikoranire ishimishije ya electrostatike cyangwa binyuze mumikorere ya covalent hagati ya sisitemu isimbuwe na Allosteric guhuza imbuga byiyongera kandi bikavamo ubufatanye. Mugihe ingaruka zimikoranire ya electrostatike ishimishije kubufatanye irashobora gukurwaho na mutation D112E, ingaruka zubusabane ntibishobora. Mu bushakashatsi bwakozwe ku mwanya wa shimi uboneka kugirango uhuze ibikomoka kuri guanidine kumuyoboro wa Hv1, twasanze 2-guanidinothiazoles nka GBTA bifite aho bihurira cyane na 2-guanidinobenzimidazoles (Igicapo 1c) .Isesengura rya coefficient de Hill ya GBTA ihuza na dimeric zombi. n'imiyoboro ya monomeric (Igishusho 2a, b) no kumuyoboro wa dimeric aho subunit imwe yabanje guhambirwa kuri inhibitor (Igishusho 4) byatumye twemeza ko kubuza Hv1 na GBTA Igikorwa ni inzira yo guhuza, kandi imbuga zihuza ibice muri subunits zombi zahujwe hamwe.Ivumburwa ryuko GBTA ihuza umuyoboro ufunguye, nkuko byerekanwe mbere kubyerekeranye na 2GBI32, byerekana ko guhuza allosteric bishobora gusuzumwa byumwihariko mugihugu cyafunguye. yashoboye gusobanura cyane irwanya HV1 na GBTA (FIG Coefficient ntarengwa ishobora kugerwaho muri poroteyine ya alloster hamwe nubumbuzi bubiri bugera kuri HB1. Ingufu zubusa, itandukaniro riri hagati yubusa bwubusa bwimbuga zo hasi kandi zisumbuye (reba uburyo), ni indege ya 1,3. ya ogisijeni kuri hemoglobine ni urugero ruzwi cyane kandi rwiga cyane rwibikorwa38.Kuri umukunzi wa muntu ufite ubumuga bwo guhambira abantu 1.5-3.0. KCAL / MOL, bitewe nibigeragezo38.Kuri, ukurikije ingufu zisi yose, mu masezerano ya GBTA ahuza na HV1 ntabwo atandukanye cyane na O2 ahuza hemoglobine iyo umubare munini wa poroteyine. Muri Moderi yacu ya Synergy, guhuza molekile ya GBTA kumutwe umwe wa subtit mu kwiyongera kwububiko bwa hegeranye buturuka ku bidukikije bihuza (bitera) biganisha ku Impinduka mubikorwa hagati ya subunts.in Igisubizo, Guverinoma yegeranye ihindura ibibanza byabo bihuza na GBTA, S1 ashinzwe gutunganiza mbere yerekanwe ko ari igice cya HV1 Proton Parmeton Urujya n'uruza rw'uruhushya no gukora nk'itoranya muyungurura27,28. Ibisubizo byacu byerekana ko muyunguruzi muri HV1 ya HV1 ya HV1 yatunganijwe muri leta ifunguye Ku nyubako ya kirisiti ya HV-CILIMERA. Icyerekezo Cyikirenga cya Alloster kuri Alloster Guhuza birimo impera za S1 zerekanwa nimyambi yumukara. . Ahantu hahanuwe GBTA ishingiye kuri ovals.black imyambi yerekana inzira ziterwa na alloster. Mu bice bibiri bitandukanye bitandukanijwe nkuko bigaragara kuva kuruhande rwindege ya membrane. Gutandukanya impera yinyuma yibisenge bibiri bya S4 (imyambi ipanga), itanga gahunda yerekanwe iburyo.ibintu bya D123 na I127 kuva kugereranya na Cyiza VSD Mubiboneza bikaba byabonetse mumiterere ya 4g80. Andika P140 mubya CIMESUjuje imyanya D123 muri HV1. Ibisubizo byacu byerekana ko imikoranire ya electrostatike iri hagati ya bisiba 123 (123d / 123d / 123r) ifitanye isano n 'amakoperativitino yo gufungura muri leta (123D / 123R) , Kongera ubufatanye (FIG. Ibisobanuro nuko ingaruka zo guhungabanya gushyira ibisigazwa hydrophobike mumashanyarazi bishobora kuvaho ingaruka zihamye hagati ya koperativitizi zibangamira amakoperativitizi. Umwanya D171 wa Ciona Gudis Ci-HV1 (bihuye na D123 muri DV1) byiyongereye ku gikorwa, gishyigikira igitekerezo kivuga ko D123 iherereye mu mashanyarazi mu buryo bweruye. Gutora imiyoboro ya HV1 bizwiho kuba mu mpinduka nyinshi19,20,39.40,40.41.61.61.QUU et al26 ihura nimpinduka za CI-HVI1 ziva mu miyoboro ikora ariko iracyafunze , hakurikiraho inzitizi zitandukanye zitera prototo kugirango ifungure munzira zombi. Urujya n'uruza rw'ibimenyetso bya fluorescent bihuye no kuba hari imikoranire ya electrostatike hagati ya D171 Ibisigisigi byegeranye.Ibisobanuro bihuye nibitekerezo byacu kuri D123 Imikoranire ya electrodata Kandi mexiateates ya allosternic guhuza hagati ya GBTA. Fujiwara et al25 yasabye ko umurongo wa Cytoplasic yakoranye-coil domain yaguye muri membrane ebyiri (figne yibumoso) .Icyitegererezo cyibumoso Isesengura rya vsd yose kandi rikora ryakarere rihuza s4 helix ya S4 na CCD. Kubura kwa Transmemmane ph Gradient, hamwe na Cystene bibaho kugirango bifungure, kandi cysteine ​​bibaho mubihe byiganjemo. Kubwibyo, interineti ya S4 yamenyekanye irashobora kwerekana ko iboneza rya leta rya leta. Ibihugu bifunze, igitekerezo gihuye nibisubizo bya Mony et al. S1 yimuka 39 mugihe cyo gutorwa. Hano, turerekana ko, muburyo bwuguruye, iherezo rya S1 Helix Yegeranye bihagije kugirango dushyigikire imikoranire ya electrostatic hagati ya sublings Usibye mugenzi wawe kugirango usangire muburyo butaziguye Hamwe nibitekerezo byacu (FIG. Nubwo Enzyme Cyermem itekereza ko ikora nk'umuntu, imiterere ya kirisiti ya VSD yafashwe muri Leta ya Dimer.Ibice byo hejuru byatanzwe muri iyi dimer muri iyi dimer bifunga hamwe nabyo bisa naho byasabwe Kuri HV1 (FIG. Kandi dimers mbonezo yerekana ko vsds ziyi poroteyine zifite imyumvire yimbere yo gukora interineti aho impera yamakuru ya S1 akorana. Uruhare rwingenzi rwa HV1 muri Spermar Revorcation ituma uyu muyoboro utera ibiyobyabwenge bishimishije Kugarura uburumbuke bwabagabo. Igikorwa cya HV1 muri Microgli stroke.Ibikorwa byabonetse ko bifitanye isano na hepfo Kurokoka abarwayi bafite amabere 12 cyangwa kanseri y'amabara 13 kandi batekereza ko bagana kuri B-selile pelignancique. Fungura HV1 Subunit, biganisha ku kongera ingwate, birashobora kuganisha ku iterambere ryibiyobyabwenge byinshi byibasiye imiyoboro ya HV1. Urubuga-ruyobowe na mutagenes ya muntu HV1 yakozwe hakoreshejwe tekiniki ya PCR isanzwe.in-96 na 228-57 ya HV1 ya HV1.in. C-termis imwe ya subunit imwe ihujwe na n-termis ya kabiri ya Sugunit ya kabiri binyuze muri GGSGSGSGGG T7 MMASSANDA MMACHINE (Ambien) .1-3 Iminsi Yibipimo bya electropysiologiya, CRNAPOS OOCYSTS, CRNOPUS OOCYTES (CLNOPUS OOCYTES (CLNAS OOCYTES (50 NL kuri. Kuva Ecocyte Biossience.Uburebure bwa RNATS, Utugari twakomeje muri ND96 muri ND96 MM NACL, 2 MM Pyruvate, 5 mm, 100 μG . 2-Guanidino-BenziMidazole [1], 2-Guanidino-Bezotiazole [2], (4-thiazol-2-thiazol-2-YL) Guanidine [5], (4 -UM-4 -3Meyl-1,3 -icyubahiro-2-yl) Guanidine) [6], Ethyl 2-Guanidino-Guanidino-Guanidino-5-Thiazole-4-Igikorikori 5-Kanseri [9] na (2-Guanidino-4-methyl-1,3-Thiazol-5-Thiazol-5] yakomokaga muri Sigma-aldrich.amotidine [7] yakomokaga muri Sigmedicals.1- -. Kuva Matrix siyanse.Ikinyago ni zo mu bucuruzi bwo hejuru mu bucuruzi .Bashongeshejwe mu gisubizo cya MM 100 byibuze 99% ubuziranenge. Kuri Guhagarika 2-4- (Trifluoromethyl) Benzenethiol Hydrochloride (1.02 G, 4.5 MG, 4.5 n yarumiwe cyane kumasaha 4.Imvugo ya reaction yakonje ku bushyuhe bwicyumba kandi itabogamye ya POTASSIM 65 ° C) Amasaha menshi, hanyuma usubiremo kuva Ethyl Acetate / perroleum ether kugirango utange igikomere gikomeye (500 mg, 48%); Mp 221-222 ° C (Umucyo 225-226 ° C) 45; 1h NMR (500 MHZ, D6): Δ [ppm] = 7.25 (3 h. , 1 H, j = 8.1 hz) .13c NMR (200 MMZ, D6), 127. 3. 3. 3. D6.2 (D. = 272 hz), 126.1 (q, j = 272 hz) = 31.6 hz), 134.8, 155.4, 175.4, 175.4, 175.4 : 261.0419. Naphtho [1,2-D] Thiazol-2-Amine (300 MG, 1.5 MMOL), yashizwe muri 200 ° yashyutswe muri 200. 1.0 ml .kukoporora ashyushye yongeyeho aside yavunitse mo uduce duto kandi yogejwe n'amazi kugirango atange Amoni Umuhondo Wumuhondo. (38 MG, 10%) Mp 246-250 ° C; 1h NMR (500 MHZ, D6, D6, D6, D6, D6, Δ [ppm] = 7.59 (t, 1 h, 8. 8 h. , J = 8.6 hz), 7.89 (d, 1 h, 8.6 hz), 8.02 (d, 8. 8 h, 80 hz) .13c NMR (150 MHZ, DMSO-D6): Δ = 119.9, 122.4, 123.6, 123.6, 129.7, 127.1.2 , habonetse: 243.0704. Imigezi ya proton yapimwe mubice byimbere kandi byo hanze bya oocytes byerekana inyubako zitandukanye ukoresheje Axpata ya Amplifier yagenzuwe na Axon ) acide ya Ethaneulfonic (Mes), 30 MM TetraethYlammonium (icyayi) Mesylal, 5 mm etlone glycol-bis (2-aminoetyeyl) -n, acide ya acide ph 6.0 hamwe nicyayi cya Hydroxide.Ibipimo byose byakorewe kuri 22 ± 2 ° C.Ububiko bwabashutse kuri 1 KHZ hanyuma usesengure kuri 5 KHZ hanyuma usesengurwa na Clampfit10.2 (ibikoresho bya molekit ) kandi ukomokamo8.1 (Inclab). Ibisubizo birimo imyumvire itandukanye ya HV1 Arimotor kandi Rimwe na rimwe MM βme yatangijwe mu bwogero bwa GC-6 Abacukuzi) Ibimenyetso.Ibimenyetso bya Perfusion yakozwe hakoreshejwe ikaramu nyinshi (byikora sciate. +120 MV Depolariring Ibipimo bya Pulse.GV byakozwe nkuko byasobanuwe mbere18.20, 200 Byakoreshejwe Kuri Gukosora Kubora Hamwe na Hill Plation: Aho