Kev nug ntawm intersubunit interface ntawm qhib Hv1 proton channel nrog kev sojntsuam sib txuas ua ke

Ua tsaug rau koj tuaj xyuas Nature.com.Qhov browser version uas koj siv tau txwv kev txhawb nqa rau CSS.Rau qhov kev paub zoo tshaj plaws, peb xav kom koj siv qhov browser tshiab (lossis tua hom kev sib raug zoo hauv Internet Explorer).Lub sijhawm no, kom paub meej txuas ntxiv kev txhawb nqa, peb yuav tso saib lub vev xaib tsis muaj qauv thiab JavaScript. Hv1 voltage-gated proton channel yog ib qho dimeric complex uas muaj ob qhov voltage-sensing domains (VSDs), txhua tus uas muaj gated proton permeation pathway.Dimerization yog tswj los ntawm cytoplasmic coiled-coil domain.Qhov kev hloov ntawm lub xeev kaw mus rau qhib lub xeev nyob rau hauv ob qho tib si VSDs yog paub tias tshwm sim cooperatively; Txawm li cas los xij, me ntsis paub txog cov txheej txheem hauv qab.Intersubunit interfaces ua lub luag haujlwm tseem ceeb hauv cov txheej txheem allosteric; Txawm li cas los xij, cov kev cuam tshuam zoo li no tsis tau txheeb xyuas nyob rau hauv qhov qhib Hv1 channels.Ntawm no peb pom tias 2-guanidinothiazole derivatives thaiv ob Hv1 VSDs hauv kev sib koom tes thiab siv ib qho ntawm cov tebchaw no ua kev sojntsuam rau allosteric coupling ntawm qhib subunits.Peb pom tias cov extracellular. Qhov kawg ntawm thawj cov khoom tawg ntawm VSD ua ib qho kev sib txuas sib txuas uas cuam tshuam kev sib txuas ntawm cov chaw sib txuas, qhov chaw coiled-coil domain tsis ncaj qha rau hauv cov txheej txheem no.Peb kuj pom muaj pov thawj tias cov channel proton-selective lim tswj kev sib koom ua ke ntawm kev sib koom ua ke. . Voltage-gated proton channels ua lub luag haujlwm tseem ceeb hauv ntau yam kab mob, los ntawm phytoplankton rau tib neeg1.Nyob rau hauv cov cell feem ntau, cov channel no kho cov proton efflux los ntawm cov proton membrane thiab tswj cov kev ua ntawm NADPH oxidase.Tsuas paub voltage-gated proton channel nyob rau hauv tib neeg. yog Hv1, uas yog cov khoom ntawm HVCN1 gene2,3.Hv1 (aka VSOP) tau pom tias ua lub luag haujlwm hauv B cell proliferation4, tsim cov pa oxygen reactive hom los ntawm lub cev tiv thaiv kab mob 5,6,7,8, phev cell. motility9 thiab pH kev cai ntawm cov pa dej saum npoo av 10. Cov channel no tau koom nrog ntau qhov Overexpressed hauv hom mob qog noj ntshav xws li B-cell malignancies4,11 thiab mob qog noj ntshav ntawm lub mis thiab cov qog nqaij hlav hauv plab 12,13.Kev ua haujlwm Hv1 ntau dhau tau pom los ua kom cov metastatic muaj peev xwm ntawm cov qog nqaij hlav cancer 11, 12 .Hauv lub hlwb, Hv1 tau qhia. los ntawm microglia, thiab nws cov haujlwm tau pom tias ua rau lub hlwb puas tsuaj hauv cov qauv ntawm ischemic stroke. Lub Hv1 protein muaj qhov voltage-sensing domain (VSD), uas muaj plaub ntu transmembrane hu ua S1 mus rau S414.VSD zoo li cov sib txuas ntawm voltage-gated Na +, K + thiab Ca2+ channels thiab voltage-sensitive phosphatase, xws li CiVSP los ntawm Ciona gutis15.Nyob rau hauv lwm cov proteins, lub C-terminus ntawm S4 yog txuas mus rau ib tug effector module, lub pore domain los yog enzyme.In Hv1, S4 yog txuas mus rau lub coiled-coil domain (CCD) nyob rau ntawm cytoplasmic sab ntawm daim nyias nyias. .Cov channel yog dimeric complex uas muaj ob VSDs, txhua tus muaj gated proton permeation pathway16,17,18.Cov ob Hv1 subunits no tau pom los qhib kev sib koom tes19,20,21,22, tawm tswv yim tias allosteric coupling thiab inter-subunit sib cuam tshuam ua si. lub luag haujlwm tseem ceeb hauv cov txheej txheem gating.Qhov kev sib tshuam ntawm cov subunits nyob rau hauv lub coiled coil domain yog zoo-txhais vim hais tias cov siv lead ua qauv ntawm ob tug cais domains yog muaj22,23.Ntawm qhov tod tes, lub interface ntawm VSDs nyob rau hauv lub membrane yog tsis. nkag siab zoo.Cov qauv siv lead ua ntawm Hv1-CiVSP chimeric protein tsis muab cov ntaub ntawv hais txog qhov kev sib tshuam no, vim tias lub koom haum trimeric ntawm crystallized channel complex yuav tshwm sim los ntawm kev hloov ntawm haiv neeg Hv1 CCD los ntawm cov poov xab leucine zipper GCN424. Kev tshawb fawb tsis ntev los no ntawm lub koom haum subunit ntawm Hv1 channel xaus lus tias ob lub S4 helices hloov mus rau hauv CCD yam tsis muaj kev cuam tshuam loj ntawm cov qauv theem nrab, uas ua rau cov helices ntev uas pib ntawm daim nyias nyias thiab qhov project mus rau hauv cytoplasm.Raws li cysteine ​​​​crosslinking tsom xam, txoj kev tshawb no qhia tias Hv1 VSDs sib cuag nrog S4 ntu.Txawm li cas los xij, lwm cov kev tshawb fawb tau tawm tswv yim lwm qhov sib cuam tshuam ntawm VSDs.Cov kev cuam tshuam no suav nrog S1 ntu 17, 21, 26 thiab sab nraud ntawm S2 ntu 21.A qhov laj thawj rau qhov tsis sib haum xeeb. Cov txiaj ntsig ntawm cov kev tshawb fawb no yog tias kev sib txuas ntawm kev sib txuas ntawm VSDs tau raug tshuaj xyuas nyob rau hauv kev sib raug zoo nrog cov txheej txheem gating, uas nyob ntawm lub xeev kaw thiab qhib, thiab qhov sib cuam tshuam ntawm VSDs tuaj yeem sib txawv hauv ntau lub xeev kev hloov pauv hauv kev sib haum xeeb. Ntawm no, peb pom tias 2-guanidinothiazole inhibits Hv1 channels los ntawm synergistically khi rau ob lub VSDs qhib, thiab siv ib qho ntawm cov tebchaw, 2-guanidinobenzothiazole (GBTA), los soj ntsuam kev sib cuam tshuam ntawm subunits hauv lub xeev qhib. interface.Peb pom tias GBTA khi nkhaus tuaj yeem piav qhia tau zoo los ntawm ntau tus qauv uas khi ntawm ib qho inhibitor rau ib lub subunit ua rau muaj kev nce hauv kev sib txuas ntawm cov subunit uas nyob ib sab.Peb kuj pom tias cov seem D112, cov lim dej xaiv rau cov channel 27, 28 thiab ib feem ntawm guanidine derivative binding site 29 tswj GBTA binding cooperativity.Peb qhia tau hais tias kev koom tes binding yog tswj nyob rau hauv lub Hv1 dimer, qhov twg CCD cais los ntawm lub S4 ntu, qhia hais tias lub intersubunit interface nyob rau hauv lub CCD tsis ncaj qha. Kev sib haum xeeb allosteric coupling ntawm GBTA-binding sites.Qhov sib txawv, peb pom tias S1 fragment yog ib feem ntawm kev sib cuam tshuam ntawm cov subunits thiab npaj ib qho kev npaj ntawm VSDs uas nyob ib sab nrog lub extracellular kawg ntawm S4 helix deb ntawm qhov chaw ntawm lub dimer kom tso cai. lub S1 fragment yuav tsum nyob rau hauv lub xeev qhib. Me me molecule inhibitors ntawm Hv1 muaj txiaj ntsig zoo li cov tshuaj tiv thaiv kab mob thiab cov tshuaj tiv thaiv neuroprotective.Txawm li cas los xij, rau hnub tim, ob peb lub tebchaw muaj peev xwm inhibit channel30,31,32,33.Ib ntawm lawv, 2-guanidinobenzimidazole (2GBI, compound [1] hauv daim duab. . yav dhau los tau pom tias inhibit Hv1 yuav luag zoo li 2GBI thaum kuaj ntawm qhov siab ntawm 200 μM (Daim duab 1b).Peb tau tshuaj xyuas lwm cov thiazole derivatives thiab pom tias qee qhov ntawm lawv inhibited cov channel uas muaj zog zoo sib xws lossis ntau dua li GBTA (Fig. 1 thiab Ntxiv. ntawv).Peb tau txiav txim siab cov ntsiab lus teb nkhaus ntawm plaub thiazole derivatives (GBTA thiab cov tebchaw [3], [6] thiab [11], Fig. 1c) thiab pom tias lawv steeper tshaj cov ntawm 2GBI.The Hill coefficients (h) ntawm thiazole derivatives ranged ntawm 1.109 ± 0.040 txog 1.306 ± 0.033 (Fig. 1c thiab ntxiv daim duab 1).Qhov sib piv, Hill coefficient rau 2GBI yog 0.975 ± 0.024 29, Fig. 2a thiab Ntxiv Fig. 1).A Hill coefficient saum toj 1 qhia txog kev sib koom tes-kev sib koom tes.Vim txhua Hv1 subunit muaj nws tus kheej. binding site, 29,32 peb xav tias kev khi ntawm thiazole derivative rau ib lub subunit tuaj yeem txhim kho qhov khi ntawm qhov thib ob inhibitor molecule rau subunit uas nyob ib sab.GBTA yog qhov sib xyaw nrog qhov siab tshaj Hill coefficient.Yog li ntawd, peb xaiv qhov sib xyaw no rau kawm ntxiv txog cov txheej txheem ntawm kev sib koom ua ke thiab siv 2GBI raws li kev siv tsis zoo. (a) Test compounds: [1] Reference Hv1 inhibitor 2-guanidino-benzimidazole (2GBI).[2] 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) Inhibition of human Hv1 kev ua los ntawm cov qhia guanidinothiazoles thiab cov ntaub ntawv siv 2GBI (xiav-ntsuab tuav) .Hv1 proton tam sim no tau ntsuas nyob rau hauv-tawm plaques ntawm Xenopus oocytes nyob rau hauv teb rau depolarization los ntawm ib tug tuav lub peev xwm ntawm -80 mV rau +120 mV.Txhua tus inhibitor tau ntxiv rau da dej ntawm ib tug concentration ntawm 200 μM.pHi = pHo = 6.0 .Cov ntaub ntawv yog txhais tau tias ± SEM (n≥4).(c) Concentration-dependent inhibition ntawm tib neeg Hv1 los ntawm cov tebchaw [2], [3], [6] thiab [11] .Txhua taw tes sawv cev qhov txhais tau tias inhibition ± SD ntawm 3 mus rau 15 ntsuas. Cov kab yog Hill haum siv kom tau txais qhov pom tseeb Kd qhov tseem ceeb tshaj tawm hauv Cov Lus Ntxiv 1.Hill coefficients tau txiav txim siab los ntawm qhov haum qhia hauv Daim duab ntxiv 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 (saib Methods). (a, b) Compounds 2GBI thiab GBTA inhibited dimeric thiab monomeric Hv1 nyob rau hauv ib tug concentration-dependent yam.Txhua point sawv cev rau qhov txhais tau tias inhibition ± SD ntawm 3 mus rau 8 kev ntsuas thiab qhov nkhaus yog Hill haum.The Hill coefficients (h) qhia hauv cov inset histograms tau txiav txim siab los ntawm qhov haum tau tshaj tawm hauv Cov Lus Qhia Ntxiv 3 thiab 4.Cov lus teb ntawm GBTA qhia hauv (a) yog tib yam li hauv daim duab 1c.Saib Cov Lus Ntxiv 1 rau qhov pom tseeb Kd qhov tseem ceeb.(c) Kev ua qauv ntawm txoj kev sib koom tes ntawm GBTA rau dimeric Hv1.Cov kab dub tawv sawv cev rau qhov haum rau cov ntaub ntawv sim los ntawm kev sib npaug (6), uas piav qhia txog cov qauv kev khi qhia hauv (d).Cov kab dashed sau npe Sub 1 thiab Sub 2 sawv cev rau lub koom haum bimolecular -dissociation equilibrium curves ntawm thawj thiab thib ob binding txheej xwm, ntsig txog (Sub 1: OO + B ⇄ BO *, Kd1 = 290 ± 70 μM; Sub 2: BO * + B ⇄ B * O *, Kd2 = 29.3 ± 2.5 μM ).(d) Schematic daim duab ntawm lub tswv yim mechanism ntawm Hv1 block.Thaum GBTA, khi rau ib qho qhib subunit nce affinity ntawm qhov qhib subunit uas nyob ib sab (Kd2 0.05) txo nyob rau hauv lub Hill coefficient ntawm GBTA khi rau GGG raws piv nrog cov qus hom (Fig. 5c), tawm tswv yim hais tias txawm nws lub luag hauj lwm nyob rau hauv ua kom cov ob lub subunits ua ke tseem ceeb, tab sis CCD tsis ncaj qha cuam tshuam intersubunit allosteric coupling ntawm GBTA binding sites. (a) Schematic daim duab ntawm Hv1 dimer nrog kev hloov pauv triglycine nyob rau sab hauv kawg ntawm S4 tsim los cuam tshuam cov intersubunit coupling mediated los ntawm cytoplasmic coiled-coil domain (xiav xub).(b) Schematic sawv cev ntawm Hv1 dimers thiab ligation dimers nrog qhia txog kev hloov pauv, tsim los sim S1 cov khoom sib txuas hauv kev sib txuas ntawm subunits (xub xub). ± SD ntawm 3 txog 10 kev ntsuas. Qhov nkhaus yog Hill haum siv kom tau txais qhov pom tseeb Kd qhov tseem ceeb (saib Daim Ntawv Ntxiv 1).Hill coefficients nyob rau hauv interpolated histograms raug txiav txim raws li tau piav nyob rau hauv cov txheej txheem (saib Ntxiv Fig 3 thiab 4).Reference h qhov tseem ceeb rau Hv1 WT tau pom raws li cov kab dashed.Asterisks qhia qhov sib txawv tseem ceeb ntawm cov mutant thiab WT cov nqe lus (p 0.05, Fig. 5d, i ) ). Ntawm qhov tod tes, kev hloov pauv D123A txo qis Hill coefficient ntawm GBTA (p 0.05/14). Txij li thaum neutralizing tus nqi ntawm txoj hauj lwm 123 ntawm ob lub subunits ua rau muaj kev hloov pauv hauv GBTA kev sib koom tes zoo, thaum thim rov qab tus nqi ntawm ob lub subunits tsuas muaj qhov cuam tshuam me me, peb tau txuas ntxiv qhov kev tshuaj xyuas kom suav nrog tsuas yog ib lub subunit nrog lub inversion channel ntawm tus nqi. generated Hv1-linked dimers nrog D123R hloov pauv nyob rau hauv C-terminal subunit (Fig. 5b) thiab ntsuas qhov concentration-response inhibition los ntawm GBTA thiab 2GBI.Peb pom tias Hill coefficient ntawm GBTA khi rau WT-D123R raws tau ntau dua li qhov ntawd. ntawm hom tsiaj qus Hv1 (p 0.05). Peb kuj pom tias thaum tsis muaj βME, Hill coefficient ntawm GBTA khi rau D112E I127C Hv1 dimer tau ntau dua li qhov ntsuas hauv qhov muaj cov neeg ua haujlwm txo qis (Daim duab 6e thiab Cov duab ntxiv 4), implying tias D112E kev hloov pauv. tsis tau tshem tawm qhov nce ntawm GBTA binding cooperativity uas tshwm sim los ntawm kev sib txuas ntawm cysteine ​​​​127.The Hill coefficient ntawm 2GBI khi rau D112E, I127C Hv1 dimers kuj tsis cuam tshuam loj heev los ntawm D112E kev hloov pauv (Daim duab 1).6d thiab ntxiv. Daim duab 3). Muab ua ke, cov kev tshawb pom no qhia tias GBTA kev khi tau txhim kho los ntawm kev sib cuam tshuam ntawm sab nrauv ntawm S1 tawg nyob rau hauv ib sab Hv1 subunits los ntawm kev sib cuam tshuam electrostatic txaus nyiam lossis los ntawm kev tsim cov ntawv cog lus ntawm cov cysteine ​​Allosteric coupling ntawm qhov chaw nce thiab ua rau muaj kev sib koom tes. Thaum cov txiaj ntsig ntawm kev sib cuam tshuam electrostatic txaus nyiam ntawm kev sib koom tes tuaj yeem raug tshem tawm los ntawm kev hloov pauv D112E, cov txiaj ntsig ntawm covalent bonds tsis tuaj yeem. Hauv peb qhov kev tshawb nrhiav ntawm cov chaw tshuaj lom neeg muaj rau kev khi guanidine derivatives rau Hv1 channels, peb pom tias 2-guanidinothiazoles zoo li GBTA muaj qhov siab tshaj qhov dependence tshaj 2-guanidinobenzimidazoles (Fig. 1c).The Hill coefficient tsom xam GBTA khi rau ob lub dimeric. thiab monomeric raws (Fig. 2a, b) thiab mus rau lub dimeric channel nyob rau hauv uas ib tug subunit twb pre-bound rau tus inhibitor (Fig. 4) coj peb mus xaus hais tias lub inhibition ntawm Hv1 los ntawm GBTA qhov kev txiav txim yog ib tug synergistic txheej txheem, thiab Cov chaw sib txuas ntawm cov khoom sib txuas hauv ob lub subunits yog allosterically ua ke.Qhov kev tshawb pom tias GBTA khi rau qhov qhib channel, raws li yav dhau los tau qhia rau cov sib txuas sib txuas 2GBI32, qhia tias kev sib txuas ntawm allosteric tuaj yeem raug soj ntsuam tshwj xeeb hauv lub xeev qhib.Peb cov qauv kev sib koom ua ke Muaj peev xwm mus rau ntau ntawm HV1 los ntawm HV1 los ntawm LAHTA (Daim duab 2C) thiab piav qhia qhov sib txawv ntawm 2GBI thiab GBTA ntawm Membrane Recolarization, uas txhawb peb txhais cov txheej txheem ntawm cov txheej txheem khi. Qhov ntau tshaj plaws toj coefficient uas tuaj yeem ua tiav hauv cov protein nrog ob qho kev ntsuas tsiaj (xws li nce li 1.88 hauv HV1 I127C.Qhov Synergistic pub dawb lub zog, qhov sib txawv ntawm cov laj thawj dawb ntawm qhov chaw qis thiab ntau tshaj plaws rau ntawm hv1 i127c.the binding ntawm cov pa rau hemoglobin yog cov qauv zoo tshaj plaws ntawm kev sib koom tes nrog plaub ntawm cov chaw sib koom tes nrog plaub ntawm 2.5-3.0, nrog qhov tseem ceeb tshaj plaws los ntawm 1.26 rau 3.64 Kcal / Mol, nyob ntawm ntawm kev sim cov yam ntxwv: Nyob rau hauv peb tus qauv synergy, tus kab nrib ntawm gbecules rau ib qho kev sib txawv ntawm cov txheej txheem uas nyob ib puag ncig uas muaj los ntawm thawj zaug kev tshwm sim) ua rau Kev hloov pauv ntawm cov kev sib cuam tshuam ntawm cov kev hloov pauv.Yog teb rau cov kev hloov pauv no tau hloov pauv ntawm lub tsev kawm ntawv uas muaj feem cuam tshuam nrog kev sib tsoo ntawm kev sib tw nrog kev sib tw nrog kev sib tsoo.d112 Yav tas los tau pom tias yog ib feem ntawm HV1 Proton proconation permay permay thiab ua raws li xaiv lim27,28. Peb cov txiaj ntsig qhia tau tias kev xaiv cov ntxaij lim dej hauv ob qho chaw seem ntawm HV1 yog cov chaw nyob ntawm GBTA cov chaw nyob ntawm HBTA cov chaw nyob ntawm HV1 VSD raws Ntawm lub crystal qauv ntawm HV1-COMSP chimera.suggic txuas nrog cov extracellular kawg ntawm S1 tau qhia nrog cov xib xub dub. (a) Schematic ntawm HV1 vsd.Based ntawm cov qauv siv lead ua Qhov kev kwv yees thaj tsam ntawm khi tau ua tiav gbta ovals.black subs qhia txoj hauv kev uas nyob rau hauv cov kev tshawb fawb ntawm HV1 Cov Lus Qhia Hauv ob qho kev sib txawv ntawm kev sib txawv raws li pom los ntawm cov khoom sib txuam ntawm cov vsds: sib cais ntawm sab nraud xaus ntawm ob lub siab pab cuam s4 ​​helices (dashed txoj kev npaj rau sab xis. (C) Kev sawv cev ntawm cov neeg Civsp Nyob rau hauv cov Dimer teeb tsa pom nyob rau hauv 4G80 Crystal qauv.PloTs P140 hauv Civsp sib raug rau txoj haujlwm D123 hauv HV1. Peb cov txiaj ntsig qhia tau tias kev sib cuam tshuam hauv hluav taws xob hauv nruab nrab ntawm thaj chaw qhib ntawm GBTA-sib tham los ntawm kev sib cuam tshuam ntawm kev ua kom zoo siab rau cov neeg nyiam (123D / 123R) , Kev koom tes ntau ntxiv (Daim duab 5G) Cov lus piav qhia yog tias txoj kev ua tau zoo ntawm tso cov nyhuv hydrothhilic yuav ua rau muaj kev cuam tshuam ntawm hydrothilic yuav ua rau muaj kev cuam tshuam ntawm hydring. Txoj Haujlwm D171 ntawm Ciona Gutis Ci-HV1 (coj mus rau D123 hauv tib neeg HV1) tau nce ntxiv thaum D123 nyob rau hauv ib puag ncig Hydrothilic hauv lub xeev qhib. Gating ntawm HV1 raws tau paub tias muaj los ntawm ntau cov kev hloov pauv, cov khoom siv volranes ntawm Ci-HV1 undergoes hloov pauv cov channel qhib tab sis tseem raug kaw , ua raws li kev hloov pauv txawv uas ua rau cov neeg muaj kev sib cav sib ceg yog kev hloov pauv ntawm cov kev hloov pauv ntawm qhov kev sib hloov ntawm txoj haujlwm D171. Kev sib txuas lus fluurity ntawm fluorescent teeb liab yog muaj kev sib txuas lus ntawm D171 cov lus txhais tau raws li peb nrhiav tau tias D123 cov khoom seem sib cuam tshuam hauv lub xeev qhib Thiab kev sib haum xeeb tag nrho cov ntaub ntawv txuas ntawm gbta cov chaw. Fujiwara thiab Al25 tau thov tias cov inster interface ntawm cytoplasmic coiled-cheles kom muaj ob lub interteine ​​hla kev sib txuas npog npog Tag nrho VSD thiab kev tsom xam ntawm thaj av txuas nrog S4 Helix thiab CCD.Thaum tsis muaj kev cuam tshuam txog kev hloov pauv, thiab cystine ntawm cov kab ke uas cov channel yog feem ntau. Yog li ntawd, cov pom tau S4-S4 interface yuav muaj kev sib txuam nrog S1 pom ntawm kev koom tes rau kev koom tes nrog kev sib tw sib txawv hauv kev qhib thiab Kaw hais tias, ib lub tswv yim raws li kev tshawb pom ntawm Mony li al. S1 Tsiv 39 thaum Vaj Tse. Ntawm no, peb qhia tias, nyob rau hauv lub xeev qhib, qhov xaus ntawm S1 Helix tau ze txaus los txhawb kev sib txuam nrog S4-SN4, S1 Helices dhau sib nrug los ntawm txhua lwm yam kom sib tham ncaj qha.Txawm li cas, 20 ° plockwicular nyob rau hauv lub dav hlau vsd ntawm ib qho kev sib cais ntawm ob s4-Sksed ib qho S1-S1 conciguration zoo sib xws Nrog peb cov kev tshawb pom (Daim duab 7B, vaj huam sib luag yog) .Peb pom zoo kom qhov kev teeb tsa no rau cov channel hauv lub xeev qhib. Txawm hais tias tus qauv civsp yog xav ua haujlwm li monomer, cov qauv siv lead ua ntawm lub siab D1 yog qhov sib txuam ntawm S1 yog qhov sib txuam ntawm cov kev sib cais ua ke, thiab kev teeb tsa txheej txheem tag nrho ua ke zoo ib yam li ntawd Rau HV1 (Daim duab 7C) Thiab civsp dimers qhia tias cov protein muaj cov neeg muaj protein muaj ib qho kev sib cuam tshuam ntawm qhov twg ntawm cov extracellular xaus ntawm S1 sib cuam tshuam. Lub luag haujlwm tseem ceeb ntawm kev ua yeeb yam phev ua rau cov channel no ua rau muaj kev tswj hwm tau los ntawm ischemic stroke.themrogremed HV1 kev ua tau pom muaj cuam tshuam nrog qis dua Kev ciaj sia rau cov neeg mob lub mis 12 lossis cov tshuaj tiv thaiv kev ua kom zoo li cov tshuaj tiv thaiv kab mob B-Cell Qhib HV1 subvit, ua rau muaj kev sib tw sib luag, tej zaum yuav ua rau kev loj hlob ntawm cov tshuaj muaj zog ntau tshaj plaws ntawm cov tshuaj HV1. Site-Qhia mutagenesis ntawm tib neeg HV1 tau ua siv PC1NCCivSp txua cov chaw nyob 1-96 thiab 240-576 ntawm HV1-txuas Diaer, C-Terminus ntawm ib tus subunit yog txuas rau n-terminus ntawm ob subsmids nrog NHE1 lossis SPH1 kev txwv tsis pub muaj (England BiolS) thiab RNA synthesis tau ua nrog tus T7 MMESSY COV KHOOM SIV (YAJ DUB) .1-3 hnub ua ntej ntsuas phoo, 0.3-1.5 μY.5 μY.5 μY.5 μY.5 μLE .stage Laevis (NASCO) tau txais Los ntawm Ecrocyte Bioscience.Strollowing RNA kev txhaj tshuaj, cov cell tau tswj hwm hauv ND96 nruab nrab muaj 96 nruab nrab muaj 96 nruab nrab, 2 hli gentamicisin, pH 7.2 18 ° C . 2-guanidino-benzimidazole [1], 2-guanidino-benzothiazole [5], (5-bromo-4 -methyl-1,3 -thiazol-2-YL) Guanidine) [6], Ethyl 2-Guanidino-4-ethyl-1,3-ethyl-1,3-methyl-1,3-thiazole- 5-carboxylate [9] thiab (2-Methyl-1,3-Thiazol-5-YL) ethyl acetate [7] yog los ntawm mp biomedicalss.1- [4 - (4-chlorophenyl) -1,3-Thiazol-2-YL] Guaranidine [11] thiab 1- D dimethoxyphenyl) -1,3- Thiazol-2-4] Guanidine [12] yog Los ntawm matrix science.these cov tebchaw yog ntawm qhov siab tshaj plaws kev daws teeb meem ntawm qhov kev xav tau kawg ntawm qhov kev xav tau kawg ntawm qhov xav tau ntawm 3 thiab 4 tau sib xyaw ua ke nrog tsawg kawg 99% purity. Rau kev ncua ntawm 2-amino-4- (trifluoromethyl) hydrotyide (1.02 g, 4.5 hli mg, 4.5 mmol), thiab lub txiaj ntsig heterogeneous tau refluxed nrog Vigorous stirring rau 4 teev.Qhov sib xyaw ua ke tau ua kom txias dua ntawm 10 toted nag los yog lim dej txias (3 × 50 ml), qhuav hauv qhov cub ( 65 ° C) Rau ob peb teev, thiab tom qab ntawd recrystallized los ntawm ethyl acetate / petroleum ether rau muab cov khoom dawb (500 mg, 48%); MP 221-222 ° C (Teeb 225-226 ° C) 45; 1h nmr (500 mhz, dmo-d6): δ [ppm] = 7.40 (d, 1 h, j = 8 h), 7.92 (d , 1 h, j = 8.1 hz) .13c NMR (200 MHz, DMSO-D6): _ HZ), 121.7 (Q, J = 272 HZ), 126.1 (Q : 261.0419. Naphtho [1,2-d] thiazol-2-amine (300 mg, 1.5 mmol), synthesized li 200 ° 1.0 ml conv.Qhov qhov cub kub tau ntxiv tau ntxiv hydrochloric acid sai thiab cov sib xyaw ua ke tau khaws cia rau chav sov kom sov thiab txias cov khoom siv tau tawg ua tej daim me me thiab ntxuav nrog dej los muab cov daj ntseg daj amorphous khoom. (38 MG, 10%) MP 246-250 ° C; 1h NMR (500 MHZ, DMOPS-D6, D2O): δ [PPM] = 7.2 H, J = 8.7 HZ), 7.77 (D, 1 h , J = 8.6 HZ), 7.89 (D, 1 h, j = 8.2 hli (d, 1 h, j = 8.3 Hz) .13c NMR (150 MHz, DMOPS-D6): Δ = 119.9, 123.H7,hrms (ESI) , pom: 243.0704. Cov tshuaj pleev prod tau ntsuas hauv cov khoom siv sab hauv thiab sab nraud uas tau ua tiav los ntawm AxoDid10.intracel cov kev daws teeb meem tau muaj cov tshuaj sib txawv: 100 hli 2- (n-morpholino ) ethanesulfonic acid (m mm tetraethylammonium (5 hli tshuaj yej) mesylene glycol-bis (2, n'-tetraacetic acid (egta), kho rau pH 6.0 nrog tea hydroxide.Yall ntsuas tau ua rau ntawm 22 khz, piv txwv li kev nkag tau ntawm 5 kHz thiab tshuaj xyuas nrog clampfit10.2 (molecular pab kiag li lawm ) Thiab keeb kwm8.1 (Keeb kwm). Cov kev daws teeb meem muaj ntau ntau yam ntau ntawm HV1 inhibitor thiab qee qhov kev sib txuas ntawm lub zog ntawm VC-6 cov lus sib txuas ntawm POSCLAMS software ttl (tristor- Kev Ntsuas Cov Tsheb Nkag) Kos Npe.Rapid Perpusion Inviments tau ua rau muaj ntau lub raj Incusion Pipenat.) nrog 360 μochronal amochronal kev ntsuas ampeptometric ameribements ntawm qhov kawg ntawm lub +120 MV repidolarizing mulache.Qhov kev ntsuas tau ua raws li yav tas los ntawm -40 MV rau +120 MV tau sau tshwj tsis yog lwm cov tes taw tes ua ntej cov mem tes ntsuas yog siv los kho rau qhov tam sim no tsis ntev tam sim no 18 .Qhov kev sib npaug ntawm Boltocmans (KD) Kev sib xyaw ua ke (KD) Nrog toj sib npaug: qhov twg [Kuv] yog lub concibitor I thiab h yog tus roob coefficient.Lau laij lub roob, kab zauv (2) raug rho tawm li: