Tlhahlobo ea sebopeho sa intersubunit sa mocha oa proton o bulehileng oa Hv1 ka probe e kopantsoeng ka mokhoa o hlakileng.

Kea leboha ka ho etela Nature.com.Mofuta oa sebatli oo u o sebelisang o na le ts'ehetso e fokolang bakeng sa CSS.Bakeng sa boiphihlelo bo botle ka ho fetisisa, re khothaletsa hore u sebelise sebatli se ntlafalitsoeng (kapa u time mokhoa oa ho lumellana ho Internet Explorer).Khabareng, ho etsa bonnete ba hore ts'ehetso e tsoelang pele, re tla bonts'a sebaka sa marang-rang ntle le mekhoa le JavaScript. The Hv1 voltage-gated proton channel is a dimeric complex e nang le libaka tse peli tsa motlakase-sensing (VSDs), e 'ngoe le e' ngoe e na le tsela ea phepelo ea proton ea gated. Dimerization e laoloa ke sebaka sa cytoplasmic coiled-coil. boemo bo bulehileng ho VSD ka bobeli bo tsejoa hore bo etsahala ka ts'ebelisano; leha ho le joalo, ha ho tsejoe hakaalo ka mekhoa ea motheo.Mahokelo a li-intersubunit a phetha karolo ea bohlokoa mekhoeng ea allosteric; leha ho le joalo, li-interfaces tse joalo ha li e-s'o tsejoe likanaleng tse bulehileng tsa Hv1. Mona re bontša hore lihlahisoa tsa 2-guanidinothiazole li thibela li-VSD tse peli tsa Hv1 ka mokhoa oa tšebelisano 'me li sebelisa e' ngoe ea metsoako ena e le probe bakeng sa ho kopanya allosteric pakeng tsa li-subunits tse bulehileng.Re fumane hore extracellular Qetello ea sekhechana sa pele sa transmembrane sa VSD se etsa sebopeho sa intersubunit se kopanyang ho kopanya lipakeng tsa libaka tse tlamang, athe sebaka sa coil-coil ha se amehe ka kotloloho ts'ebetsong ena. . Liteishene tsa proton tse nang le motlakase oa motlakase li phetha karolo ea bohlokoa linthong tse fapaneng, ho tloha ho phytoplankton ho ea ho batho1.Liseleng tse ngata, litsela tsena li buisana le proton efflux ho tloha lera la proton le ho laola mosebetsi oa NADPH oxidase.Ke eona feela mocha o tsejoang oa voltage-gated proton ho batho. ke Hv1, e leng sehlahisoa sa HVCN1 gene2,3.Hv1 (aka VSOP) e bontšitsoe hore e na le karolo ea B cell proliferation4, tlhahiso ea mefuta ea oksijene e sebetsang ka mokhoa oa ho itšireletsa mafung5,6,7,8, sperm cell. motility9 le pH regulation ea airway surface fluid10. Seteishene sena se kenyelelitsoe ho tse 'maloa Ho feta tekano mefuteng ea mofetše e kang B-cell malignancies4,11 le kankere ea matsoele le colorectal12,13.Mosebetsi o feteletseng oa Hv1 o fumanoe ho eketsa bokhoni ba metastatic ba lisele tsa kankere 11, 12. Bokong, Hv1 e hlalosoa. ka microglia, mme ts'ebetso ea eona e bonts'itsoe hore e mpefatsa tšenyo ea boko mefuteng ea ischemic stroke. Protheine ea Hv1 e na le "voltage-sensing domain" (VSD), e nang le likarolo tse 'nè tsa transmembrane tse bitsoang S1 ho ea ho S414. VSD e tšoana le libaka tse tsamaellanang tsa li-channel tsa Na+, K+ le Ca2+ le li-phosphatase tsa voltage-sensitive phosphatase, tse kang CiVSP ho tloha. Ciona gutis15.Liprotheineng tsena tse ling, C-terminus ea S4 e khomaretsoe ho module ea effector, pore domain kapa enzyme.Ho Hv1, S4 e amahanngoa le sebaka sa coiled-coil (CCD) se karolong ea cytoplasmic ea lera. .Lekanale ke dimeric complex tse nang le li-VSD tse peli, e 'ngoe le e 'ngoe e na le tsela ea phepelo ea proton e gated16,17,18. Li-subunit tsena tse peli tsa Hv1 li fumanoe li butsoe ka kopanelo19,20,21,22, tse fanang ka maikutlo a hore ho kopana ha allosteric le ho sebelisana ha li-inter-subunit ho bapala. karolo ea bohlokoa ts'ebetsong ea ho betla.Sehokelo pakeng tsa li-subunits ka har'a sebaka se koetsoeng sa coil se hlalositsoe hantle hobane libopeho tsa kristale tsa libaka tse peli tse arohaneng li fumaneha22,23.Ka lehlakoreng le leng, sehokelo pakeng tsa VSD ka har'a lera ha se. ho utloisisoa hantle.Mohaho oa kristale oa protheine ea chimeric ea Hv1-CiVSP ha e fane ka tlhahisoleseding mabapi le sebopeho sena, kaha mokhatlo oa trimeric oa mochine o khabisitsoeng oa kristale o ka bakoa ke ho nkela sebaka sa Hv1 CCD sebaka ka tomoso leucine zipper GCN424. Phuputso ea morao-rao ea mokhatlo oa subunit oa mocha oa Hv1 o fihletse qeto ea hore li-helice tse peli tsa S4 li fetela ho CCD ntle le tšitiso e matla ea sebopeho sa bobeli, e leng se hlahisang li-helice tse telele tse qalang ho tloha lera le morero ho ea ho cytoplasm. phuputso ena e sisinya hore Hv1 VSDs ikopanye hammoho le karolo ea S4. Leha ho le joalo, liphuputso tse ling li sisintse mekhoa e meng pakeng tsa VSDs.Mahokelo ana a kenyelletsa likarolo tsa S1 17, 21, 26 le lipheletsong tse ka ntle tsa S2 karolo ea 21. Lebaka le ka 'nang la e-ba teng bakeng sa likhohlano liphetho tsa lithuto tsena ke hore allosteric coupling pakeng tsa VSDs e ile ea hlahlojoa mabapi le ts'ebetso ea gating, e itšetlehileng ka libaka tse koetsoeng le tse bulehileng, 'me sebopeho se pakeng tsa VSD se ka fapana ka liphetoho tse fapaneng tsa boemo ba conformation. Mona, re fumane hore 2-guanidinothiazole e thibela likanale tsa Hv1 ka ho tlama li-VSD tse peli tse bulehileng ka synergistically, 'me e sebelisitse e' ngoe ea metsoako, 2-guanidinobenzothiazole (GBTA), ho hlahloba tšebelisano pakeng tsa li-subunits sebakeng se bulehileng. segokanyimmediamentsi sa sebolokigolo.Re fumane hore GBTA e tlamang curve e ka hlalosoa hantle ka mohlala oa palo eo ho eona ho tlamaha ha inhibitor ho subunit e le 'ngoe ho fellang ka keketseho ea kamano e tlamang ea subunit e haufi.Re boetse re fumane hore masala a D112, li-filters tsa khetho mocha oa 27, 28 le karolo ea sebaka se tlamang sa guanidine se tsoang ho 29 taolo ea GBTA e tlamang cooperativity.Re bontša hore tlamahano ea tšebelisano-'moho e bolokiloe Hv1 dimer, moo CCD e arohanang le karolo ea S4, e fana ka maikutlo a hore sebopeho sa intersubunit ho CCD ha se etse ka ho toba. ho kopanya allosteric ho kopanya pakeng tsa libaka tse tlamang tsa GBTA. Ka lehlakoreng le leng, re fumana hore sekhechana sa S1 ke karolo ea sehokelo pakeng tsa li-subunits le ho etsa tlhahiso ea tlhophiso ea li-VSD tse haufi le pheletso ea extracellular ea S4 helix hole le bohareng ba dimer ho lumella. sekhechana sa S1 ho ba sebakeng se bulehileng. Li-inhibitors tse nyenyane tsa molek'hule tsa Hv1 li na le thuso e le lithethefatsi tsa anticancer le li-neuroprotective agents. 1a) le lihlahisoa tsa eona li fumanoe ho thibela ho phunyeletsa ha VSD29,32 ea protons ka mocha.Ho tlama ha metsoako e joalo ho nahanoa hore ho etsahala ka boikemelo likarolong tse peli tse bulehileng.2-guanidinobenzothiazole (GBTA, motsoako [2] setšoantšong sa 1a) e ne e le pele e bontšitsoe ho thibela Hv1 hoo e ka bang ka katleho joaloka 2GBI ha e lekoa ka bongata ba 200 μM (Setšoantšo sa 1b) .Re ile ra hlahloba lihlahisoa tse ling tsa thiazole 'me ra fumana hore tse ling tsa tsona li thibela mocha ka matla a tšoanang kapa a maholo ho feta GBTA (Figure 1 le Supplementary). mongolo).Re ile ra etsa qeto ea likhahla tsa likarabo tsa metsoako e mene ea thiazole (GBTA le metsoako [3], [6] le [11], Fig. 1c) 'me ra fumana hore li ne li le matla ho feta tsa 2GBI. The Hill coefficients (h) tsa thiazole derivatives ho tloha 1.109 ± 0.040 ho 1.306 ± 0.033 (sa feiga. 1c le Supplementary Fig. 1).Ka lehlakoreng le leng, Hill coefficient bakeng sa 2GBI e ne e le 0.975 ± 0.024 29, Fig. 2a le Supplementary Fig. 1).A Hill coefficient e ka holimo ho 1 e bontša tšebelisano e tlamang.Hobane Hv1 subunit ka 'ngoe e na le inhibitor ea eona. sebaka se tlamang,29,32 re ile ra beha mabaka a hore ho tlamaha ha motsoako oa thiazole ho subunit e le 'ngoe ho ka matlafatsa tlamahano ea molek'hule ea bobeli ea inhibitor ho subunit e haufi.GBTA e ne e le motsoako oa teko o nang le coefficient e phahameng ka ho fetisisa ea Hill.Ka hona, re khethile motsoako ona ho ho tsoela pele ho ithuta mokhoa oa ho kopanya synergy le ho sebelisa 2GBI e le taolo e mpe ea litšupiso. (a) Metsoako ea liteko: [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) Ho thibela mosebetsi oa Hv1 oa motho ka li-guanidinothiazoles tse bontšitsoeng le motsoako oa 2GBI (li-bars tse botala bo botala) .Hv1 proton currents e ile ea lekanyetsoa ka har'a li-plaque tsa Xenopus oocyte e le karabelo ea depolarization ho tloha ho -80 mV ho ea ho +120 mV. Inhibitor ka 'ngoe e ile ea kenngoa bateng ka motsoako oa 200 μM.pHi = pHo = 6.0 .Data e bolela ±SEM (n≥4) .(c) Thibelo e itšetlehileng ka ho tsepamisa mohopolo ea Hv1 ea motho ka metsoako [2], [3], [6] le [11].Ntlha e' ngoe le e 'ngoe e emela thibelo e bolelang ± SD ea 3 ho isa ho litekanyo tse 15. Mohala ke Hill fit e sebelisetsoang ho fumana boleng bo hlakileng ba Kd bo tlalehiloeng ho Supplementary Table 1. Li-coefficients tsa Hill li ile tsa khethoa ho tloha ho lumellana ho tlalehiloeng ho Supplementary Fig. 1: h(1) = 0.975 ± 0.024 h(2) = 1.306 ± ± 1.306 ± 0.033, h (3) = 1.25 ± 0.07, h (6) = 1.109 ± 0.040, h (11) = 1.179 ± 0.036 (bona Mekhoa). (a,b) Compounds 2GBI le GBTA inhibited dimeric and monomeric Hv1 ka mokhoa o itšetlehileng ka mahloriso. Ntlha ka 'ngoe e emela ho thibela ho bolelang ± SD ea litekanyo tse 3 ho isa ho tse 8 'me mothinya ke Hill fit. The Hill coefficients (h) e bontšitsoeng ho li-histograms tsa inset li ile tsa khethoa ho tsoa ho tse tlalehiloeng ho Tlatsetso Figs 3 le 4. Karabo ea mahloriso ea GBTA e bontšitsoeng ho (a) e tšoana le e hlahang setšoantšong sa 1c.Sheba Tlatsetso ea 1 bakeng sa litekanyetso tse hlakileng tsa Kd.(c) Mohlala oa tlamahano e kopanetsoeng ea GBTA ho dimeric Hv1. Mohala o motšo o tiileng o emela ho lekana ho data ea liteko ka equation (6), e hlalosang mohlala o tlamang o bontšitsoeng ho (d) . -dissociation equilibrium curves ea liketsahalo tse tlamang tsa pele le tsa bobeli, ka ho latellana (Sub 1: OO + B ⇄ BO *, Kd1 = 290 ± 70 μM; Sub 2: BO * + B ⇄ B * O *, Kd2 = 29.3 ± 2.5 μM ) .(d) Setšoantšo sa moralo oa mochine o reriloeng oa block ea Hv1. Tabeng ea GBTA, ho tlama karolo e le 'ngoe e bulehileng ho eketsa kamano ea karolo e haufi e bulehileng (Kd2 Ho hlalosa ka bongata tlamo ea tšebelisano-'moho ea GBTA ho mocha, re sebelisitse mohlala oo subunit e ka tlamang molek'hule ea pele ea inhibitor ho latela karabelo ea bimolecular ka ho arohana kamehla Kd1 (Fig. 2c, Sub 1, OO+ B ⇆ BO * ). Ho tlama ho etsa hore mocha o amohele boemo boo likaroloana tse setseng tse se nang letho li tlamella ho inhibitor ho latela mokhoa o ikhethang oa bimolecular reaction ka ho arohana kamehla Kd2, moo Kd2 Hang ha molek'hule ea bobeli ea inhibitor e tlameletsoe, li-subunits ka bobeli li na le ho arohana kamehla Kd2 (setšoan. OO ho Setšoantšo sa 2d) pele ho fumanoe ho kenya letsoho ka mokhoa o sa tsotelleng ho Hv1 hona joale tlas'a maemo ana19,20 'me kahoo ha baa ka ba kenyelletsoa ka mokhoa o tlamang. Mohala o motšo o tiileng ho Setšoantšo sa 2c o lekana le equation ea mohlala ho mokoloko oa karabelo oa mahloriso oa liteko, o fanang ka Kd1 ea ~ 290 μM le Kd2 ea ~ 29 μM (karolo ea mekhoa, equation (6)). ho tlama ha 2GBI, moo Kd2 ≈ Kd1 = Kd (Fig. 2d).Ho monomeric Hv1, ho na le sebaka se le seng se tlamang le Kdm e le 'ngoe (O ° + B ⇆ B °). Tabeng ea GBTA, Kdm e haufi le 54 μM, e ts'oanang haholo le Kd1 ho feta Kd2 (Fig. 2d) .Ka mantsoe a mang, sebaka se tlamang sa monoma se boemong ba tlhophiso (O °) se ts'oanang le boemo bo phahameng ba kamano (BO *) ho feta boemo bo tlaase- Affinity state (OO) ea dimer, mohlomong ka lebaka la ho felisoa ha sehokelo lipakeng tsa li-subunits. Joalokaha ho bontšitsoe pele bakeng sa 2GBI32, re fumane hore GBTA e hatelletse maqhubu a Hv1 ka ho thibela mocha ha o ne o butsoe ho e-na le ho etsa hore ho be thata ho bula (Supplementary Fig. 2a,b,d) .2GBI e boetse e tsejoa ka ho susumetsa maqhubu a mohatla butle-butle karabelo ea membrane repolarization, empa ketsahalo ena ha ea ka ea hlokomeloa ho GBTA (Supplementary Fig. 2c) .Boteng ba Hv1 inactivation ka pel'a 2GBI, liheke tsa subunit e 'ngoe le e' ngoe li ke ke tsa koala ho fihlela thibela a tloha sebakeng se tlamang (the " " foot gate"), 'me 2GBI e tlamolla butle ho feta ha liheke li koala.Haeba subunit e le 'ngoe ea Hv1 e koetsoe 'me e koetsoe, ha karolo e haufi e ntse e koetsoe, ho tlamoha ha limolek'hule tse setseng tsa 2GBI ho ba butle (blocker capture). subunit e le 'ngoe feela e koetsoeng e tsamaisa protons nakoana pele e koala le ho kenya letsoho haholo ho mohatla o ntseng o bola butle. Ho fumana hore ho bola ha maqhubu a mohatla oa Hv1 ha hoa ka ha fokotseha haholo ka pel'a GBTA (Supplementary Fig. 2c) e lumellana le mochine o tlamang oa synergistic bakeng sa blocker ena (Setšoantšo sa 2d) . , kamano ea blocker le subunit e haufi e theoha ka makhetlo a ka bang 10, e ratang mokhoa o sa tlameng.Sena se bolela hore molek'hule oa bobeli oa GBTA o na le monyetla o moholo oa ho phutholoha pele o tšoasoa mocha. hlahisa subunit e le 'ngoe feela e thibelang ho lebeletsoe hore e be ngata haholo ka pel'a GBTA ho feta boteng ba 2GBI, e leng se bakang ho bola ha mohatla ka potlako. E le hore GBTA e tle e kopane ka kopanelo le li-subunit tsa Hv1 ka bobeli, libaka tse tlamang li tlameha ho kopanngoa ka mokhoa o fapaneng. phetoho ho tloha ho kamano e tlase ho ea ho tlamahano e phahameng.Re ile ra beha mabaka a hore haeba masala a itseng ka har'a sebaka se tlamang a tlatselitse ts'ebetsong ea ts'ebelisano, phetoho ea bona e lokela ho fetola Hill coefficient ea concentration-response curve ea GBTA inhibition of Hv1.Residues D112, F150 , S181 le R211 li ne li bontšitsoe pele e le karolo ea tikoloho e tlamang ea 2GBI29 'me re nahana hore le bona ba tla ameha ka tsela e tšoanang ho tlamang GBTA (Fig. 3A) .Re lekanya inhibitory concentration-response curves of GBTA for mutant channel D112E, F150A, S181A , le R211S (setšoantšo sa 3) le ho bapisa li-coefficients tsa bona tsa Hill le tsa Hv1 wild-type, ho sebelisa 2GBI e le tšupiso.Residue V109 e sefahlehong se le seng sa karolo ea S1 le D112 mme e na le phetoho e le 'ngoe e eketsehileng ea helical ka hare ho sele. V109 e ne e sa ameha ho tlamong ha 2GBI29, re sebelisitse V109A mutant e le taolo (Feie. 3b). (a) Litlhahiso tse hlahisitsoeng tsa Hv1 tse amehang ho tlamang ha guanidine derivative. Li-curve tse putsoa tse pota-potiloeng li pota-potile liketane tsa mahlakoreng tse neng li reriloe pele tse sebelisanang le likarolo tse fapaneng tsa motsoako oa 2GBI29.(bf) Thibelo e itšetlehileng ka concentration ea liphetoho tse bontšitsoeng tsa Hv1 ka metsoako 2GBI (cyan) le GBTA ( bofubelu bo lefifi).Ntlha e 'ngoe le e' ngoe e emela thibelo e bolelang ea litekanyo tse 3 ho isa ho tse 12 ± SD V109 e le taolo e mpe.Curves are Hill fits used to find apparent Kd values ​​​​(sheba Supplementary Table 1). The Hill coefficients (h) bontšitsoeng ho the inset histograms li ile tsa khethoa ho tsoa ho fits tse tlalehiloeng ho Supplementary Figs 3 le 4. Reference h values ​​​​bakeng sa Hv1 WT li bonts'oa e le mela e nchocho.Asterisks e bonts'a phapang e kholo lipakeng tsa litemana tsa mutant le WT (p 0.05) ho coefficient ea Hill ea GBTA e tlamang likanale tsa GGG ha e bapisoa le mofuta o hlaha (Fig. 5c), e fanang ka maikutlo a hore ho sa tsotellehe karolo ea eona ho boloka li-subunit tse peli hammoho tsa bohlokoa, empa CCD ha e kenelle ka ho toba khokahano ea li-intersubunit allosteric lipakeng tsa libaka tse tlamang tsa GBTA. 20 liphatsa tsa lefutso tse bontšitsoeng, tse etselitsoeng ho leka likaroloana tsa S1 tse amehang ho kopantseng li-subunits (metsu e putsoa).(c–h) 2GBI (cyan) le GBTA (bofubelu bo lefifi) li thibela lihahi tse bontšitsoeng ka mokhoa o itšetlehileng ka mahloriso.Ntlha e' ngoe le e 'ngoe e emela ho thibela ho boleloang. ± SD ea litekanyo tse 3 ho isa ho tse 10. Mokokotlo e ne e le Hill fit e sebelisetsoang ho fumana boleng bo hlakileng ba Kd (bona Supplementary Table 1). Li-coefficients tsa Hill ho li-histograms tse kopantsoeng li ile tsa khethoa joalokaha ho hlalositsoe karolong ea Methods (sheba Supplementary Figs 3 le 4). Reference h values Hv1 WT e bontšoa e le mela e nchocho.Linaleli tsa linaleli li bontša phapang e khōlō ea lipalo lipakeng tsa litemana tse fetotsoeng le tsa WT (p 0.05, Fig. 5d, i ). ). Ka lehlakoreng le leng, phetoho ea phetoho ea D123A e fokolitse haholo Hill coefficient ea GBTA (p 0.05/14). Kaha ho fokotsa tefiso sebakeng sa 123 ho li-subunit tse peli ho ile ha fella ka phetoho e matla ho tšebelisano e tlamang ea GBTA, ha ho khutlisa tefiso ho li-subunits ka bobeli ho bile le phello e nyane feela, re atolositse tlhahlobo ho kenyelletsa subunit e le 'ngoe feela le mocha oa inversion oa tefo. e hlahisitse li-dimers tse amanang le Hv1 tse nang le sebaka sa D123R sebakeng sa C-terminal subunit (Fig. 5b) le ho lekanya ho thibela ho arabela ka mahloriso ka GBTA le 2GBI. ea mofuta o hlaha oa Hv1 (p 0.05). Hape re fumane hore ha ho se na βME, Hill coefficient ea GBTA e tlamang ho D112E I127C Hv1 dimer e ne e phahame haholo ho feta e lekantsoeng ka boteng ba li-agent tse fokotsang (setšoan 6e le Supplementary Fig. 4), e bolelang hore phetoho ea D112E ha ea ka ea felisa keketseho ea tšebelisano-'moho e tlamang ea GBTA e bakoang ke ho hokahanya ha cysteine ​​​​127. The Hill coefficient ea 2GBI e tlamang ho D112E, I127C Hv1 dimers le eona ha ea ka ea ameha haholo ke phetoho ea D112E (Setšoantšo sa 1) 6d le Tlatsetso. Setšoantšo sa 3). Ha li kopane, liphuputso tsena li fana ka maikutlo a hore tlamahano ea GBTA e matlafatsoa ke tšebelisano pakeng tsa likarolo tse ka ntle tsa S1 likarolong tse haufi tsa Hv1 ka litšebelisano tse khahlehang tsa electrostatic kapa ka ho thehoa ha maqhama a covalent pakeng tsa substituted cysteine ​​​​ho kopanya Allosteric pakeng tsa libaka ho eketseha le ho fella ka ho tlama tšebelisano. Le hoja phello ea litšebelisano tse khahlehang tsa electrostatic mabapi le tšebelisano-'moho li ka felisoa ke phetoho ea D112E, phello ea litlamo tsa covalent e ke ke ea khona. Phuputsong ea rona ea sebaka sa lik'hemik'hale se fumanehang bakeng sa ho tlama lihlahisoa tsa guanidine ho li-channel tsa Hv1, re fumane hore 2-guanidinothiazoles e kang GBTA e na le ts'episo e matla ea mahloriso ho feta 2-guanidinobenzimidazoles (Fig. 1c) .The Hill coefficient analysis of GBTA e tlamang ka bobeli dimeric le liteishene tsa monomeric (setšoantšo sa 2a, b) le mocha oa dimeric oo subunit e le 'ngoe e neng e tlameletsoe ho inhibitor (setšoan 4) e ile ea etsa hore re fihlele qeto ea hore ho thibela Hv1 ka GBTA Ketso ke ts'ebetso ea synergistic, le libaka tse tlamang tsa metsoako ea li-subunits tse peli li kopantsoe ka mokhoa o fapaneng.Ho sibolloa hore GBTA e tlamella mocha o bulehileng, joalokaha ho bontšitsoe pele bakeng sa motsoako o amanang le 2GBI32, e fana ka maikutlo a hore ho kopanya allosteric ho ka hlahlojoa ka ho khetheha sebakeng se bulehileng.Mohlala oa rona o tlamang tšebelisano-mmoho. E ne e khona ho hlalosa ka bongata ho hlalosa ho HV1 ka GBTA (sa feiga. 2c) mme o hlalosetse litlamorao tse fapaneng tsa ts'ebetso ea tlamo. E bophahamo bo phahameng bo lekaneng e ka fihlelleheng protheine e nang le chelete e ngata e nang le li-gbta tse peli tsa ho tlama. Mofuta oa HV1) matla a mahala a seriti ea oksijene ho hemoglobin ke mohlala o tsebahalang haholo le o neng o ithutile ka ho fetisisa oa ts'ebetso ea ho sebelisana KCal / Mol, ho latela maemo a liteko38.ths, mesebetsing e fapaneng ea Gbaglobin ha palo e fapaneng ea li-protein li ntse li nkoa ka lits'ebetso tse peli. Ka har'a mohlala oa rona oa Synergy, o tlamang oa limolek'hule tse le mong ho hlahisa mofuta oa tikoloho e tlamang. Liphetoho lipakeng tsa li-eckines.in karabelo ea liphetoho tsa bona tse tlamahaneng, S1 aspartate ea sebaka se amanang le ho hlophisa bocha E ne e bontšoa pele e ne e le karolo ea tsela ea ho nyopa ea HV1 ea phetetso ea HV1 le ho etsa joalo ka firater ea khethollo27,28. Liphetho tsa rona li bontša hore li-file tsa ho khetha li-tsackity tse peli li lula li le haufi le sebaka sa marang-rang sa GBTA. Ka sebopeho sa Crystal sa TO HV1-CHAPSSPSSS e nang le bokhabane bo nang le botsoalle bo kenyelletsang pheletso ea S1 e bonts'itsoe ka metsu e metsu e metsu e ntšo. . Libaka tse boletsoeng esale pele tsa GBTA li bontšoa e le li-ovals tse putsoa. Ka tatellano e 'meli e fapaneng e bonoang ka lehlakoreng le leng la lifofane tsa membrane karohano ea lipheletso tsa ka ntle tsa litlolo tse peli tsa li-ar4 tse peli (lijalo tse halikiloeng), li hlahisa tlhophiso e bontšitsoeng. Ka tlhophisong ea dimer e fumaneha mohahong oa 4G80. Positions P140 ho baahi ba Civ e tsamaellana le maemo D123 ho HV1. Liphetho tsa rona li bontša hore ho ba le tšebelisano ea Eleke ea elektrome pakeng tsa masala 123 (123d / 123r kapa 123r) e amanang le ho phetoa (123d / 123r) , tšebelisano e eketsehang (figa). Tlhaloso ke hore phello e solang ea ho beha hydrophophic e ka feta tekanyetso e matlafatsang ka lebaka la ho tlamahana le maemo a mangata Sebaka D171 of Co3 _ham (e tsamaellanang le D123 Hv1) ​​e ntse e eketseha tikolohong ea D123 e fumanehang tikolohong ea hydrophphilic setsing se bulehileng. Gating of HV1 Tencens e hlahang ka mekhoa e mengata ea TVento9,20,46,46,46,36,46 , e lateloang ke phetoho e ikhethileng e etsang hore ho bululeloa ke lits'oants'o tsa voltage. Morero oa lets'oao la fluorescent ha a lumellane le boteng ba mabenkele a ho ba teng ka bongata bo haufi le ho fumana chelete ea rona ea D113 le medias e media e meholo lipakeng tsa li-cindA tse ngata tsa GBTA. Fujiwara et al25 e reriloeng hore sebopeho sa Dimere sa Cytop-Coil e na le mekhabiso e 'meli e amanang le lihlahlobo tsa Cysteine. Tlhahlobo eohle ea sebaka le ts'ebetso ea sebaka se hokahanyang sa S4 le CCD.Ka ho ba sieo ha transmembation, le Cysteine ​​cross Crowings tlasa moo mocha o notleloa haholo. Ka hona, sebopeho se fumanoeng ka s4-S4 se bonts'a moelelo oa tlhahiso-leseling. Lithuto tsa thuto ea STA17,21,26 linaha tse koetsoeng, mohopolo o fumanehang le seo o se fumaneng oa Mony et al. S1 e tsamaisa 399 nakong ea gaio. Mona, re bontša hore, naheng e bulehileng, lipheletso tse ling tsa s1 helix li lekana ho tšehetsa li-cofferactions tse nang le mekhahlelo e nang le li-s4-S4 ka thoko ho e mong le e mong ho bua ka ho toba.however Ka seo re se fumaneng (sa feiga (feiga. 7B, panel e nepahetseng) Le ha ho nahanoa hore Anzyme e sebetsa e le monotor, sebopeho sa kristale sa masapo a eona a ikhethileng a hapiloeng ka seretse. bakeng sa HV1 (sa feiga ea 7c). 'Me ba boholo-holo ba sechaba ba fana ka maikutlo a hore liprothekeng tsa liprotheine li na le tloaelo ea ho etsa sebopeho moo lipheletso tsa S1 li amanang le tsona. Karolo ea bohlokoa ea HV1 ho ts'ebetso ea seretse sa spem e etsa hore seterata se phetehang sa taolo ea taolo ea li-microglia. Ho pholoha ho bakuli ba nang le mofets'e oa makhapetla a 12 kapa creserate. Bula HV1 Bubbinit, e lebisang ho tlama kholo ea ho tlama, ho ka lebisa ho nts'etsopele ea lithethefatsi tse matla tse eketsehileng li shebile liteishene tsa HV1. Sebaka sa polokelo ea sebaka sa hv1 se ne se etsoa ka mekhoa e tloaelehileng ea prusi. Jos C-Ferminus ea konopo e le 'ngoe e hokahane le n-chelminus ea bobeli ka li-enzgsggsggsgggs T7 MMESSESSASE CHEME KHEKENG (AMBILE) .1-3 matsatsi a bosholu Ho tloha Ecocyte Bioscience. ha a ne a imeloa ke ND96 a nang le CACLY . 2-ganidino-Benzimadazole [1], 2-genidino-Benzozle [2], (4-Methyl-1-Tiazol-1-Brono-4 -Mo-4 -Mo-4 -eazol-2-yl) Guanadine) [6], Ethyl 2-Gulinoxl-1-granidino-4-thethyl-1,3-thiazole- 5-cmboxylate [9] le (2-Guanidino-Methyl-1,3-thiazol-1-thiazol-1-yele) ethyl acta-aldrich.1- [4 - (4-chlorophenyl) -1,3-thiazol-2] Guanidine [11] Ho tloha ho metsoako ea Matrix.these ke ea bohloeki bo phahameng ka ho fetisisa khoebong e omeletseng ea DMSO ho hlahisa tharollo ea ho rekota ea 100 le 4 e ne e sa ngolisoe ka tlase ho Bonyane bohloeki ba 99%. Ho emisoa ha 2-4- (trifluoromethyl) anzenethiol hydrokeride (380 mg, motsoako oa 4.5 e ne e tsoetse pele ka ho hlohlelletsa ka thata bakeng sa lihora tse 4.Thente e ne e tlositsoe ka mocheso oa likamore tsa 10n. 65 ° C) Ka lihora tse 'maloa ebe o pheta-pheta ho tloha Ethyl actate / Petroleum ho fa sota e tšoeu e le tšoeu. mp'S22 ° C (E bobebe 225-226 ° C.5; 1h nmr (500 mhz, DMSso-D6): Δ [PPM] = 7.25 (J, 1 H), 7.92 (D), 7.92 (d , 1 h, J = hz) .13c nmr (200.5 (D, J =5 HZ), 121.7 - 124.6 (D, 124.6 = 272 HZ), 126.1 (Q, J, J, J, J, J, J, JO3 HZ), 134.1, C9HCF3n414 : 2611419. Naphthoh [1,2-D] Thiazol-2 -mine (300 mg, 1.5 mg 1.0 ml conc.to the e chesang e ekelitsoe ka potlako mme ka nako eo boholo ba eona bo boholong. o ne a robehile likotoana tse nyane 'me a hlatsuoa ka metsi ho fana ka' mala o mosehla o mosehla. (38 mg, 10%) MP 246-250 ° 1h nmr (500 mhz, DMSso-D6, D2O): Δ [PPM]: 7.2 HZ), 7.77 (D, 1 h , J =,6 HZ), 7.89 (D, 1 h, J, 1.6 HZ), 8.35 (D, 1 h, 1 h MHZ, DMSso-D6): Δ = 119.9, 123.4, 136.1.5, 136.1.5, 156.1.5, 1003.1.0. H) , e fumanoe: 243.0704. Li-curts tsa proton li ne li lekantsoe ka har'a li-oocytes tse bonts'ang li-amplifier tsa axon.int ) Ethanesulfnic acid (Mes), 30 ise Tearede, 5 mm i-tutraactic acid PH 6.0 e nang le tee ea TeayE.Le litekete tse hapiloeng ho la 1 ° C.E.TR ) le tšimolohong8.1 (pepesitsoeng). Litharong tse nang le meetso e fapaneng ea HV1 Inhibitor le maemong a mang ka ho hlahella ka matlakano ka " Tlhahiso ea Transistor) +120 MV Likhakanyo tsa mela tsa pulse.gv li ne li tlalehiloe ka har'a lintlafatso tse fapaneng. E sebelisoa ho e lokisa bakeng sa ho bola ha hona joale. The DV Ka equation equation: na [I]