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Njira ya proton ya Hv1 voltage-gated proton ndi dimeric complex yomwe ili ndi madera awiri okhudzidwa ndi magetsi (VSDs), iliyonse yomwe ili ndi njira yowonongeka ya proton. Dimerization imayendetsedwa ndi cytoplasmic coiled-coil domain. malo otseguka m'ma VSD onse amadziwika kuti amachitika mogwirizana; komabe, ndi zochepa zomwe zimadziwika za njira zomwe zili pansi. komabe, mawonekedwe oterowo sanadziwike mumayendedwe otseguka a Hv1. Apa tikuwonetsa kuti zotumphukira za 2-guanidinothiazole zimatsekereza ma Hv1 VSD awiri mothandizana ndikugwiritsa ntchito imodzi mwazinthuzi ngati kafukufuku wa kulumikizana kwa allosteric pakati pa ma subunits otseguka. Kumapeto kwa chidutswa choyamba cha transmembrane cha VSD chimapanga mawonekedwe a intersubunit omwe amagwirizanitsa kulumikizana pakati pa malo omangirira, pamene dera lozungulira-coil silimakhudzidwa mwachindunji ndi izi. .
Njira za proton zokhala ndi mphamvu yamagetsi zimagwira ntchito zofunika pazamoyo zosiyanasiyana, kuchokera ku phytoplankton kupita kwa anthu1.M'maselo ambiri, njirazi zimagwirizanitsa proton efflux kuchokera ku membrane ya proton ndikuyendetsa ntchito ya NADPH oxidase. Njira yokhayo yodziwika ya proton-gated proton mwa anthu ndi Hv1, yomwe imapangidwa ndi HVCN1 gene2,3.Hv1 (aka VSOP) yasonyezedwa kuti imathandizira pakukula kwa maselo a B4, kupanga mitundu ya okosijeni yogwira ntchito ndi chitetezo cha m'thupi5,6,7,8, umuna. motility9 ndi pH malamulo a airway pamwamba madzimadzi10. Njirayi imakhudzidwa ndi mitundu ingapo Yambiri ya khansa monga B-cell malignancies4,11 ndi khansa ya m'mawere ndi colorectal12,13.Ntchito yowonjezereka ya Hv1 inapezeka kuti iwonjezere mphamvu ya metastatic ya maselo a khansa 11, 12. Mu ubongo, Hv1 imasonyezedwa. ndi microglia, ndipo ntchito yake yasonyezedwa kuti imawonjezera kuwonongeka kwa ubongo mu zitsanzo za ischemic stroke.
Puloteni ya Hv1 ili ndi dera lotchedwa voltage-sensing domain (VSD), lomwe lili ndi magawo anayi a transmembrane otchedwa S1 mpaka S414. VSD imafanana ndi madera omwe ali ndi magetsi a magetsi a Na +, K + ndi Ca2 + ndi phosphatase ya voltage-sensitive phosphatases, monga CiVSP kuchokera. Ciona gutis15.Mumapuloteni enawa, C-terminus ya S4 imamangiriridwa ku gawo la effector, pore domain kapena enzyme.Mu Hv1, S4 imagwirizanitsidwa ndi coiled-coil domain (CCD) yomwe ili kumbali ya cytoplasmic ya nembanemba. .Chaneliyo ndi dimeric complex yopangidwa ndi ma VSD awiri, iliyonse ili ndi njira yopititsira pulotoni16,17,18.Magawo awiriwa a Hv1 adapezeka kuti amatsegula mogwirizana19,20,21,22, kutanthauza kuti kulumikizana kwa allosteric ndi ma inter-subunit amasewera. gawo lofunika kwambiri pamayendedwe a gating.Mawonekedwe apakati pa ma subunits mkati mwa chigawo chophimbidwa ndi coil amatanthauzidwa bwino chifukwa mawonekedwe a kristalo a madera awiri akutali akupezeka22,23.Kumbali inayi, mawonekedwe pakati pa ma VSD mkati mwa nembanemba sali. kumveka bwino.Mapangidwe a kristalo a Hv1-CiVSP chimeric mapuloteni sapereka chidziwitso chokhudza mawonekedwe awa, monga gulu la trimeric la crystallized channel complex lingabwere chifukwa cholowa m'malo mwa Hv1 CCD ndi yisiti leucine zipi GCN424.
Kafukufuku waposachedwapa wa gulu la subunit la Hv1 njira anamaliza kuti awiri S4 helices kusintha mu CCD popanda kusokoneza kwambiri dongosolo yachiwiri, kuchititsa helices yaitali kuti kuyambira nembanemba ndi polojekiti mu cytoplasm. Kafukufukuyu akuwonetsa kuti ma Hv1 VSD alumikizana wina ndi mnzake m'gawo la S4. Zotsatira za maphunzirowa ndikuti kulumikizana kwa allosteric pakati pa ma VSD kudawunikidwa pokhudzana ndi njira yolowera, zomwe zimadalira malo otsekedwa ndi otseguka, ndi mawonekedwe pakati pa VSDs angasiyane mukusintha kosiyanasiyana kwa boma.
Apa, tinapeza kuti 2-guanidinothiazole linalake ndipo tikulephera Hv1 njira ndi synergistically kumanga kwa VSDs awiri lotseguka, ndipo ntchito imodzi mwa mankhwala, 2-guanidinobenzothiazole (GBTA), kufufuza mogwirizana subunits mu poyera boma. Tidapeza kuti ma curve omangirira a GBTA amatha kufotokozedwa bwino ndi chitsanzo chochulukira momwe kumangiriza kwa inhibitor ku gawo limodzi kumapangitsa kuti pakhale mgwirizano womangirira wa subunit yoyandikana nayo. njira 27, 28 ndi mbali ya guanidine otumphukira kumanga malo 29 kulamulira GBTA kumanga cooperativity.Timasonyeza kuti mgwirizano kumanga anakhalabe mu Hv1 dimer, kumene CCD amalekanitsa S4 gawo, kutanthauza kuti intersubunit mawonekedwe mu CCD si mwachindunji. Mosiyana ndi izi, timapeza kuti chidutswa cha S1 ndi gawo la mawonekedwe pakati pa ma subunits ndikupereka makonzedwe a ma VSD oyandikana ndi mathero owonjezera a S4 helix kutali ndi pakati pa dimer kuti alole. chidutswa cha S1 kuti chikhale poyera.
Ma molekyulu ang'onoang'ono oletsa Hv1 ndi othandiza monga mankhwala oletsa khansa ndi neuroprotective agents. .1a) ndi zotumphukira zake zinapezeka kuti zimatsekereza ma protoni a VSD29,32 kudzera munjira.Kumangirira kophatikizana kotereku kumaganiziridwa kuti kumachitika paokha pamagulu awiri otseguka.2-guanidinobenzothiazole (GBTA, kompositi [2] mu Chithunzi 1a) chinali Zomwe zimawonetsedwa kale kuti ziletsa Hv1 pafupifupi mogwira mtima ngati 2GBI poyesedwa pamagulu a 200 μM (Chithunzi 1b) .Tinapenda zotumphukira zina za thiazole ndipo tidapeza kuti zina mwazomwe zimalepheretsa njirayo ndi mphamvu yofananira kapena yayikulu kuposa GBTA (mkuyu 1 ndi Supplementary). malemba) .Tinatsimikiza zokhotakhota zoyankha zamagulu anayi a thiazole (GBTA ndi mankhwala [3], [6] ndi [11], Fig. 1c) ndipo tinapeza kuti anali okwera kwambiri kuposa a 2GBI. The Hill coefficients (h) za zotumphukira za thiazole zinali kuchokera ku 1.109 ± 0.040 mpaka 1.306 ± 0.033 (Mkuyu. 1c ndi Supplementary Fig. 1) Mosiyana, Hill coefficient ya 2GBI inali 0.975 ± 0.024 29, Fig. 2a ndi Supplementary Fig. 1) . kumanga malo,29,32 tinaganiza kuti kumanga kwa thiazole kumachokera ku subunit imodzi kungapangitse kumangirira kwa molekyulu yachiwiri ya inhibitor kupita ku subunit yoyandikana nayo.GBTA inali gawo loyesa lomwe lili ndi mphamvu yapamwamba kwambiri ya Hill. pitilizani kuphunzira njira yomangira ma synergy ndikugwiritsa ntchito 2GBI ngati chiwongolero cholakwika.
(a) Kuyeza mankhwala: [1] Buku 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) Kuletsa ntchito ya Hv1 ya anthu ndi guanidinothiazoles yosonyezedwa ndi 2GBI (mipiringidzo yobiriwira ya buluu) .Mphepo ya proton ya Hv1 inayesedwa mkati mwa zolembera za Xenopus oocyte poyankha kutsika kwa mphamvu kuchokera ku mphamvu yogwira ya -80 mV mpaka +120 mV. Inhibitor iliyonse inawonjezedwa ku bafa pamtunda wa 200 μM.pHi = pHo = 6.0 .Deta ndikutanthauza ± SEM (n≥4) .(c) Kulepheretsa kudalira kwa anthu Hv1 ndi mankhwala [2], [3], [6] ndi [11] Mfundo iliyonse ikuyimira kulepheretsa ± SD ya 3 mpaka 15 miyeso. Mzerewu ndi wokwanira wa Phiri lomwe limagwiritsidwa ntchito kupeza zowoneka bwino za Kd zomwe zafotokozedwa mu Supplementary Table 1. Ma coefficients a Hill adatsimikiziridwa kuchokera pazomwe zafotokozedwa mu 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 (onani Njira).
(a,b) Compounds 2GBI ndi GBTA inhibited dimeric and monomeric Hv1 m'njira yodalira ndende. Mfundo iliyonse imayimira kuletsa kwapakati ± SD kwa miyeso ya 3 mpaka 8 ndipo mpiringidzo ndi Hill fit. The Hill coefficients (h) akuwonetsedwa mu ma histograms a inset adatsimikiziridwa kuchokera ku zoyenera zomwe zafotokozedwa mu Supplementary Figs 3 ndi 4. Mayankho okhudzidwa a GBTA omwe akuwonetsedwa mu (a) ndi ofanana ndi mkuyu 1c. kumanga mgwirizano wa GBTA ku dimeric Hv1. Mzere wakuda wolimba umayimira kuyenerera kwa deta yoyesera ndi equation (6), yomwe ikufotokoza chitsanzo chomangirira chomwe chikuwonetsedwa mu (d). -Dissociation equilibrium curves ya zochitika zoyambirira ndi zachiwiri zomangirira, motsatira (Sub 1: OO + B ⇄ BO *, Kd1 = 290 ± 70 μM; Sub 2: BO * + B ⇄ B * O *, Kd2 = 29.3 ± 2.5 μM ) .(d) Chithunzi chojambula cha njira yomwe ikuyembekezeredwa ya chipika cha Hv1. Pankhani ya GBTA, kumangiriza ku gawo limodzi lotseguka kumawonjezera kuyanjana kwa gawo lotseguka loyandikana nalo (Kd2
Kuti tifotokoze mochulukira kugwirizanitsa kwa GBTA ku njira, tinagwiritsa ntchito chitsanzo chomwe subunit ikhoza kumangirira molekyulu yoyamba ya inhibitor kutsatira bimolecular reaction ndi dissociation constant Kd1 (Fig. 2c, Sub 1, OO+ B ⇆ BO * ). Kumangiriza kumapangitsa kuti tchanelo likhazikike m'malo momwe ma subunits opanda kanthu otsala amamangiriza ku choletsa kutsatira mawonekedwe apadera a bimolecular ndi dissociation mosalekeza Kd2, pomwe Kd2
Mzere wakuda wakuda mu Chithunzi 2c ndi wolingana ndi equation yachitsanzo ku curve yoyeserera-yankho, yopereka Kd1 ya ~ 290 μM ndi Kd2 ya ~ 29 μM (gawo la Njira, equation (6)). kumanga kwa 2GBI, kumene Kd2 ≈ Kd1 = Kd (Mkuyu 2d). Mu monomeric Hv1, pali malo amodzi omangiriza ndi Kdm imodzi (O ° + B ⇆ B °). Pankhani ya GBTA, Kdm ili pafupi ndi 54 μM, yomwe ili yofanana kwambiri ndi Kd1 kuposa Kd2 (mkuyu 2d). Mwa kuyankhula kwina, malo omangiriza a monomer ali mu kasinthidwe (O °) mofanana kwambiri ndi chikhalidwe chapamwamba (BO *) kuposa chochepa- affinity state (OO) ya dimer, makamaka chifukwa cha kuchotsedwa kwa mawonekedwe pakati pa ma subunits.
Monga momwe tawonetsera kale 2GBI32, tapeza kuti GBTA inapondereza mafunde a Hv1 mwa kutsekereza njira pamene inali yotseguka m'malo mopangitsa kuti ikhale yovuta kutsegula (Supplementary Fig. 2a,b,d) . poyankha repolarization ya membrane, koma chodabwitsa ichi sichinawonedwe mu GBTA (Supplementary Fig. 2c) .Pamaso pa Hv1 inactivation pamaso pa 2GBI, zipata mu subunit iliyonse sangathe kutseka mpaka blocker atachoka pamalo omangiriza (the " foot gate” mechanism), ndipo 2GBI imamasula pang’onopang’ono kuposa kutseka kwa zipata. gawo limodzi lokha lotsekeka limayendetsa ma protoni pang'onopang'ono asanatseke ndipo amathandizira kwambiri pakuwola pang'onopang'ono kwa mchira.
Kupeza kuti kuwola kwa mafunde a mchira wa Hv1 sikunachedwe kwambiri pamaso pa GBTA (Supplementary Fig. 2c) imagwirizana ndi njira yomangirira yolumikizira iyi (mkuyu 2d) .Kamodzi molekyu ya GBTA imakhala yosasunthika kuchokera ku subunit ya Hv1 , kuyanjana kwa blocker ku subunit yoyandikana nayo kumatsika pafupifupi 10-fold, kukomera ndondomeko yosagwirizana.Izi zikutanthauza kuti molekyulu yachiwiri ya GBTA ili ndi mwayi wochuluka kwambiri wovundukuka isanatsekedwe mu njira. kutulutsa gawo limodzi lokha lotsekereza likuyembekezeka kukhala lochuluka kwambiri pamaso pa GBTA kuposa kukhalapo kwa 2GBI, zomwe zimapangitsa kuwola kwakanthawi kwa mchira.
Kuti GBTA imangirire mogwirizana ndi ma subunits onse a Hv1, malo omangirira ayenera kukhala ophatikizana. kusintha kuchokera ku chiyanjano chochepa kupita ku chiyanjano chapamwamba.Tidaganiza kuti ngati zotsalira zinazake mkati mwa malo omangirira zimathandizira kuti ntchitoyo ikhale yogwirizana, kusintha kwawo kuyenera kusintha Hill coefficient of concentration-response curve of GBTA inhibition of Hv1.Residues D112, F150 , S181 ndi R211 m'mbuyomu adawonetsedwa kuti ndi gawo la 2GBI29 yomangiriza chilengedwe ndipo tidaganiza kuti nawonso angatenge nawo gawo pakumanga kwa GBTA (mkuyu 3A) . , ndi R211S (Chithunzi 3) ndipo anayerekezera ma coefficients awo a Phiri ndi amtundu wa Hv1 zakutchire, pogwiritsa ntchito 2GBI monga chofotokozera.Residue V109 ili pa nkhope yomweyo ya gawo la S1 monga D112 ndipo ili ndi kutembenuka kwina kowonjezera mkati mwa selo. V109 sinatenge nawo gawo pakumanga kwa 2GBI29, tidagwiritsa ntchito V109A mutant ngati chiwongolero (Chith. 3b ndi).
(a) Zotsalira za Hv1 zomwe zimakhudzidwa ndi kumangidwa kwa guanidine. Ma curve opindika a buluu amazungulira maunyolo am'mbali omwe adapangidwa kale omwe amalumikizana ndi magawo osiyanasiyana a 2GBI29. chofiyira chakuda). Mfundo iliyonse ikuyimira kulepheretsa kwa 3 mpaka 12 miyeso ± SD V109 monga kuwongolera koyipa. Ma Curves ndi Mapiritsi omwe amagwiritsidwa ntchito kupeza ma Kd owoneka (onani Supplementary Table 1). The Hill coefficients (h) akuwonetsedwa mu ma histograms adatsimikiziridwa kuchokera pazokwanira zomwe zafotokozedwa mu Supplementary Figs 3 ndi 4. Reference h values za Hv1 WT zikuwonetsedwa ngati mizere yodukaduka. .
Tinapeza kuti kusintha kwa D112E kunachepetsa kwambiri mphamvu ya Hill (p Kupeza kuti Hill coefficient ya GBTA yomangirira ndi yapamwamba kuposa 1 mu Hv1 dimer ndipo imakhala ~ 1 mu njira ya monomeric (mkuyu 2b) ikugwirizana ndi kukhalapo kwa kuyanjana kwa allosteric pakati pa malo omangiriza m'magulu awiriwa. nkhaniyo, Hill coefficient ya GBTA yomangirira ku dimer komwe blocker wamanga ku subunit imodzi iyenera kukhala ~ 1.Tidayesa kulosera uku mu Hv1 F150A-WT yolumikizidwa dimer (mkuyu 4a), pomwe kuyanjana kwa F150A subunit ya GBTA inali yoposa maulamuliro a 2 apamwamba kuposa a WT subunit (Figs. 1c ndi 3d) . Kenako tinayesa mipiringidzo-mayankhidwe oletsa kuletsa kwa WT subunit pa basal concentration ya 2 μM GBTA. , blocker ikuyembekezeka kumanga pafupifupi 99% ya subunit ya F150A ndi (a) Schematic diagram ya F150A-WT junction dimer yomwe imagwiritsidwa ntchito popanga chotchinga hemichannel ({F150A}b-WT/BAO*).Madamondi oyera amawonetsa malo osinthira. M'maboma a BAO* ndi BA*B*, mgwirizano wa F150A subunit ukuyembekezeka kukhala wapamwamba kuposa wa subunit ya WT.(b) Mafunde a proton kuchokera kumayendedwe a F150A-WT amayezedwa potengera kusintha kwa nembanemba kuchokera ku -80 mV mpaka +120 mV.pHi = pHo = 6.0 .Grey traces imayimira mafunde omwe amayezedwa ngati palibe inhibitor.Kufufuza kwakuda (kuwongolera) kumayimira kuyesedwa kwamakono pambuyo pa kuwonjezera kwa 2 μM GBTA. Pa ndondomeko iyi, gawo la WT silinalepheretsedwe kwambiri, pamene F150A subunit inamangidwa pafupifupi kwathunthu ku GBTA. ({F150A}b-WT) .Kuwonjezeka kwina kwa ndende ya GBTA kunachititsa kutsekeka kwa gawo la WT (malalanje ndi zofiira).(c) Kuletsa kwa Concentration-dependent kwa {F150A}b-WT dimer (inhibitor pre-bound to to F150A subunit).Mfundo iliyonse imayimira kulepheretsa kwapakati ± SD kwa miyeso 3 mpaka 7. Ma curve anali a Hill fit omwe amagwiritsidwa ntchito kuti apeze ma Kd owoneka bwino omwe adanenedwa mu Supplementary Table 1. Ma coefficients a Hill omwe akuwonetsedwa mu histogram yamkati adatsimikiziridwa kuchokera pazomwe zanenedwa mu Supplementary Fig 3 ndi 4. Miyezo ya Hv1 WT ndi kuwonetsedwa ngati mizere yodukaduka. Nyenyezi zimasonyeza kusiyana kwakukulu kowerengera poyerekeza ndi WT dimer Hv1 (p Popeza GBTA inhibition ya Hv1 idayesedwa panjira yotseguka, tidaganiza kuti mgwirizano womanga wa GBTA ungagwiritsidwe ntchito pophunzira momwe amalumikizirana ma intersubunit poyera. 22 ndi intersubunit kugwirizana nawo gating ankafuna kuti mkhalapakati ndi cytoplasmic coiled-koyilo ankalamulira (CCD) 22,25. Choncho, tinafunsa ngati CCD nawonso kugwirizana pakati GBTA-zomangira malo poyera boma.Triglycine kusintha kwa mawonekedwe pakati pa chidutswa cha S4 transmembrane fragment ndi coiled-coil domain adawonetsedwa m'maphunziro am'mbuyomu kuti achepetse domain iyi kuchokera panjira yonseyo ndikusunga umphumphu wa dimerization. Chifukwa chake, tidayesa kusintha kwa V220G, K221G ndi T222G . 5a, GGG mutant) pa GBTA yomangirira mgwirizano.Tinapeza kuchepa kwachiwerengero (p> 0.05) mu Hill coefficient ya GBTA yomangirira kumayendedwe a GGG poyerekeza ndi mtundu wakutchire (Fig. 5c), kutanthauza kuti ngakhale ntchito yake yosunga zigawo ziwiri pamodzi zofunika, koma CCD si mwachindunji mkhalapakati intersubunit allosteric lumikiza pakati GBTA kumanga malo.
(a) Chithunzi chojambula cha Hv1 dimer chokhala ndi masinthidwe a triglycine kumapeto kwamkati kwa S4 opangidwa kuti asokoneze kulumikizana kwa intersubunit komwe kumalumikizidwa ndi dera la cytoplasmic coiled-coil (mivi yabuluu). masinthidwe owonetsedwa, opangidwa kuti ayese zidutswa za S1 zomwe zikuphatikizidwa polumikizana pakati pa ma subunits (mivi ya buluu).(c-h) 2GBI (cyan) ndi GBTA (yofiyira yakuda) imalepheretsa mapangidwe omwe akuwonetsedwa modalira. ± SD ya 3 mpaka 10 miyeso. Mpenderoyo unali wofanana ndi Phiri womwe umagwiritsidwa ntchito kupeza ma Kd owonekera (onani Supplementary Table 1). Ma coefficients a Hill mu histograms olowetsedwa adatsimikiziridwa monga momwe zafotokozedwera mu gawo la Njira (onani Supplementary Figs 3 ndi 4). Hv1 WT ikuwonetsedwa ngati mizere yodukaduka. Nyenyezi zimasonyeza kusiyana kwakukulu pakati pa ndime zosinthika ndi za WT (p Popeza tinagamula kuti CCD ngati mkhalapakati wachindunji wa GBTA cooperative kumanga, tinafunsa ngati kucheza pakati VSDs nawo allosteric lumikiza pakati kumanga malo. mu gawo la S1 transmembrane.S1 ili ndi zotsalira zina ziwiri zowonongeka, E119 ndi D123, zomwe zili mbali imodzi ya helix monga D112, koma pafupi ndi mapeto akunja a 24 a chidutswacho. kupita ku D112 kudzera pa waterlines36,37.Choncho, tinayesa ngati E119 ndi D123 akukhudzidwa ndi kugwirizana kwa allosteric pakati pa malo omangirira a GBTA (mkuyu 5b) . koma kumbali ina ya S1 helix (mkuyu 5b).
Tidayeza kuletsa kodalira kwa mayendedwe a E119A, D123A ndi K125A ndi 2GBI ndi GBTA ndipo tidapeza kuti kusintha kwa E119A ndi K125A sikunasinthe kwambiri kuchuluka kwa Hill ya inhibitor kuyerekeza ndi mtundu wakutchire (p > 0.05, Fig. 5d, i ). ). Komano, masinthidwe D123A kwambiri kuchepetsa Hill koyefila wa GBTA (p 0.05/14).
Popeza kusokoneza mtengo pamalo 123 pamagulu onse awiri kunapangitsa kusintha kwakukulu kwa mgwirizano womangirira wa GBTA, pomwe kubweza mlandu pamagawo onse awiri kunali ndi zotsatira zochepa, tidakulitsa kuwunikaku kuti aphatikize gawo limodzi lokha ndi njira yosinthira. adapanga ma dimers olumikizidwa ndi Hv1 ndi D123R m'malo mwa C-terminal subunit (mkuyu 5b) ndikuyesa kulepheretsa kuyankha kwa GBTA ndi 2GBI. wamtundu wamtundu wa Hv1 (p Tinapezanso kuti kusintha kwa D112E kunathetsa kuwonjezeka kwa GBTA-binding Hill coefficient yopangidwa ndi maziko a WT-D123R (Fig. 5b, h ndi Supplementary Fig. 4) . Chithunzi cha 5h ndi Supplementary Fig. 3) .Zofukufukuzi zikusonyeza kuti kuwonjezereka kwa allosteric kugwirizana pakati pa magulu ang'onoang'ono omwe amayamba chifukwa cha milandu yosiyana pa malo a 123 sikumasulira kugwirizanitsa kolimba kwambiri pamene malo omangirira pa malo D112 asokonezedwa.
Tidaganiza kuti ngati zotsalira za D123 zomwe zili pafupi ndi ma subunits otseguka zili pafupi kwambiri kuti zigwirizane ndi wina ndi mzake, kuyanjana konyansa pakati pa milandu yolakwika kungasinthidwe kukhala kuphatikiza kwa milandu yoyipa ndi yabwino polowa m'malo mwa aspartic acid kukhala arginine. kuyanjana kokongola pakati pawo.One subunit (WT-D123R dimer) .Kuyanjana kochititsa chidwi kungathe kulimbikitsa mawonekedwe pakati pa malekezero akunja a chidutswa cha S1, zomwe zimatsogolera ku kugwirizana kwamphamvu kwa allosteric pakati pa ma subunits ndi kuwonjezeka kwa GBTA-kumanga mgwirizano.
Kuti tithandizire lingaliro ili, tidayang'ana njira yolimbikitsira mawonekedwe pakati pa malekezero akunja a gawo la S1, lomwe ndi losiyana ndi kusintha kwa ndalama pa malo 123.Lee et al.Kusintha kwa Hv1 zotsalira I127 kupita ku cysteine kunapezeka kale. kupangitsa kuti pakhale intersubunit cross-linking17. Komanso, mawonekedwe a kristalo a Hv1-CiVSP chimeric VSD adawonetsa kuti I127 imasiyanitsidwa ndi malekezero akunja a S1 helix ndi chotsalira chimodzi chokha24. ma cysteine pa udindo 127, omwe akuyembekezeka kuchititsa kuti pakhale mgwirizano wamphamvu wa S1-S1, ali ndi zotsatira pa GBTA yomangirira mgwirizano wofanana ndi womwe umawonedwa mwa kusintha malipiro pa malo 123.
Tinayesa kulepheretsa kudalira kwa ndende ya Hv1 I127C ndi GBTA ndi 2GBI pokhalapo komanso kusowa kwa 10 mM β-mercaptoethanol (βME) (mkuyu 6a) . Wothandizira amawonjezedwa ku yankho la extracellular. Dimer yolumikizidwa ndi cysteine m'malo mwa subunit imodzi yokha (WT-I127C) idagwiritsidwa ntchito ngati kuwongolera koyipa (mkuyu 6a) . pakati pa ma I127C ma subunits pa 2GBI inhibition, chifukwa kuchuluka kwa Hill kwa I127C kumangiriza ku Hv1, kapena popanda βME, kunali kosiyana kwambiri ndi kumangiriza kwa monocysteine substituted dimers. Phiri la Hill silingathe kusiyanitsa WT-I127C (Mkuyu 6b ndi Supplementary Fig. 3) . Mosiyana kwambiri, tinayesa zotsatira zochititsa chidwi za intersubunit crosslinking pa GBTA inhibition. The Hill coefficient yomanga ku Hv1 I127C popanda βME (crosslink on) anali apamwamba kwambiri kuposa momwe amayezera pamaso pa βME (crosslink off) kapena kugwiritsa ntchito WT-127C kuti agwirizane ndi dimer (popanda crosslink) (mkuyu 6c ndi Supplementary Fig. 4), kusonyeza kuti kugwirizana kwa allosteric pakati pa malo omangiriza kumalimbikitsidwa kwambiri ndi kupanga zomangira za disulfide pakati pa malekezero akunja a zidutswa za S1. Zotsatirazi zimathandizira kutanthauzira kwathu kuti kuyanjana kwa electrostatic kwa aspartate ndi arginine pa malo 123 mu WT-D123R dimer kumabweretsa kuwonjezeka kwa allosteric pakati ma subunits.
Kulumikizana kwa boma kumayanjanitsidwa ndi kuyanjana kwachindunji pakati pa malekezero akunja a gawo la S1 m'magulu awiriwa.
(a) Kuyimilira kwadongosolo kwa ma mutant Hv1 dimers ndi ma linker dimers, opangidwa kuti afufuze momwe ma intersubunit cysteine crosslinks amakhudzira mgwirizano womanga wa GBTA. e) kukhalapo kapena kusakhalapo kwa 10 mM βME mu njira yowonjezera yowonjezera.Mfundo iliyonse imayimira kulepheretsa ± SD ya 3 mpaka 10 miyeso. Mpenderoyo unali wofanana ndi Hill womwe umagwiritsidwa ntchito kupeza ma Kd (onani Supplementary Table 1). Ma coefficients a Hill mu histograms ya inset adatsimikiziridwa kuchokera ku zoyenera zomwe zafotokozedwa mu Supplementary Figs 3 ndi 4. Asterisks mu (c,e) zimasonyeza kusiyana kwakukulu pakati pa mikhalidwe yoletsa ya GBTA yosiyana (p 0.05).
Tinapezanso kuti popanda βME, Hill coefficient ya GBTA yomangirira ku D112E I127C Hv1 dimer inali yaikulu kwambiri kuposa yomwe inayesedwa pamaso pa ochepetsera (mkuyu 6e ndi Supplementary Fig. 4), kutanthauza kuti The D112E mutation Sizinathetse kuwonjezeka kwa mgwirizano womangirira wa GBTA chifukwa cha kulumikizana kwa cysteine 127. The Hill coefficient ya 2GBI yomanga ku D112E, I127C Hv1 dimers sichinakhudzidwenso kwambiri ndi kusintha kwa D112E (Chithunzi 1).6d ndi Supplementary Chithunzi 3).
Kuphatikizidwa, zomwe zapezazi zikuwonetsa kuti kumangirira kwa GBTA kumakulitsidwa ndi kuyanjana pakati pa malekezero akunja a zidutswa za S1 m'magawo oyandikana ndi Hv1 kudzera pamachitidwe owoneka bwino a electrostatic kapena kupanga mapangidwe ogwirizana pakati pa ma cysteines Kulumikizana kwa Allosteric pakati pamasamba kumawonjezeka ndipo kumapangitsa kuti mgwirizano ukhale wogwirizana. Ngakhale zotsatira za kuyanjana kokongola kwa electrostatic pa cooperativity zitha kuthetsedwa ndi masinthidwe a D112E, zotsatira za ma covalent bonds sangathe.
Pakufufuza kwathu kwa malo opangira mankhwala omwe alipo kuti amangire zotumphukira za guanidine kumayendedwe a Hv1, tapeza kuti 2-guanidinothiazoles ngati GBTA ili ndi kudalira kopitilira muyeso kuposa 2-guanidinobenzimidazoles (mkuyu 1c) ndi njira za monomeric (mkuyu 2a, b) ndi njira ya dimeric yomwe subunit imodzi idamangidwa kale ku inhibitor (mkuyu. Kupeza kuti GBTA imamangiriza ku njira yotseguka, monga momwe zasonyezedwera kale pamagulu okhudzana ndi 2GBI32, zikusonyeza kuti mgwirizano wa allosteric ukhoza kuyesedwa makamaka poyera. adatha kufotokoza mochulukira kuletsa kwa Hv1 ndi GBTA (mkuyu 2c) ndikufotokozera zotsatira zosiyanasiyana za 2GBI ndi GBTA pakuwola kwa mafunde a mchira pambuyo pa kukonzanso kwa membrane, komwe kumathandizira kutanthauzira kwathu njira yomangiriza.
The maximum Hill coefficient yomwe ingapezeke mu allosteric protein yokhala ndi malo awiri omangirira (monga Hv1) ndi 2.Tinayesa GBTA kuti imangire Hv1-mtundu wamtundu wa 1.31, womwe unakwera kufika 1.88 mu Hv1 I127C. mphamvu ya synergistic yaulere, kusiyana pakati pa mphamvu zomangira zaulere za malo otsika kwambiri komanso apamwamba kwambiri (onani Njira), anali 1.3 kcal/mole pankhani ya Hv1 zakutchire ndi 2.7 kcal/mole pankhani ya Hv1 I127C. wa okosijeni kupita ku hemoglobin ndi chitsanzo chodziwika bwino komanso chophunziridwa bwino cha njira yogwirira ntchito38. Kwa hemoglobin yamunthu (tetramer yokhala ndi malo anayi omangika a allosterically), Phiri la coefficient limachokera ku 2.5-3.0, ndi miyeso kuyambira 1.26 mpaka 3.64 kcal/mol, malingana ndi mikhalidwe yoyesera38.Motero, ponena za mphamvu zapadziko lonse, mgwirizano wa GBTA yomangiriza ku Hv1 sikusiyana kwambiri ndi wa O2 kumanga ku hemoglobin pamene ziwerengero zosiyana za mapuloteni amagulu mu machitidwe awiriwa akuganiziridwa.
Muchitsanzo chathu cha synergy, kumangirira kwa mamolekyu a GBTA ku gawo limodzi kumabweretsa kuwonjezeka kwa mgwirizano womangirira wa subunit yoyandikana nayo. Kusintha kwa kuyanjana pakati pa magulu ang'onoang'ono.Potsatira kusintha kumeneku, ma subunits oyandikana nawo amasintha malo awo omangirira, zomwe zimapangitsa kuti GBTA ikhale yolimba kwambiri.Panthawiyi, S1 aspartate D112 imayang'anira kukonzanso malo omangirira omwe akugwirizana ndi kuwonjezeka kwa mgwirizano.D112.D112 idawonetsedwa kale kuti ndi gawo la njira ya Hv1 proton permeation ndikuchita ngati selectivity filter27,28. Zotsatira zathu zikuwonetsa kuti zosefera za kusankha m'magawo awiri a Hv1 zimaphatikizidwa momasuka. Chithunzi 7a chikuwonetsa pafupifupi malo omangirira a GBTA ndi malo otsalira D112, D123, K125 ndi I127 pa schematic ya Hv1 VSD yochokera. pa mawonekedwe a kristalo a chimera cha Hv1-CiVSP.Malangizo opangira ma allosteric ophatikizana ndi mapeto a extracellular a S1 akuwonetsedwa ndi mivi yakuda.
(a) Schematic ya Hv1 VSD. Malingana ndi mawonekedwe a kristalo a chimera cha Hv1-CiVSP, zigawo za helical zikuwonetsedwa kuti ndi cylindrical.Pakapangidwe kameneka, mapeto a mkati mwa gawo la S4 akuphatikizana ndi CCD (osasonyezedwa). Malo onenedweratu a GBTA yomangika akuwonetsedwa ngati zozungulira zotuwa. Mivi yakuda ikuwonetsa njira zomwe zimaphatikizidwa ndi kulumikizana kwa allosteric pakati pa masamba omangika m'magawo awiri oyandikana. Malo a zotsalira zomwe zafufuzidwa za S1 zimayikidwa ndi zigawo zamitundu. mu masinthidwe awiri osiyana dimer monga taonera kuchokera extracellular mbali ya nembanemba ndege. kulekanitsidwa kwa malekezero akunja a ma helice awiri a S4 (mivi yophwanyika), kumapanga dongosolo lomwe likuwonetsedwa kumanja.Mu kasinthidwe uku, zotsalira za D123 ndi I127 zochokera kumagulu oyandikana nawo zimaloledwa kuyandikira pamodzi.(c) Kuyimilira kwadongosolo kwa CiVSP VSD mu kusintha kwa dimer komwe kumapezeka mu 4G80 crystal structure.Position P140 mu CiVSP ikugwirizana ndi udindo D123 mu Hv1.
Zotsatira zathu zikuwonetsa kuti kuyanjana konyansa kwamagetsi pakati pa zotsalira 123 (123D/123D kapena 123R/123R) kumalumikizidwa ndi “zabwinobwino” za mgwirizano womanga wa GBTA pamalo otseguka ndikusintha kuyanjanako kuchoka ku kunyansidwa kupita ku Attracted (123D/123R) , kuwonjezeka kwa mgwirizano (mkuyu 5g) .Zikuyembekezeka kuti kuchotsedwa kwa kuyanjana konyansa ndi kulowetsedwa kwa alanine kungapangitsenso kuwonjezereka kwa mgwirizano. Kufotokozera ndikuti zotsatira zowonongeka za kuyika zotsalira za hydrophobic m'malo a hydrophilic zikhoza kupitirira mphamvu yokhazikika chifukwa cha kuthetsa kuyanjana konyansa pakati pa zotsalira za D123, zomwe zimapangitsa kuti kuchepetsa kugwirizanitsa mgwirizano.Mony et al.39 posachedwapa adanena kuti kupezeka kwa zosungunulira pa Udindo wa D171 wa Ciona gutis Ci-Hv1 (wogwirizana ndi D123 mu Hv1 yaumunthu) umachulukitsidwa pakutsegula, kuchirikiza lingaliro lakuti D123 ili m'malo a hydrophilic poyera.
Gating of Hv1 channels imadziwika kuti imachitika kudzera pakusintha kangapo19,20,26,39,40,41.Qiu et al26 adapeza kuti pa depolarization ya membrane, sensor yamagetsi ya Ci-Hv1 imasinthanso kusintha komwe kumasiya tchanelo chitsegulidwa koma chotsekedwa. , kutsatiridwa ndi kusintha kosiyana komwe kumapangitsa kuti ma protoni atsegule conduction mu njira zonse ziwiri. Kusokoneza kwa siginecha ya fulorosenti kumagwirizana ndi kupezeka kwa ma electrostatic interactions pakati pa zotsalira za D171 za ma subunits oyandikana panthawi inayake pamodzi ndi zomwe zimagwirizanitsa kusintha kwa conformational. ndikuyimira kulumikizana kwa allosteric pakati pa masamba omangira a GBTA.
Fujiwara et al25 adanena kuti mawonekedwe a dimer a cytoplasmic coiled-coil domain apitirire mu nembanemba kuti azikhala ndi ma helice awiri a S4 (mkuyu 7b, gulu lakumanzere) . VSD lonse ndi kusanthula ntchito za dera kulumikiza S4 helix ndi CCD. Popeza transmembrane pH gradient, Hv1 njira amafuna ndithu nembanemba depolarization kutsegula, ndi cysteine crosslinking kumachitika pansi pa zinthu pamene njira makamaka chatsekedwa. Chifukwa chake, mawonekedwe odziwika a S4-S4 mwina akuwonetsa kasinthidwe kagawo kakang'ono.Kafukufuku wina wapeza umboni wokhudzana ndi kukhudzidwa kwa S1 ndi S2 muzolumikizana zapakati pa gating17,21,26 zomwe zikuwonetsa kuti njira zitha kutengera masinthidwe osiyanasiyana a subunit poyera komanso. mayiko otsekedwa, lingaliro logwirizana ndi zomwe Mony et al. S1 imasuntha 39 panthawi yolowera.
Pano, tikuwonetsa kuti, poyera, malekezero amtundu wa S1 ali pafupi mokwanira kuti athandizire kuyanjana kwa electrostatic komwe kumagwirizanitsa mgwirizano wa allosteric pakati pa ma subunits. Komabe, kusinthasintha kwa 20 ° molunjika kwa ma subunits awiri a VSD mozungulira axis perpendicular kwa ndege ya nembanemba, kuphatikizapo kupatukana kwa malekezero akunja a ma helice awiri a S4, kunapereka kasinthidwe ka S1-S1. ndi zomwe tapeza (mkuyu 7b, gulu lakumanja) .Timalimbikitsa kasinthidwe kameneka kameneka kakutsegula.
Ngakhale kuti puloteni ya CiVSP imaganiziridwa kuti imagwira ntchito ngati monomer, mawonekedwe a kristalo a VSD yake yakutali amatengedwa mu dimer state. kwa Hv1 (mkuyu 7c) .Mu dimer ya CiVSP, zotsalira zapafupi kwambiri kuchokera ku subunit yoyandikana inali proline pa malo a 140 (mkuyu 7c) .Chochititsa chidwi, P140 mu CiVSP ikugwirizana ndi malo D123 mu Hv1.Kufanana kwa makonzedwe a subunit pakati pa Hv1 ndi CiVSP dimers zimasonyeza kuti ma VSD a mapuloteniwa ali ndi chizolowezi chamkati chopanga mawonekedwe pomwe malekezero a extracellular a S1 amalumikizana.
Udindo wofunikira wa Hv1 poyambitsa ma cell a umuna umapangitsa kuti njira iyi ikhale chandamale chowoneka bwino chamankhwala oletsa kubereka kwa amuna. Kuphatikiza apo, kuyambitsa kwa Hv1 mu microglia kwawonetsedwa kuti kukuwonjezera kuchira ku stroke. kupulumuka kwa odwala omwe ali ndi khansa ya m'mawere 12 kapena khansa yapakhungu 13 ndipo akuganiza kuti amathandizira ku B-cell malignancies 11. Chifukwa chake, mankhwala ang'onoang'ono a molekyulu omwe amayang'ana Hv1 angagwiritsidwe ntchito ngati neuroprotective agents kapena anticancer therapeutics. kutsegulira kwa Hv1 subunit, zomwe zimapangitsa kuti pakhale mgwirizano wowonjezereka, zitha kupangitsa kuti pakhale mankhwala amphamvu kwambiri omwe amalunjika kumayendedwe a Hv1.
Mutagenesis wotsogozedwa ndi tsamba wa Hv1 wamunthu adachitidwa pogwiritsa ntchito njira zokhazikika za PCR.Mukumanga kwa Hv1NCCiVSP, zotsalira 1-96 ndi 228-273 za Hv1 zidasinthidwa ndi zotsalira 1-113 ndi 240-576 za CiVSP18.Mu dimer yolumikizidwa ndi Hv1, C-terminus ya subunit imodzi imagwirizanitsidwa ndi N-terminus ya subunit yachiwiri kupyolera mu mgwirizano wa GGSGGSGGSGSGGSGG. Ma plasmids a pGEMHE omwe ali ndi mapangidwe osiyanasiyana anali opangidwa ndi Nhe1 kapena Sph1 restriction enzymes (New England Biolabs) ndi RNA synthesis inachitidwa ndi T7 mMessage mMachine transcription kit (Ambion) .1-3 masiku asanayezedwe ndi electrophysiological, cRNA inabayidwa mu Xenopus oocytes (50 nl pa selo, 0.3-1.5 μg/μl). Stage V ndi VI oocytes kuchokera ku Xenopus laevis (NASCO) anapezedwa kuchokera ku Ecocyte Bioscience.Kutsatira jekeseni wa RNA, maselo anasungidwa mu ND96 sing'anga yomwe ili ndi 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl, 1 mM MgCl, 10 mM HEPES, 5 mM pyruvate, 100 μg / ml 7 ° C7 ° 1 gentamicin, pH8 ° 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- 5-carboxylate [9] ndi (2-guanidino-4-methyl-1,3-thiazol-5-yl) ethyl acetate [10] anali ochokera ku Sigma-Aldrich.Famotidine [7] anali ochokera ku MP Biomedicals.1-[4] -(4-Chlorophenyl)-1,3-thiazol-2-yl]guanidine[11] ndi 1-[4-(3,4-dimethoxyphenyl)-1,3- Thiazol-2-yl]guanidine [12] anali Kuchokera ku Matrix Scientific.Mapangidwe awa ndi oyeretsedwa kwambiri pamalonda omwe amapezeka pamalonda.Anasungunuka mu DMSO youma kuti apange 100 mM stock solution, yomwe inachepetsedwa muzojambula zojambulira pamtengo womaliza wofunidwa.Mipangidwe 3 ndi 4 inapangidwa pansipa ndi osachepera 99% chiyero.
Kuti kuyimitsidwa kwa 2-amino-4-(trifluoromethyl)benzenethiol hydrochloride (1.02 g, 4.5 mmol) mu 25 ml ya aqueous hydrochloric acid (2.5 N) anawonjezedwa olimba dicyandiamide (380 mg, 4.5 mmol), ndipo chifukwa heterogeneous osakaniza osakaniza. anali refluxed ndi kusonkhezera mwamphamvu kwa maola 4. The osakaniza anachita utakhazikika kutentha firiji ndi neutralized ndi pang'onopang'ono kuwonjezera 10N potaziyamu hydroxide. Madzi oyera anapangidwa ankasefedwa, otsukidwa ndi madzi ozizira (3 × 50 ml), zouma mu uvuni. 65 °C) kwa maola angapo, ndiyeno recrystallized kuchokera ethyl acetate/petroleum ether kupereka olimba woyera (500 mg, 48%); mp 221–222 °C (kuwala 225–226 °C) 45; 1H NMR (500 MHz, DMSO-d6): δ [ppm] = 7.25 (yotakata kwambiri 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 mtengo wowerengedwa. Pakuti C9H8F3N4S (M + H)+0 41, 261. Chithunzi: 261.0419.
Naphtho[1,2-d]thiazol-2-amine (300 mg, 1.5 mmol), yopangidwa monga momwe tafotokozera kale, inatenthedwa kufika ku 200 °C posamba mafuta mu chubu laling'ono loyesera.300 mg (kuchuluka kwakukulu) cyanamide ndi 1.0 ml conc.To pawiri otentha anawonjezedwa hydrochloric asidi mofulumira ndi osakaniza anali kusungidwa mu osamba mafuta kwa mphindi 2, pamene ambiri a madzi evaporated.The anachita osakaniza ndiye utakhazikika kwa firiji ndi chifukwa solidified zakuthupi. anathyoledwa mu tiziduswa tating'ono ting'ono ndikutsukidwa ndi madzi kuti apereke cholimba chachikasu cha amorphous.(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 Hz) , 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/4 mtengo wa H60: H +1 9 , anapeza: 243.0704.
Mafunde a proton ankayezedwa mkati ndi kunja kwa ma oocyte omwe amawonetsa mapangidwe osiyanasiyana pogwiritsa ntchito Axopatch 200B amplifier yoyendetsedwa ndi Axon Digidata 1440A (Molecular Devices) yokhala ndi pClamp10 software. ) 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), yosinthidwa kukhala pH 6.0 yokhala ndi TEA hydroxide. Miyezo yonse inachitidwa pa 22 ± 2 ° C. Pipette ili ndi kukana kofikira kwa 1.5-4 MΩ. Zotsatira zamakono zinasefedwera pa 1 kHz, sampuli pa 5 kHz ndikuwunikidwa ndi Clampfit10.2 (Molecular Devices). ) ndi Origin8.1 (OriginLab).
Mayankho omwe ali ndi mitundu yosiyanasiyana ya Hv1 inhibitor ndipo nthawi zina 10 mM βME adalowetsedwa mu bafa ndi mphamvu yokoka kudzera munjira zambiri zolumikizidwa ndi VC-6 perfusion valve system (Warner Instr.), yomwe idadutsa pClamp software TTL (Transistor- Transistor Logic) zizindikiro.Kuyesa kofulumira kwa kutulutsa madzi kunachitidwa pogwiritsa ntchito pensulo yamachubu ambiri (AutoMate Sci.) yokhala ndi nsonga yobweretsera ya 360 μm yoyikidwa patsogolo pa chigamba pipette.Kuletsa njira kunatsimikiziridwa ndi miyeso ya isochronal amperometric kumapeto kwa + 120 mV depolarizing pulse.GV miyeso idachitidwa monga momwe idafotokozedwera kale18,20.Mafunde a mchira adalembedwa pa -40 mV pambuyo pa masitepe a depolarization pamagetsi osiyanasiyana kuchokera ku -20 mV mpaka +120 mV pokhapokha atanenedwa mwanjira ina. amagwiritsidwa ntchito kukonza kuwonongeka kwamakono 18 .Grafu ya GV ikugwirizana ndi equation ya Boltzmann:
Mawonekedwe a dissociation constants (Kd) amitundu yosiyanasiyana ya ma tchanelo ndi zoletsa (Supplementary Table 1) adatsimikiziridwa ndikuyika kudalira koletsa (kutanthauza % inhib values) ndi Hill equation:
pamene [I] ndi kuchuluka kwa inhibitor I ndi h ndi phiri coefficient. Kuti muwerengere kuchuluka kwa Phiri, equation (2) imasanjidwanso monga:
Nthawi yotumiza: Jun-07-2022
