Nīnau ʻana o ke kikowaena intersubunit o ke kaila proton Hv1 hāmama me kahi ʻimi allosterically i hui pū ʻia.

Mahalo iā ʻoe no kou kipa ʻana iā Nature.com. ʻO ka polokalamu kele pūnaewele āu e hoʻohana nei he kākoʻo liʻiliʻi ia no CSS. No ka ʻike maikaʻi loa, paipai mākou e hoʻohana ʻoe i kahi polokalamu kele pūnaewele hou (a i ʻole e hoʻopau i ke ʻano hoʻohālikelike ma Internet Explorer). kākoʻo mau, e hōʻike mākou i ka pūnaewele me ka ʻole o nā styles a me JavaScript. ʻO ka Hv1 voltage-gated proton channel he dimeric complex i loaʻa i ʻelua mau kikowaena voltage-sensing domains (VSDs), kēlā me kēia mea i loaʻa kahi alahele proton permeation gated. Hoʻomalu ʻia ka dimerization e ka cytoplasmic coiled-coil domain. ʻO ka hoʻololi ʻana mai ka mokuʻāina pani a hiki ʻike ʻia ke ʻano ākea o nā VSD ʻelua e hui pū ʻia; akā naʻe, liʻiliʻi ka ʻike e pili ana i nā mīkini kumu. akā naʻe, ʻaʻole i ʻike ʻia kēlā mau pilina ma nā kaha Hv1 hāmama. Eia mākou e hōʻike nei i nā derivatives 2-guanidinothiazole e kāohi i ʻelua Hv1 VSD ma ke ʻano hui like a hoʻohana i kekahi o kēia mau pūhui ma ke ʻano he ʻimi no ka hui allosteric ma waena o nā subunits hāmama. Ua ʻike mākou ʻo ka extracellular. ʻO ka hopena o ka ʻāpana transmembrane mua o VSD e hana i kahi interface intersubunit e hoʻopili ana i ka hoʻopili ʻana ma waena o nā pūnaewele paʻa, ʻoiai ʻaʻole pili pono ka domain coiled-coil i kēia kaʻina. . He kuleana koʻikoʻi nā kahawai proton-gated i nā ʻano mea ola like ʻole, mai ka phytoplankton a hiki i ke kanaka1. I ka hapa nui o nā cell, ua hoʻoponopono kēia mau ala i ka proton efflux mai ka membrane proton a hoʻoponopono i ka hana o NADPH oxidase. ʻO ia ka Hv1, ʻo ia ka huahana o ka HVCN1 gene2,3.Hv1 (aka VSOP) ua hōʻike ʻia e pāʻani i ka hana i ka hoʻonui ʻana o ka cell B4, ka hana ʻana o nā ʻano oxygen reactive e ka ʻōnaehana immune innate5,6,7,8, sperm cell. ka motility9 a me ka hooponopono pH o ka wai ili o ke ea10. Hoʻopili ʻia kēia kanal i kekahi mau Overexpressed i nā ʻano maʻi kanesa e like me B-cell malignancies4,11 a me ka umauma a me ka colorectal cancers12,13. ʻO ka nui o ka hana Hv1 i ʻike ʻia e hoʻonui i ka hiki ke metastatic o nā maʻi maʻi maʻi 11, 12 .Ma ka lolo, hōʻike ʻia ka Hv1 e microglia, a ua hōʻike ʻia kāna hana e hoʻonui i ka pōʻino o ka lolo ma nā ʻano o ka maʻi ischemic stroke. Aia i loko o ka protein Hv1 kahi kikowaena voltage-sensing (VSD), nona nā ʻāpana transmembrane ʻehā i kapa ʻia ʻo S1 a i S414. Ua like ka VSD me nā kikowaena pili o nā kaha uila Na +, K + a me Ca2 + a me nā phosphatases-sensitive, e like me CiVSP mai. ʻO Ciona gutis15. Ma kēia mau protein ʻē aʻe, pili ka C-terminus o S4 i kahi module effector, ka pore domain a i ʻole enzyme. Ma Hv1, pili ʻia ka S4 i ka domain coiled-coil (CCD) aia ma ka ʻaoʻao cytoplasmic o ka membrane. .He paʻakikī dimeric ka kanal i loaʻa i ʻelua VSD, aia kēlā me kēia me ka gated proton permeation pathway16,17,18. Ua ʻike ʻia kēia mau subunits ʻelua Hv1 e wehe like ʻia19,20,21,22, e hōʻike ana e pāʻani ana ka hui allosteric a me nā pilina waena. He kuleana koʻikoʻi i ke kaʻina hana. ʻAʻole i hāʻawi ʻia ka ʻike e pili ana i kēia interface ka ʻano aniani o ka Hv1-CiVSP chimeric protein, no ka mea, ʻo ka hui trimeric o ka crystallized channel complex ma muli o ka hoʻololi ʻana o ka Hv1 CCD maoli e ka hū leucine zipper GCN424. ʻO kahi noiʻi hou o ka hui subunit o ke kahawai Hv1 i hoʻoholo i ka hoʻololi ʻana o ʻelua mau helices S4 i ka CCD me ka ʻole o ka hoʻohaunaele nui ʻana o ka hale kiʻekiʻe, e hopena i nā helices lōʻihi e hoʻomaka ana mai ka membrane a me ka papahana i loko o ka cytoplasm. Ke manaʻo nei kēia haʻawina e hoʻopili nā Hv1 VSD i kekahi i kekahi ma ka māhele S4. Akā naʻe, ua manaʻo nā noiʻi ʻē aʻe i nā pilina ʻokoʻa ma waena o nā VSD. ʻO kēia mau mea hoʻopili i loaʻa i nā ʻāpana S1 17, 21, 26 a me nā ʻaoʻao o waho o ka māhele S2 21. He kumu kūpono no ka hakakā. ʻO nā hopena o kēia mau haʻawina ʻo ia ka hoʻopili ʻana o ka allosteric ma waena o nā VSD i nānā ʻia e pili ana i ke kaʻina hana, e hilinaʻi ana i ka pani a me ka wehe ʻana, a ʻokoʻa paha ka pilina ma waena o nā VSD i nā loli mokuʻāina like ʻole i ka conformation. Maʻaneʻi, ua ʻike mākou ua kāohi ʻo 2-guanidinothiazole i nā ala Hv1 ma ka synergistically hoʻopaʻa ʻana i ʻelua VSD hāmama, a hoʻohana i kekahi o nā pūhui, 2-guanidinobenzothiazole (GBTA), e ʻimi i ka pilina ma waena o nā subunits i ka mokuʻāina ākea. interface.Ua ʻike mākou e hiki ke wehewehe maikaʻi ʻia ka pihi hoʻopaʻa GBTA e kahi kumu hoʻohālike e hoʻopaʻa ʻia ai ka mea paʻa i hoʻokahi subunit e hopena i ka hoʻonui ʻana i ka pili pili o ka subunit pili. Ua ʻike pū mākou i nā koena D112, nā kānana koho channel 27, 28 a me ka hapa o ka guanidine derivative binding site 29 control GBTA binding cooperativity.We hōʻike i ka hoʻopaʻa ʻana i ka hoʻopaʻa ʻana i mālama ʻia i ka dimer Hv1, kahi i hoʻokaʻawale ai ka CCD mai ka māhele S4, e hōʻike ana ʻaʻole pololei ke kikowaena intersubunit i ka CCD. hoʻopili i ka hui allosteric ma waena o nā pūnaewele paʻa GBTA. ʻO ka hoʻohālikelike, ʻike mākou ʻo ka ʻāpana S1 kahi ʻāpana o ke kikowaena ma waena o nā subunits a manaʻo i kahi hoʻonohonoho o nā VSD e pili ana me ka hopena extracellular o ka S4 helix mai ke kikowaena o ka dimer e ʻae. ʻo ka ʻāpana S1 i ke kūlana ākea. ʻO nā mea hoʻopaneʻe mole liʻiliʻi o Hv1 he mea pono e like me nā lāʻau anticancer a me nā mea neuroprotective. Eia naʻe, i kēia lā, ua hiki i nā mea hoʻohui ke kāohi i ke kahawai30,31,32,33. Ma waena o lākou, 2-guanidinobenzimidazole (2GBI, hui [1] ma Fig .1a) a me kona mau mea i loaa i ka VSD29,32 permeation o protons ma o ke ala. i hōʻike muaʻia e kāohi i ka Hv1 kokoke e like me ka 2GBI i ka wā i ho'āʻoʻia ma kahi o ka 200 μM (Figure 1b). kikokikona) Ua hoʻoholo mākou i nā kaʻe pane pane o nā thiazole derivatives ʻehā (GBTA a me nā pūhui [3], [6] a me [11], Fig. 1c) a ʻike mākou ua ʻoi aku ka steeper ma mua o ka 2GBI. The Hill coefficients (h) o ka thiazole derivatives mai ka 1.109 ± 0.040 a i ka 1.306 ± 0.033 (Fig. 1c a me ka Fig. kahua paʻa, 29,32 ua manaʻo mākou ʻo ka hoʻopaʻa ʻana i kahi derivative thiazole i hoʻokahi subunit hiki ke hoʻonui i ka hoʻopaʻa ʻana o kahi molekele inhibitor lua i ka subunit pili. ʻO GBTA ka pūhui hoʻāʻo me ka helu kiʻekiʻe Hill. No laila, koho mākou i kēia hui e aʻo hou i ke ʻano o ka hoʻopaʻa ʻana i ka synergy a hoʻohana iā 2GBI ma ke ʻano he mana maikaʻi ʻole. (a) Nā pūhui hoʻāʻo: [1] Kuhikuhi 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) Ke kaohi ʻana i ka hana Hv1 kanaka e nā guanidinothiazoles i hōʻike ʻia a me ka hui kuhikuhi 2GBI (nā lāʻau uliuli-ʻōmaʻomaʻo) .Hv1 proton au i ana i loko-waho plaques o Xenopus oocytes i pane i ka depolarization mai ka paʻa hiki o -80 mV i +120 mV. kela mea paʻa hoʻopaʻa 'ia i ka 'auʻau 'auʻau ma kahi o 200 μM.pHi = pHo = 6.0 .He mean ± SEM (n≥4) ka ikepili.(c) Hoʻopaʻa ʻia ka hoʻopaʻa ʻana o ke kanaka Hv1 e nā pūhui [2], [3], [6] a me [11] .Ke hōʻike nei kēlā me kēia kiko i ka mean inhibition ± SD o 3 i 15 ana. ʻO ka laina kahi Hill kūpono i hoʻohana ʻia no ka loaʻa ʻana o nā koina Kd i hōʻike ʻia ma ka Pākuʻi Pākuʻi 1. Ua hoʻoholo ʻia nā coefficients Hill mai nā kūpono i hōʻike ʻia ma ka Fig. 1: h(1) = 0.975 ± 0.024 h(2) = 1.306 ± 0.033, h(3) = 1.25 ± 0.07, h(6) = 1.109 ± 0.040, h (11) = 1.179 ± 0.036 (e nānā i nā Hana Hana). (a,b) Hoʻopaʻa ʻia nā hui 2GBI a me GBTA i ka dimeric a me ka monomeric Hv1 ma ke ʻano hilinaʻi. ua hoʻoholo ʻia nā histograms inset mai nā kūpono i hōʻike ʻia ma nā Figs 3 a me 4. ʻO ka pane ʻana o ka GBTA i hōʻike ʻia ma (a) ua like ia me ka Fig. ka hoʻopaʻa ʻana o GBTA i ka dimeric Hv1. ʻO ka laina ʻeleʻele paʻa e hōʻike ana i ke kūpono i ka ʻikepili hoʻokolohua ma ka hoohalike (6), e wehewehe ana i ke kumu hoʻohālikelike i hōʻike ʻia ma (d). - nā ʻāpana kaulike hoʻokaʻawale o nā hanana paʻa mua a me ka lua, i kēlā me kēia (Sub 1: OO + B ⇄ BO*, Kd1 = 290 ± 70 μM; Sub 2: BO* + B ⇄ B*O*, Kd2 = 29.3 ± 2.5 μM ).(d) Hoʻomāhuahua ka pilina o ka ʻāpana hāmama e pili ana (Kd2 0.05) i ka helu Hill o GBTA e hoʻopaʻa ana i nā kahawai GGG i hoʻohālikelike ʻia me ke ʻano hihiu (Fig 5c), e hōʻike ana ʻoiai ʻo kāna kuleana i ka mālama ʻana i ka ʻelua mau ʻāpana he mea koʻikoʻi, akā ʻaʻole ʻo CCD e hoʻopili pololei i ka hoʻohui ʻana o nā allosteric intersubunit ma waena o nā pūnaewele paʻa GBTA. (a) Hoʻolālā kiʻi kiʻi o ka dimer Hv1 me kahi hoʻololi triglycine ma ka ʻaoʻao o loko o S4 i hoʻolālā ʻia e hoʻopau i ka hoʻopili intersubunit i hoʻopili ʻia e ka cytoplasmic coiled-coil domain (nā pua uliuli). hōʻike ʻia nā hoʻololi ʻana, i hoʻolālā ʻia e hoʻāʻo i nā ʻāpana S1 i pili i ka hoʻopili ʻana ma waena o nā subunits (nā pua uliuli). ± SD o 3 a 10 ana. ʻO ka pihi kahi Hill kūpono i hoʻohana ʻia no ka loaʻa ʻana o nā waiwai Kd (e nānā i ka Papa Hoʻohui 1). Hōʻike ʻia ʻo Hv1 WT ma ke ʻano he mau laina kaha. Ma muli o ko mākou hoʻokuʻuʻana i ka CCD ma keʻano he mea hoʻoponopono pololei o ka hui pūʻana o GBTA, ua nīnau mākou inā pili ka pilina ma waena o nā VSD i ka hui allosteric ma waena o nā pūnaewele hoʻopaʻa. i loko o ka S1 transmembrane segment.S1 loaʻa i ʻelua mau koena ʻino ʻē aʻe, E119 a me D123, aia ma ka ʻaoʻao hoʻokahi o ka helix me D112, akā kokoke i ka hopena o waho 24 o ka ʻāpana. i D112 ma o waterlines36,37. No laila, ua hoʻāʻo mākou inā pili ʻo E119 a me D123 i ka hui allosteric ma waena o nā pūnaewele paʻa GBTA (Fig. 5b). Ma ke ʻano he mana maikaʻi ʻole, ua hoʻāʻo mākou i ke kūlana i hoʻopaʻa ʻia ʻo K125, aia ma kahi kokoke i D123 akā ma kēlā ʻaoʻao o ka S1 helix (Fig. 5b). Ua ana mākou i ka inhibition hilinaʻi o ka E119A, D123A a me K125A e 2GBI a me GBTA a ʻike ʻia ʻaʻole i hoʻololi nui ʻia nā hoʻololi ʻana o E119A a me K125A i ka helu Hill o kēlā me kēia inhibitor i hoʻohālikelike ʻia me ke ʻano hihiu (p> 0.05, Fig. 5d,i). ). Ma ka ʻaoʻao ʻē aʻe, ua hoʻemi nui ka mutation D123A i ka helu Hill o GBTA (p 0.05/14). Ma muli o ka hoʻokaʻawale ʻana i ka hoʻopiʻi ma ke kūlana 123 ma nā ʻāpana ʻelua i hopena i ka hoʻololi ikaika ʻana i ka hui pū ʻana o ka GBTA, ʻoiai ʻo ka hoʻohuli ʻana i ka uku ma nā ʻāpana ʻelua he hopena liʻiliʻi wale nō, ua hoʻolōʻihi mākou i ka nānā ʻana e hoʻokomo i hoʻokahi subunit wale nō me ke kahawai hoʻohuli o ka uku. i hoʻokumu i nā dimer Hv1-linked me D123R substitution i loko o ka C-terminal subunit (Fig. 5b) a ana i ka hoʻopaʻa ʻana-pane inhibition e GBTA a me 2GBI. Ua ʻike mākou ua ʻoi aku ka kiʻekiʻe o ka helu Hill o GBTA e hoʻopaʻa ana i nā kahawai WT-D123R ma mua o kēlā. o ka wild-type Hv1 (p