1
bPlum Island Animal Disease Cen e , ARS, USDA, G eenpo , NY 11944, USA.
cLabo a o y o Molecula Biophysics. Depa men o Physics. Uni e si y Jaume I,
12071 Cas ellón, Spain
*Co esponding au ho :
E-mail: [email p o ec ed]
Mu a ion-induced changes o ansmemb ane po e size e ealed by combined ion-
channel conduc ance and single esicle pe meabiliza ion analyses.
Eneko La goa, Douglas P. Gladueb, Johana To albaa, Vicen e M. Aguilellac, An onio
Alca azc, Manuel V. Bo cab, and José L. Nie aa*
aBio isika Ins i u e (CSIC, UPV/EHU) and Biochemis y and Molecula
Biology Depa men , Uni e si y o he Basque Coun y (UPV/EHU), P.O. Box
644, 48080 Bilbao, Spain.
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Biochimica e Biophysica Ac a (BBA) - Biomemb anes
1860(5) : 1015-1021 (2018), which has been published in inal o m a h ps://doi.o g/10.1016/j.bbamem.2018.01.012. © 2018
Else ie unde CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
2
Abs ac :
Pe meabiliza ion o he Endoplasmic Re iculum (ER) is ins umen al in he p og ession
o hos -cell in ec ion by many i al pa hogens. We ha e desc ibed ha pe meabiliza ion
o ER model memb anes by he po e- o ming domain o he Classical Swine Fe e
Vi us (CSFV) p7 p o ein depends on wo sequence de e minan s: he C- e minal
ansmemb ane helix, and he p eceding pola loop ha egula es i s ac i i y. He e, by
combining ion-channel ac i i y measu emen s in plana lipid bilaye s wi h imaging o
single Gian Unilamella Vesicles (GUVs), we demons a e ha poin subs i u ions
di ec ed o conse ed esidues wi hin hese egions a ec ER-like memb ane
pe meabiliza ion ollowing dis inc mechanisms. Whe eas he pola loop appea ed o be
in ol ed in p o ein inse ion and oligome iza ion, subs i u ion o esidues p edic ed o
ace he lumen o he po e inhibi ed la ge conduc ing channels (> 1nS) o e smalle ones
(120 pS). Quan i a i e analyses o he ER-GUV dis ibu ion as a unc ion o he solu e
size e ealed a selec i e inhibi ion o he pe mea ion o solu es wi h sizes la ge han 4
kDa, u he demons a ing ha he mu a ion a ge ing he ansmemb ane helix
p e en ed o ma ion o he la ge po es. Collec i ely, ou da a suppo he idea ha he
po e- o ming domain o p7 may assemble in o ini e po es wi h app oxima e diame e s
o 1 and 5 nm. Mo eo e , he obse a ion ha he mu a ion in e e ing wi h o ma ion o
he la ge po es can hampe i us p oduc ion wi hou a ec ing ER localiza ion o homo-
oligome iza ion, sugges s p ospec i e s a egies o block/a enua e pes i i uses.
Key wo ds: po e- o ming pep ide; memb ane pe meabiliza ion; ion channel; ER
memb ane; pep ide-lipid in e ac ion
3
1. In oduc ion
Un egula ed po e o ma ion in cell memb anes esul s in he al e a ion o he ionic
homeos asis, he loss o elec ochemical g adien s and ul ima ely cell dea h. Sec e ed
po e- o ming p o eins o pep ides a ge ing memb anes cons i u e molecula weapons
p oduced by o ganisms o di e se o igin, and a e also pa o ances al immune
sys ems, which a e unc ional in de ending agains pa hogen in asion [1-6]. Memb ane
speci ici y o po e o ma ion can be in addi ion exploi ed as he basis o an ibio ic and
an i-cance d ug de elopmen [7-9]. Thus, un a elling he molecula mechanisms ha
sus ain he o ma ion o pe mea ing po es and go e n hei unc ioning in lipid bilaye s
emains a highly ele an esea ch issue [3, 10-13].
Po a ion o he plasma memb ane and he di e en componen s o he
endomemb ane sys em is also a signi ican issue in he cell in ec ion cycle o many
i uses. Fo ma ion by i ally encoded po e- o ming p o eins (designa ed as
“ i opo ins”[14]), o poo ly-selec i e, ion-conduc i e channels has been p o ed
ins umen al in uncoa ing, anspo and ma u a ion o i al pa icles, bu can also
in luence sp ead and pa hogenici y [15, 16]. A conspicuous o ganelle a ge ed by
i opo ins is he Endoplasmic Re iculum (ER) [17]. Besides i s aking pa in he
di e en s ages o i al eplica ion and assembly, pe meabiliza ion o his o ganelle can
modula e se e al i al unc ions and plays an impo an ole in hos cell-dea h [15, 17].
Thus, ER pe meabiliza ion by po e- o ming i al p oduc s is a biologically ele an
phenomenon, bu s udies add essing he mechanisms o po e o ma ion in ER model
memb anes a e o a g ea ex en missing.
To add ess his issue, we ha e ecen ly modelled he ER pe meabiliza ion
phenomenon combining: (i) memb anes ha emula ed he ER lipid composi ion; and
4
(ii) he po e- o ming domain o he Classical Swine Fe e Vi us (CSFV) p7 p oduc
[18-20]. In he one hand, e en hough he ER is he main si e o syn hesis o s e ols and
complex sphingolipids, he ER memb ane displays only low concen a ions o hese
lipid species a localized si es [21]. Acco dingly, he o e all ER memb ane can be ai ly
modelled by a combina ion o i s main cons i uen phospholipids: zwi e ionic PC and
PE plus he anionic PI mixed in a oughly 5:3:2 mola a io [21]. On he o he hand,
CSFV p7 is a small, hyd ophobic p o ein o app oxima ely 60-70 amino acids, which
displays memb ane-po a ing ac i i y and o ms homo-oligome s ha mainly localize o
he ER [15, 18, 19, 22]. La ge Unilamella esicle (LUV) pe meabili y assays mapped
he p7 po a ing domain o i s C- e minal ansmemb ane helix, while he addi ion o he
p eceding pola segmen con e ed pH dependence and sensi i i y o channel blocke s
[19]. These obse a ions we e u he suppo ed by unc ional measu emen s o ion-
conduc ing channel (IC) ac i i y in ER-like plana lipid memb anes [20], which
con i med ha a sequence combining he pola segmen and he C- e minal helix,
designa ed as p7C, comp ises he po e- o ming domain o he p o ein.
He e, o gain insigh s in o he mechanisms unde lying ER pe meabiliza ion by i al
p oduc s and i s po en ial ela ion o i ulence, we ha e ocused on he e ec o poin
mu a ions in ol ing s ic ly conse ed esidues wi hin he cy osolic loop and
ansmemb ane helix o CSFV p7C [18]. We ha e ollowed an unp eceden ed
combined app oach o analyze po e o ma ion, namely, IC ac i i y measu emen s in
plana bilaye s and de e mina ion o he pe meabiliza ion deg ees o indi idual esicles
by luo escence mic oscopy imaging. Ou combina ional app oach p o ided e idence
sus aining he o ma ion in ER-like memb anes o wo ypes o p7C po es wi h
app oxima e diame e s o 1 and 5 nm. Mo eo e , he obse a ion ha a mu a ion
a o ing he small po es hampe ed i us p oduc ion wi hou a ec ing ER localiza ion,
5
poin s o he la ge po e s uc u es as po en ial a ge s o blocking pes i i us
p opaga ion and pa hogenici y.
2.Ma e ials and Me hods
2.1 Ma e ials – Syn he ic pep ides p7C-w , p7C-FH, and p7C-KK (sequences
displayed in Fig 1A) we e p oduced as p e iously desc ibed [18, 19].
Phospha idylcholine (PC), phospah idyle hanolamine (PE), phospah idyle hanolamine-
N-(lissamine hodamine B sul onyl) (Rho-PE), and phospha idylinosi ol (PI) we e
pu chased om A an i Pola Lipids (Bi mingham, AL, USA). The 8-aminonaph alene-
1,3,6- isul onic acid sodium sal (ANTS), p-xylenebis(py idinium)b omide (DPX),
phenol 4-[5-(4-me hyl-1-pipe azinyl)[2,5'-bi-1H-benzimidazol]-2'-yl]- ihyd ochlo ide
(Hoechs 33258), and Alexa Fluo 488 we e ob ained om Molecula P obes (Junc ion
Ci y, OR, USA). Rabbi polyclonal an ibody agains GFP (FL) conjuga ed wi h
ho se adish pe oxidase (HRP) was ob ained om San a C uz Bio echnology (Dallas,
TX, USA). Plasmid con aining mCh-Sec61 be a was a gi om Gia Voel z (Addgene
plasmid # 49155) [23].
2.2 Monolaye pene a ion assays – Pene a ion in o lipid monolaye s was measu ed
o compa e he capaci y o p7 pep ides o inse ing in o memb anes ha mimic he ER
[19]. In b ie , maximal changes in su ace p essu e we e moni o ed as a unc ion o
ini ial su ace p essu e (π0) in a ixed-a ea ci cula ough (μT ough S sys em, Kib on,
Helsinki) measu ing 2 cm in diame e and wi h a olume o 1.25 ml. The aqueous phase
consis ed o 1 ml o 5 mM NaOAc, 100 mM NaCl (pH 5.0). Lipids, dissol ed in
chlo o o m, we e sp ead o e he su ace and he desi ed π0 was a ained by changing
he amoun o lipid applied o he ai -wa e in e ace. Fo allowing inco po a ion in o
6
he monolaye , pep ides we e injec ed in o he aqueous subphase wi h a Hamil on
mic osy inge.
2.3 Vesicle pe meabili y assays – ER-like la ge unilamella esicles (LUV) we e
p epa ed acco ding o he ex usion me hod [24]. Vesicle pe meabiliza ion was assayed
by moni o ing he elease o he medium o encapsula ed luo escen ANTS (ANTS-
DPX assay) [25]. LUV con aining 12.5 mM ANTS, 45 mM DPX, 20 mM NaCl and 5
mM Hepes we e ob ained by sepa a ing he unencapsula ed ma e ial by gel- il a ion in
a Sephadex G-75 column ha was elu ed wi h 5 mM Hepes and 100 mM NaCl (pH
7.4). In e nal and ex e nal osmola i ies we e measu ed in a c yoscopic osmome e
(Osmoma 030, Gono ec, Be lin, Ge many) and adjus ed by adding NaCl. Fluo escence
measu emen s we e pe o med in an SLM Aminco 8100 spec o luo ime e (Spec onic
Ins umen s, Roches e , NY) by se ing he ANTS emission a 520 nm and he
exci a ion a 355 nm. A cu o il e (470 nm) was placed be ween he sample and he
emission monoch oma o . The baseline leakage (0%) co esponded o he luo escence
o he esicles a ime 0, while 100% leakage was he luo escence alue ob ained a e
addi ion o T i on X-100 (0.5% / ).
2.4 Plana lipid memb anes o ma ion –. Two monolaye s we e made om 5 mg/ml
pen ane solu ions o lipid mix u e bu e ed wi h 5 mM NaOAc wi h 150 mM KCl a
bo h sides o Te lon chambe s pa i ioned by a 15 m hick Te lon ilm wi h 70-100 m
diame e o i ices. Plana lipid bilaye s we e o med by monolaye apposi ion on he
o i ices p e iously ea ed wi h a 1% solu ion o hexadecane in pen ane. P o ein and
pep ides dissol ed in DMSO we e supplemen ed o he lipid solu ions p io o
monolaye o ma ion only in one o he chambe sides, he cis side. Bilaye o ma ion
was di ec ly de ec ed and i s hickness can be es ima ed by capaci ance measu emen s.
7
2.5 Channel conduc ance measu emen s – An elec ic po en ial was applied using
Ag/AgCl elec odes in 2 M KCl, 1.5% aga ose b idges assembled wi hin s anda d 250
µl pipe e ips. Po en ial is de ined as posi i e when i is highe a he side o he p o ein
addi ion ( he cis side), while he ans side is se o g ound. An Axopa ch 200B
amplifie (Molecula De ices, Sunny ale, CA) in he ol age-clamp mode was used o
measu ing he cu en and applying po en ial. The memb ane chambe and he head
s age we e isola ed om ex e nal noise sou ces wi h a double me al sc een (Amuneal
Manu ac u ing Co p., Philadelphia, PA). Fo each sample, a leas 50 di e en aces
we e ypically eco ded ( eco ding ime o each ace was 200 s).
A ough i s es ima ion o po e diame e conside ed a cylind ical neu al po e. Thus,
channel conduc ance G can be w i en in e ms o solu ion conduc i i y
, and po e
dimensions, acco ding o he ollowing equa ion:
2
4
D
GL
(1)
whe e L and D s and o leng h and diame e , espec i ely.
2.6 Single esicle pe meabiliza ion –.Fo he single esicle app oach, Gian
Unilamella Vesicles (GUVs) made o PC:PE:PI:Rho-PE (50:30:20:0.1 mole a io)
we e p epa ed acco ding o he elec o o ma ion me hod as desc ibed in p e ious wo ks
[26, 27]. Con ocal luo escence mic oscopy images o indi idual GUVs we e ob ained
in a comme cial Nikon D Eclipse TE2000-U luo escence mic oscope (Nikon
Ins umen s, Tokyo, Japan). Image p ocessing and analyses we e ca ied ou wi h
ImageJ ( sb.in o.nih.go /ij/). Ex en s o pe meabiliza ion we e calcula ed a e
incuba ion wi h Alexa Fluo 488 and 0.2 μM o p7 pep ides om he a io o
luo escence in ensi y inside and ou side each esicle. Exposu e o GUVs o pep ide o
8
30 min o 2 hou s ende ed essen ially he same p opo ion o esicles pe meabilized,
and he same le els o pe meabiliza ion pe esicle, he e o e indica ing ha he sys em
was a equilib ium a he imes selec ed o pe o ming he measu emen s (usually 30
min).
2.7 Cell exp ession o ecombinan p7 –.Fo cell exp ession o GFP-p7 usion
p o eins, 293T cells (2 x 105 cells) we e co- ans ec ed wi h plasmids encoding GFP-p7
usions and mCh-Sec61 be a (1 µg each) using calcium phospha e [17]. Unde hese
condi ions 70-80% o he cells in he cul u e we e success ully ans ec ed. A 36 h
pos - ans ec ion, cells we e ixed wi h 4% o maldehyde in phospha e saline bu e
(PBS) and incuba ed wi h Hoechs dye. Con ocal images we e acqui ed on a Leica
TCS SP5 II mic oscope (Leica Mic osys ems GmbH, We zla , Ge many), using a x63
wa e -imme sion objec i e. Fo he oligome iza ion assays, 293T cells (1.5 x 106 cells)
we e ans ec ed wi h 10 µg o plasmid encoding GFP-p7 cons uc s. A 36 h pos -
ans ec ion, cells we e collec ed in cold PBS, sonica ed o 1 min on ice wi h a p obe
ip sonica o (MSE Sonip ep 150, MSE, UK) and dissol ed in SDS-PAGE loading
bu e . Oligome s we e de ec ed by immunoblo analysis using an i-GFP an ibody.
2.8 Cons uc ion o CSFV mu an s –. A ull-leng h in ec ious clone o he i ulen
B escia s ain (pBIC) [28] was used as a empla e o ob ain all cDNA as desc ibed [18].
pBIC -p7-KK (p7 esidues 39KK40 o EE) and pBIC -p7-FH (p7 esidues 46FH47 o
AA) cons uc s con aining desi ed mu a ions in he genomic a ea encoding o p7
p o ein we e ob ained using he QuickChange XL Si e-Di ec ed Mu agenesis ki
(S a agene, San Diego, CA). P esence o i us in ec ed cells was de ec ed by
immunope oxidase s aining u ilizing he CSFV monoclonal an ibody WH303 (mAb
9
WH303) [29] and he Vec as ain ABC ki (Vec o Labo a o ies, Bu lingame, CA).
16
EE mu a ion a he cy osolic loop e ealed a mo e di use in acellula luo escen
labeling, a pa e n esembling cells exp essing he GFP con ol. Acco dingly, his la e
cons uc did no co-localize signi ican ly wi h mCh-Sec61 (Fig 5B).
To es he oligome ic s a e o he exp essed p o eins, SDS-PAGE analyses we e
subsequen ly pe o med on ex ac s o he ans ec ed cells (Fig 5C). Immunoblo s
p obed wi h he an i-GFP an ibody showed bands consis en wi h he o ma ion o high-
o de homo-oligome s in cells exp essing he GFP-p7 and GFP-p7-FH cons uc s
(a ow). E en hough a band consis en wi h he o ma ion o homo-oligome s was also
ound in cells exp essing he GFP-p7-KK cons uc , he band co esponding o he
highe -o de homo-oligome s was absen in hese samples. F om he esul s displayed
in Figu es 5A-C he ollowing conclusion can be d awn: whe eas he FH x AA
mu a ion does no al e he biogenesis o he i opo in p7, he KK x EE mu a ion
appea s o in e e e wi h inse ion in o he ER memb ane and oligome iza ion he ein.
Finally, o e alua e he e ec o he amino acid subs i u ions 39KK40-EE and 46FH47-
AA in he eplica ion o CSFV, wo ecombinan CSF i uses con aining he
co esponding amino acid subs i u ions in he po e- o ming domain o p7 we e
p oduced using he cDNA in ec ious clone o he B escia s ain (BIC ) as a empla e.
Mu a ed cDNA in ec ious clones pBIC -p7-KK and pBIC -p7-FH we e de eloped by
si e di ec ed mu agenesis.
In ec ious RNA was in i o ansc ibed om each mu a ed ull-leng h cDNA and
used o ans ec SK6 cells [18]. In ec ious i us was escued om ans ec ed cells by
day 4 pos - ans ec ion using he pBIC cons uc encoding o he pa en al i us BIC
wi h i e s eaching almos 107 TCID50/ml. In con as , a e ou independen
ans ec ion p ocedu es, pBIC -p7-KK and pBIC -p7-FH cons uc s did no p oduce
17
in ec ious i uses. Real- ime RT-PCR analysis o o al RNA ex ac ed om cells
ans ec ed wi h pBIC -p7-KK and pBIC -p7-FH cons uc s e ealed genomic RNA
eplica ion, (da a no shown). In addi ion, immunohis ochemis y analysis o ans ec ed
cell monolaye s was pe o med using a monoclonal an ibody ecognizing CSFV
s uc u al glycop o ein E2 (Fig. 5D). As expec ed, cell monolaye s ans ec ed wi h
pBIC-p7 showed a la ge numbe o cells exp essing massi e le els o E2 ( igh -mos
panel). Con e sely, pBIC -p7-FH ans ec ed cells showed he p esence o small and
isola ed oci o cells exp essing s uc u al glycop o ein E2, while he e was a comple e
absence o E2 exp ession in cells ans ec ed wi h pBIC -p7-KK cons uc (mid
panels). A emp s o escue in ec i e i us om cell cul u es ans ec ed wi h ei he
pBIC -p7-KK o pBIC -p7-FH we e pe o med by ou successi e blind passages in
esh monolaye s o SK6 cells. No in ec ious i us could be de ec ed in any o he ou
passages o ei he o he cons uc s es ed. The e o e, esidues 46FH47 and 39KK40 in he
po e- o ming domain o p7 appea o be c i ical o CSFV eplica ion.
18
4. Discussion
ER pe meabiliza ion is a pi o al e en equi ed o he p og ession o he cell
eplica ion cycle o many i uses [15, 17]. The CSFV p7 p oduc has been shown o
pe meabilize ER-like memb anes ollowing a lipid-dependen pa e n [18, 19], and o
localize in he ER upon exp ession [22], and hence cons i u es an op imal model o
s udy his phenomenon. Following s a egies desc ibed p e iously [33, 35], in a
p eceding wo k we es ablished a minimal channel s uc u e ep esen ed by p7C-w [19].
Those s udies pinpoin ed he pola , cy osolic loop 33MRDEPIKK40, as a egula o y
elemen ha con e ed pH and inhibi o sensi i i y o he po e, and he C- e minal helix
41WILLLFHAMTNNPVKTITVALLMVSGV67 as he ac ual po e- o ming domain. The
p7C-w pep ide combined bo h elemen s and was simul aneously compe en in
conduc ing ions ac oss lipid bilaye s mimicking he ER, and in allowing elease o
small solu es (ANTS/DPX) om ER-LUVs [20]. Mo i a ed by he obse a ion ha he
in a ian esidues 39KK40 and 46FH47 we e loca ed wi hin hese unc ional egions [18],
we sough o analyze he e ec o non-conse a i e KK x EE and FH x AA double
subs i u ions on he po e- o ming ac i i y o p7C-w .
Since he cy osolic loop in es s he po e domain o p7 wi h sensi i i y o pH and
inhibi o s [19, 20], we expec ed assembly o less egula ed po es in memb anes be he
consequence o subs i u ing he conse ed Lys39-Lys40 esidues by he nega i ely
cha ged Glu-Glu dipep ide. Howe e , con a y o ou expec a ions, inco po a ing he
KK x EE mu a ion in he p7C-KK pep ide esul ed in de ec i e monolaye pene a ion
and absence o memb ane pe meabiliza ion (Figs 1-4). These indings sugges ha he
loop no only egula es pe meabili y bu also condi ions inse ion in o ER memb anes.
19
In consonance wi h his limi ing de ec , he same mu a ion esul ed in an anomalous
dis ibu ion and de ec i e homo-oligome iza ion o p7 upon cell exp ession (Fig. 5A-
C). In e es ingly, he supp essing e ec o a cogna e mu a ion in HCV p7 has been
ela ed o al e a ions in memb ane- opology [36]. F om molecula dynamics
simula ions, i was in e ed ha he basic esidues si ing in he memb ane in e ace
could engage elec os a ic in e ac ions wi h nega i ely cha ged lipid pola head g oups
o p ope ly o ien he TM helices [33, 37]. Thus, we in e ha pe u bing he se o
in e ac ions be ween basic Lys-Lys esidues and nega i ely cha ged PI a he memb ane
in e ace, may also esul in he ine icien inse ion/po e- o ma ion o CSFV p7.
Finally, highligh ing he c ucial ole o po e- o ming p7 p o ein in he CSFV eplica ion
cycle, he KK x EE mu a ion also supp essed p oduc ion o in ec ious i us (Fig 5D),
an obse a ion in line wi h p e ious esul s ob ained o p7 o he BVDV pes i i us [38]
and ou own Ala-scanning mu agenesis [18].
In con as , he p7C-FH pep ide, displayed less capaci y o inse ing in o monolaye s
han p7C-w , bu e ained he memb ane-pe meabilizing capaci y o he pa en al
sequence measu ed in ANTS/DPX-LUV assays (Fig. 1). Howe e , complemen a ion o
hese bulk measu emen s wi h IC ac i i y and single ER-GUV pe meabili y
de e mina ions e ealed unc ional di e ences be ween bo h sequences (Figs. 2-4).
Elec ophysiogical eco dings disclosed wo dis inc pheno ypic ai s: co-exis ence o
small and la ge conduc ance channels in p7C-w , and p edominance o small ape u es
in he p7C-FH. Simila ly, single-channel eco dings o Hepa i is C i us p7 p o ein by
he pa ch clamp echnique e ealed dis inc conduc ance alues o 35, 57, 120, and 184
pS [33]. I was a gued ha la ge conduc ance le els could ep esen ei he inse ion o
simul aneous channels, o an inc ease in he numbe o p7 monome s pe channel and
hence, a wide cu en passage. Unde ou measu ing condi ions, we obse e
20
compa able conduc ance le els o 120 pS o bo h p7C-w and p7C-FH, which would
be compa ible wi h channel sizes allowing elease o ANTS/DPX, explaining he
simila po a ing capaci ies obse ed in he LUV-based assay. Howe e , in he case o
he p7C-w pep ide, hese s uc u es co-exis ed wi h channels gi ing ise o highe
conduc ance (> 1 nS) whose o igin was unce ain.
To add ess he o igin o he di e en s ages obse ed in he memb ane
pe meabiliza ion induced by p7C-w , he p ocess was imaged a he indi idual esicle
le el by luo escence mic oscopy o ER-GUVs (Figs 3 and 4). These objec s a ain cell
dimensions and, when applied o pe meabili y assays, he single GUV app oach o e s
se e al ad an ages as compa ed o measu emen s in bulk (i.e., LUV-based ANTS/DPX
assays). Fi s ly, owing o he luo escen lipid label, hei su ounding lipid bilaye can
be eadily obse ed by con ocal mic oscopy, which allows moni o ing i s o e all
s abili y upon ea men wi h he pe meabilizing agen , i.e., one can disce n memb ane
pe meabiliza ion h ough disc e e po es s. lipid bilaye disagg ega ion by de e gen -
like mechanisms [3, 10]. Secondly, GUV memb anes can be conside ed as la planes
de oid o cu a u e s ess, i.e., hey mimic he condi ions o he plana bilaye s used in
elec ophysiological expe imen s mo e closely han LUVs. And hi dly, one can disce n
solu e-encapsula ion he e ogenei ies ha may a ise in he popula ion o pe meabilized
esicles, which go unno iced in bulk measu emen s [26].
Explici ly, in he case o o ally pe meabilized GUVs, he open s a e o he po e las s
enough as o allow he equilib a ion o he p obe wi h he ex e nal medium. Bo h, p7C-
w and p7C-FH pe meabilized ER-GUVs o alexa-488 and 4 kDa-dex an acco ding o
his mechanism (Fig. 4B). Howe e , he numbe o o ally pe meabilized ER-GUVs
d opped o la ge dex ans in he case o p7C-FH, bu no in he case o p7C-w (Fig.
21
4C). This obse a ion sugges s ha s ably opened po es o la ge dimensions we e only
accessible o he w sequence. On he o he hand, he pa ial pe meabiliza ion p ocess
seems o be ela ed o ansien memb ane lesions o he bilaye pe meabili y ha do no
allow apid equilib a ion wi h he ex e nal solu ion. The ac ha he numbe o ER-
GUVs pa ially pe meabilized was ma ginal in he p7C-w and p7C-FH samples (Fig
4C), unde sco es he p e alen ole o s able channel-po e s uc u es in media ing IC
ac i i y and dye in lux in o GUVs.
22
5. Conclusions
In conclusion, ou IC ac i i y and GUV pe meabiliza ion da a p o ide a clea
demons a ion o assembling channel-po es wi h dis inc ape u e wid hs by he
po a ing domain o p7 CSFV in ER-mimicking memb anes. To ou bes knowledge,
his is he i s ime ha elec ophysiological and single- esicle app oaches a e
combined o sol e he pe meabiliza ion mechanism o a po e- o ming p oduc
unc ioning in he con ex o ER memb anes. No ably, he FH x AA mu a ion a o ing
small-size po es ab oga ed i us p oduc ion wi hou al e ing he cell dis ibu ion o
oligome iza ion deg ee o p7 upon exp ession (Fig. 5). Fu he esea ch e o s will be
equi ed o ge insigh in o he dis inc physiological oles o he p7C capaci y o o m
po es o di e en dimensions, and we o esee ha he FH x AA mu a ion desc ibed in
his wo k may p o ide a use ul ool in he con ex o cell in ec ion by pes i i uses. I is
emp ing o specula e ha manipula ing po e- o ming unc ion o p7 by mu agenesis
may p o ide in he u u e new app oaches o accine and an i- i al de elopmen o ea
pes i i us in ec ions.
23
Acknowledgemen s
This s udy was in pa suppo ed by he Ag icul u al Resea ch Se ice o he US (ARS-
USDA P ojec 8064-32000- 056-18S o EL and JLN) and he Basque Go e nmen
(P ojec IT838-13 o JLN). Financial suppo om he Minis y o Economy and
Compe i i eness o Spain (p ojec s no. FIS2013-40473-P and FIS2016-75257-P), and
Uni e si a Jaume I (p ojec no. P1.1B2015-28) is also acknowledged.
24
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