ERK'2021, Portorož, 407-410 407
Initial steps of pore formation  in planar lipid bilayers made of 
phospholipids and archaeal lipids  
 
Alenka Maček Lebar, Damijan Miklavčič, Peter Kramar 
Fakulteta za elektrotehniko, Tržaška 25, 1000 Ljubljana  
E-pošta: peter.kramar@fe.uni-lj.si 
 
 
Vzpostavitev vodnih por v ravninskih 
lipidnih dvoslojih zgrajenih iz fosfolipidnih 
in arheolipidnih molekul 
 
Povzetek. Plazemska membrana je biološka membrana, 
ki ločuje notranjo celično vsebino od zunanjega okolja. 
Zato je zgradba plazemske membrane odvisna od okolja, 
v katerem se celica običajno nahaja. Arheje so se tudi z 
zgradbo plazemske membrane prilagodile na ekstremne 
pogoje v katerih živijo. V študiji smo preučevali proces 
nastajanja vodnih por med elektroporacijo ravninskih 
lipidnih dvoslojev zgrajenih iz lipidnih molekul 
pridobljenih iz arheje Aeropyrum pernix K1 in 
ravninskih lipidnih dvoslojev zgrajenih iz fosfolipidnih 
molekul 1-pamitoyl 2-oleoyl phosphatidylcholine 
(POPC), 1-pamitoyl 2-oleoyl phosphatidylserine (POPS) 
in mešanice POPC:POPS v razmerju 1:1. Izmerili smo 
specifično kapacitivnost ravninskih lipidnih dvoslojev ter 
porušitveno napetost,; i.e. napetost, ki povzroči porušitev 
ravninskega lipidnega dvosloja ob prisotni 
transmembranski napetosti. Porušitveno napetost smo 
določili z izpostavitvijo ravninskih lipidnih dvoslojev 
linearno naraščajoči napetosti. Specifična kapacitivnost 
ravninskih lipidnih dvoslojev je v veliki meri odvisna od 
dolžine verige ogljikovodikov, saj najmanjšo specifično 
kapacitivnost izkazujejo ravninski lipidni dvosloji 
narejeni iz lipidov arheje Aeropyrum pernix K1, največjo 
pa ravninski lipidni dvosloji narejeni iz POPC. 
Porušitvena napetost ravninskih lipidnih dvoslojev 
zgrajenih iz lipidov arheje Aeropyrum pernix K1 je bila 
pri vseh uporabljenih naklonih linearno naraščajoče 
napetosti večja, kot porušitvena napetost ravninskih 
lipidnih dvoslojev narejenih iz POPC, POPS ali 
POPC:POPS (1:1). Pri vseh obravnavanih ravninskih 
lipidnih dvoslojih zavzamejo vodne pore manj kot 1% 
površine ravninskega lipidnega dvolsoja. Na podlagi 
zgradbe obravnavanih ravninskih dvoslojev sklepamo, 
da je proces nastajanja vodnih por odvisen od urejenosti 
lipidnih molekul v ravninskem lipidnem dvosloju. 
 
1 Introduction 
The composition of plasma membrane is not the same in 
all cells. Phospholipids, glycolipids and sterols are the 
most common lipid molecules in plasma membranes of 
eukaryotic cells and bacteria. But archaeal membranes, 
for example, contain glycerol ether lipids with saturated 
chains containing methyl branches [1]. Unique 
characteristic of archaeal membranes are the reason for 
diversity of studies suggesting their use in various 
biomedical applications [2]. Among others, 
archaeosomes are proposed for using as a drug carrier. In 
this case drug release can be enhanced by electroporation 
[3]. Considering such application, the behaviour of the 
archaeal lipid membrane in electric field is important in 
addition to membrane’s mechanical and chemical 
properties. 
 In this study we focused on the initial processes of 
pore formation during electroporation in planar lipid 
bilayers that constitute the membrane of the aerobic 
hyperthermophilic archaeon Aeropyrum pernix K1. The 
detailed structure of constituents, 2,3-di-O-
sesterterpanyl-sn-glycerol-1-phospho-1’-(2’-O- -D-
glucosyl)-myo-inositol (AGI) and 2,3-di-O-
sesterterpanyl-sn-glycerol-1-phospho-myo-inositol (AI), 
was elucidated by Morii et al. in 1999 [4]. These two 
lipids usually compose archaeal membrane in the mol% 
ratio 91:9. The important feature of both lipids is C25-
isopranoid as a hydrophobic part, while in the head of the 
lipid molecule inositol is linked on the phosphate group 
in AI and glucosylinositol in AGI. Hydroxyl groups are 
present on all available C-atoms in the sugar rings. 
 Archaeosomes prepared from lipids isolated form A. 
pernix K1 exhibit large negative surface charge (-50 to -
110 mV, increasing with diameter) in broad pH range 
(2.5 to 12) [5]. Typical gel to liquid phase transition in 
the temperature range from 0
o
C to 100
o
C has not been 
detected [5]. Electron paramagnetic resonance spectra 
have shown that the archaeosome membranes are 
heterogeneous, and are composed of several regions with 
three types of fluidity characteristics. Presence of each 
fluidity type depends on pH and temperature. In general, 
increase in membrane fluidity with temperature has been 
also noticed [5]. 
 To explore the possible differences in initial steps of 
pore formation in planar lipid bilayers of different 
compositions, we formed planar lipid bilayers using 
lipids extracted from archaea A. pernix K1, 1-pamitoyl 2-
oleoyl phosphatidylcholine (POPC) - lipid molecules 
with a zwitterionic head group and almost zero 
spontaneous curvature, negatively charged 1-pamitoyl 2-
oleoyl phosphatidylserine (POPS) molecules with 
negative spontaneous curvature and a mixture of POPC 
and POPS in 1:1 ratio. For each planar lipid bilayer 
composition we determined the capacitance C of planar 
lipid bilayer. Then, planar lipid bilayers were exposed to 

408
 
linearly rising voltage signals of different slopes k u and 
the voltage U br and time t br at which planar lipid bilayer 
rupture occurs were measured. From these measurements 
we evaluated the reduction of C that corresponds to 
presence of water pores in planar lipid bilayer and is a 
consequence of significant difference in dielectric 
constant values of lipid bilayer and water. We also 
estimated the fraction of planar lipid bilayer area that is 
occupied by water pores.the fraction of planar lipid 
bilayer area that is occupied by water pores. 
 
2 Materials and Methods 
2.1 Chemicals  
The extraction of archaeal lipids was done at University 
of Ljubljana, Biotechnical Faculty, Slovenia. The lipids 
POPC and POPS were purchased in powder form (Avanti 
Polar-Lipids Inc. USA). A solution of 10 mg/ml of each 
lipid was prepared in a 9:1 mixture of hexane and ethanol. 
POPS:POPS (1:1) mixture was prepared by pipetting the 
same amount of both lipid solutions in a small plastic 
tube and mixing by repeated pipetting immediately 
before the experiments. The mixture of hexadecane and 
pentane in the ratio of 3:7 was used for torus formation. 
The electrolyte was prepared from 0.1 M KCl and 0.01 
M Hepes in the volume ratio of 1:1. A few drops of 1 M 
NaOH were added to obtain a pH of 7.4.  
 
2.2 Experimental setup and measurements 
Planar lipid bilayers were formed by the Montal-Muller 
method across a circular hole 117 m in diameter (d) as 
described previously [6]. All experiments were 
performed at room temperature (24°C). 
 The measurement protocol consisted of planar lipid 
bilayer formation and C, U br, and t br determination. C was 
measured by the planar lipid bilayer discharge method  
and normalized to the area of the hole (A = 1.075 ٠10
-8
 
m
2
) to calculate the specific capacitance of the planar 
lipid bilayer c BLM. Transmembrane voltage on planar 
lipid bilayer was buid-up using a linearly rising voltage 
signal. Seven different slopes of the linearly rising 
voltage k u (4.8 kV/s, 5.5 kV/s, 7.8 kV/s, 11.5 kV/s, 16.7 
kV/s, 21.6 kV/s and 48.1 kV/s) were used. At the time t br, 
when a sudden increase in transmembrane current was 
detected, U br was measured. 
 
2.3 Experimental data analysis  
The specific capacitance c BLM was determined for at least 
18 planar lipid bilayers of each lipid composition. Mean 
value and standard deviation were calculated for each 
experimental group.  
 Using nonlinear regression, a two-parameter 
strength-duration curve 
𝑈 =
√
𝑎 +
𝑏 𝑡 4
                                (1) 
based on the viscoelastic model of Dimitrov [7] and 
proposed by Sabotin et al. [8] was fitted to 
experimentally obtained points (t br, U br) measured at 
different slopes of the linearly rising voltage. The 
parameter √ 𝑎 4
 is an asymptote of the strength-duration 
curve corresponding to minimal breakdown voltage 
U brmin for planar lipid bilayers of a given lipid 
composition. The parameter b determines the inclination 
of the strength-duration curve. As in the case of strength-
duration curve for excitable cell membranes of nerves 
and muscles, we can define for each lipid composition a 
kind of a chronaxie time constant t c = b/( 15 ٠a) as the 
minimum time required for a stimulus of twice U brmin to 
cause rupture of the planar lipid bilayer. 
 In the short interval before t br we can assume that non-
conductive water pores are already present in the planar 
lipid bilayer [9]. The capacitance of planar lipid bilayer 
C br can be represented as 
𝐶 𝑏𝑟
= ∑ 𝐶 𝑝𝑖
+ ∑ 𝐶 𝑙𝑗
𝑚 𝑗 =1
𝑛 𝑖 =1
                 (2) 
where C pi =  p  0A pi/D is the capacitance of the i-th water 
pore and C lj =  l  0A lj/D is the capacitance of j-th patch of 
the still intact planar lipid bilayer.  p is a dielectric 
constant of water (  p = 80),  l  is a dielectric constant of 
planar lipid bilayer (  l = 2) and  0 is vacuum permittivity 
(  0 = 8.854 253 ٠10
-12
 Fm
-1
). The thickness D of the 
planar lipid bilayer was considered 6.00, 3.75, 4.32 and 
4.00 nm for A. pernix K1, POPC, POPS, and 
POPC:POPS 1:1 respectively. The values were obtained 
from molecular dynamic (MD) simulation as the head to 
head distance between two electron density profiles [10, 
11].  
 Change in planar lipid bilayer capacitance ΔC due to 
the water pore formation is C br – C, if we assume that 
capacitance of intact planar lipid bilayer at the beginning 
of the experiment is C =  l  0A/D. To obtain the fraction 
of the planar lipid bilayer that is occupied by pores 
(A wat/A), Σ iA pi is calculated from ΔC and normalized on 
the whole area of planar lipid bilayer A. ΔC was 
evaluated from experiments as described previously [6]. 
  
3 Results and discussion 
In Table 1 c BLM and U brmin determined for planar lipid 
bilayers made of different lipid molecules are given. The 
specific capacitance of planar lipid bilayer is related to its 
geometrical dimensions; i.e. planar lipid bilayer area and 
thickness. All planar lipid bilayers were formed on the 
hole of the same size, therefore the thickness is the main 
geometrical parameter that affects c BLM. Planar lipid 
bilayers made of A. pernix K1 have considerably smaller 
c BLM then planar lipid bilayers made of POPC, POPS or 
POPC:POPS (1:1) mixture. It seems that the reason is in 
longer hydrocarbon chains in both archaeal lipids (C 25,25) 
than in POPC and POPS lipids (C 16, 18) that leeds to 
thicker planar lipid bilayer made of A. pernix K1.  
 

409
 
Tabel 1. Specific capacitance (cBLM) and minimal breakdown 
voltage (Ubrmin) for planar lipid bilayers made of different lipid 
molecules. The values are given as mean ± standard deviation 
(number of measurements). 
Lipid composition cBLM [ F/cm
2
] Ubrmin [V] 
A. pernix K1 
POPC  
POPS 
POPC:POPS 1:1. 
0.16 ± 0.06 (18) 
0.51 ± 0.17 (80)  
0.41 ± 0.14 (76) 
0.31 ± 0.07 (60)  
0.61±0.36 
0.42 ± 0.23 
0.53 ± 0.24 
0.42 ± 0.23 
 
 Application of linearly rising voltage of different 
slopes k u resulted in typical “strength-duration” behavior 
of U br. The steeper the slope of the linearly rising 
stimulus, the higher transmembrane voltage is needed for 
planar lipid bilayer rupture. We fitted the strength–
duration curves (1) to (t br, U br) points measured for each 
and every lipid compositions (Figure 1). Archeal planar 
lipid bilayers exhibit the highest U br at all slopes k u of 
linearly rising voltage. The lowest values of U br were 
obtained for POPC planar lipid bilayers, where, 
according to MD simulations, hydrophilic pores occur 
before final membrane rupture, and planar lipid bilayers 
formed from a POPC:POPS (1:1) mixture.  
  
 
 
Figure 1. Breakdown voltages of specific lipid compositions 
measured during voltage-controlled experiments. The gray 
lines show different slopes ku of linearly rising signal. Colored 
curves represent two-parameter strength-duration curves (1) 
fitted to the data obtained by voltage-controlled measurements 
on POPS, POPC, POPC:POPS mixture 1:1 and A. pernix K1, 
respectively. 
 
 
 In Figure 2, the fraction of the planar lipid bilayer area 
that is occupied by water pores at the moment of planar 
lipid bilayer rupture is presented as a function of  U br. 
Aqueous fractional area in the planar lipid bilayer due to 
an exponential pulse, bipolar square pulse, or a square 
pulse was theoretically evaluated by Freeman et al. in 
1994 [12]. According to their calculations, only 0.1% of 
the membrane is occupied by pores, which is in good 
agreement with our experimental results obtained when 
the transmembrane voltage was built up slowly. MD 
simulations have shown that in the case of planar lipid 
bilayers made of archaeal lipids and in the case of POPS 
planar lipid bilayers only hydrophobic pores are created 
[11, 13]. Wodzinska et al. claim that formation of the 
pore must keep the overall area of the membrane constant 
[14], which means that hydrophobic pores cannot expand 
and consequently lead to final planar lipid bilayer 
breakdown. 
 The properties of lipids which contribute to the 
stability of lipid bilayers when subject to transmembrane 
voltage are numerous and diverse, and include at least the 
structure of the lipid tails, the chemical nature and charge 
of the head group and the nature of the head to tail linkage 
(ester or ether) [11]. Measurements based on 2H nuclear 
magnetic resonance [15] have shown that the lipid 
hydrocarbon chains in pure PC and PS membranes have 
different order parameters at room temperatures. 
Namely, although both were in a lamellar liquid 
crystalline phase, PS was more ordered than PC. On the 
other hand, it was shown that the archaeosome 
membranes are heterogeneous, and are composed of 
nanodomains with different fluidity characteristics at 
room temperatures [5]. Archaeal lipids also have special 
head groups carrying inositol and glucose functions. In 
comparison to simple phosphatidylcholine (PC) head 
groups these carbohydrates are larger moieties that move 
much more slowly and are also involved in hydrogen 
bonds that contribute to stability and order of these 
bilayers. It can be assumed that planar lipid bilayers made 
of A. pernix K1 and POPS are more ordered than POPC 
planar lipid bilayers which leads to harder formation of 
water pores and lipid bilayer rupture. 
 
 
Figure 2. The fraction of the planar lipid bilayer area that is 
occupied by water pores as a function of Ubr. 
 
 
4 Conclusions 
 
In the study planar lipid bilayers were formed using lipids 
extracted from archaea A. pernix K1, zwitterionic POPC 
lipids, negativly charged POPS lipids and POPC:POPS 
mixture in ratio 1:1. For each planar lipid bilayer 
capacitance was measured to monitor quality of the 
membrane model at forming. Planar lipid bilayers were 
exposed to linealy rising voltage and U br and t br were 

410
 
determined. Using an analytical method of planar lipid 
bilayer substitute circuit we estimated the change of the 
capacitance at the breakdown and the fraction of planar 
lipid bilayer area ocupied by water. Our results confirm 
the results of pore formation theories and MD models. 
The fraction of area occupied by water pores at the 
moment of planar lipid bilayer rupture and U br is smaller 
when the transmembrane voltage is built up slower. In 
more disordered planar lipid bilayers, water pores are 
easily formed.  
 
 
 
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