Phase-State Dependent Current Fluctuations in Pure Lipid Membranes

B. Wunderlich,

C. Leirer,

A.-L. Idzko,

U. F. Keyser,

A. Wixforth,

V. M. Myles,

T. Heimburg,

and M. F. Schneider

University of Augsburg, Experimental Physics I, Augsburg, Germany;

Universita t Leipzig, Experimentalphysik I, Leipzig, Germany; and

Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark

ABSTRACT Current uctuations in pure lipid membranes have been shown to occur under the inuence of transmembrane
electric elds (electroporation) as well as a result from structural rearrangements of the lipid bilayer during phase transition
(soft perforation). We demonstrate that the ion permeability during lipid phase transition exhibits the same qualitative temperature
dependence as the macroscopic heat capacity of a D15PC/DOPC vesicle suspension. Microscopic current uctuations show
distinct characteristics for each individual phase state. Although current uctuations in the uid phase show spikelike behavior
of short timescales (~2 ms) with a narrow amplitude distribution, the current uctuations during lipid phase transition appear in
distinct steps with timescales of ~20 ms. We propose a theoretical explanation for the origin of timescales and permeability based
on a linear relationship between lipid membrane susceptibilities and relaxation times near the phase transition.
INTRODUCTION
The permeability of cell membranes to ions, proteins, and
other transmitters, and the regulation of these processes, is
of crucial importance in maintaining basic cell functions. In
this context, ion channel proteins have been found to play
a major role in the regulation of ionic transport across the
membrane (1). In addition to the characteristics of the
channel, proteins themselves as an inuence of the membrane
phase state on channel function have been identied (2,3),
indicating the importance of the lipid bilayer for transmem-
brane currents. Boheim et al., for example, reported on the
slowing down of protein induced ion channels during the lipid
phase transition (4,5). The rst indication of thermodynami-
cally induced changes in permeability was already given by
Papahadjopoulos et al. in 1973 (6), who found a maximum
leakage of radioactive labeled sodium ions during lipid phase
transition.
The application of high electric elds (typically ~10
7

10
8
V/m) presents another mechanism for overcoming the
lipid membrane barrier, inducing current uctuations in the
absence of channel forming peptides (electroporation) (711).
A third mechanism (in addition to ion channels, pores,
and electroporation) to cross the lipid membrane barrier
has been elucidated by Antonov et al. (12). They reported
that quantized ion-conducting channels appear in unmodi-
ed 1,2-distearoyl-sn-glycero-3-phosphocholine membranes
close to the phase transition and demonstrated more recently,
that the conductance of different ions is in accordance with
the well-known Hofmeister series (13). Similarly, Kaufmann
and Silmann (14) as well as the literature (2,1518) were
able to demonstrate a close relationship between the physical
state of the bilayer and ion current uctuations. This relation-
ship is also the subject of an accompanying article by Blicher
et al.(19).
In this article, we study the origin of ion current uctuations
in protein free lipid bilayers by a thorough comparison of
timescales and amplitudes of ion current uctuations with
the heat capacity prole of lipid membranes. Although current
uctuations in the uid phase appear as short (~2 ms) spikes,
they last for ~20 ms as discrete steplike currents in the phase
transition regime including long-lasting events of ~100 ms.
Based on a model recently proposed by Grabitz et al. (20),
we suggest that the increased timescales at T
m
have their
origin in the extended relaxation times generally found during
lipid membrane phase transitions and originating fromthe at
thermodynamic potential and consequent weak restoring
forces driving the system back to its equilibrium near T
m
.
MATERIALS AND METHODS
Lipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, T
m
4

C) and
1,2 dipentadecanoyl-sn-glycero-3-phosphocholine (D15PC, T
m
33

C)
dissolved in chloroform were purchased from Avanti Polar Lipids (Birming-
ham, AL) and used without further purication. Hexadecane was dissolved
in pentane to achieve a nal concentration of 2.5% for the prepainting
solution.
We performed current measurements using a patch-clamp amplier
(Cornerstone Series) from Dagan (Minneapolis, MN). The temperature
was controlled during the experiments with the aid of a standard heat bath
(Julabo, Seelbach, Germany). Heat capacity proles of small unilamellar
vesicles were recorded using a VP-DSCcalorimeter fromMicrocal (Avestin,
Ottawa, Canada).
To obtain lipid mixtures, lipids dissolved in chloroform were mixed in the
desired proportion. Aqueous solution of lipids for calorimetric measure-
ments were obtained as described previously (21). The experiments on the
articial membranes (in this case, black lipid membranes, or BLMs) were
performed by using a setup consisting of two Teon chambers separated
by a Teon foil of 25-mm thickness with a 150-mm diameter hole. The
hole was produced by drilling it into the foil. Lipid membranes were
obtained by prepainting the Teon foil with the hexadecane solution and
15 min of waiting until the pentane was evaporated. Subsequently, both
Submitted August 2, 2008, and accepted for publication February 20, 2009.
*Correspondence: matthias.schneider@physik.uni-augsburg.de
Editor: Joshua Zimmerberg.
2009 by the Biophysical Society
0006-3495/09/06/4592/6 $2.00 doi: 10.1016/j.bpj.2009.02.053
4592 Biophysical Journal Volume 96 June 2009 45924597
chambers were lled up to a level sufciently far below the hole with a
200-mM NaCl solution. Ten microliters of lipid solution was spread on
the water surface and after a waiting time of 15 min, the electrolyte level
was raised above the hole. Before experiments, the bilayer was characterized
by measuring the resistance and the capacity of the membrane.
RESULTS AND DISCUSSION
Theory
In thermodynamics, the different functions of state (enthalpy,
energy, etc.), the susceptibilities (heat capacity, compressibil-
ities, etc.) and the response times are coupled via the
uctuation-dissipation theorem (22). For lipid membranes,
it has been shown by Grabitz et al. (20) and Seeger et al.
(23) that the relaxation timescale t after a pressure pertur-
bation is proportional to the excess heat capacity Dc
p
such that
t
T
2
L
Dc
p
; (1)
where T is the temperature in Kelvin and L is a phenomenolog-
ical coefcient independent of temperature (see Grabitz et al.
(20), and Seeger et al. (23), for details). For 1,2-dimyristoyl-
sn-glycero-3-phosphocholine (DMPC) multilamellar vesicles
(MLV), for instance, L 6.6 x 10
8
J K/mol and for DMPC
large unilamellar vesicle (LUV) it is L 15.7 x 10
8
J K/mol.
This equation implies that relaxation is slow if the system
is at the heat capacity maximum, which should be very
pronounced in lipid membranes exhibiting a phase transition.
For multilamellar dipalmitoylphosphatidlycholine (DPPC)
vesicles, this time can be as long as 30 s. According to Onsager
(24,25), each uctuation can be considered as a perturbation
of the system away from the entropy maximum. Therefore,
uctuation lifetimes and relaxation times are the same.
According to Einstein (26), uctuations in the thermody-
namic variable x
i
are related to the curvature of the entropy
potential S by

dx
i
dx
j

k
B

v
2
S
vx
i
vx
j

1
;
or for x
i
x
j
, in the often experimentally more convenient
free energy (G) expression
hdxdxi k
B
T

v
2
G
vx
2

1
;
where k
b
is the Boltzmann constant and T the temperature.
This can be used to show that heat capacity and uctua-
tions in enthalpy H are proportional,

d H
2

H
2

hHi
2
RT
2
c
p
;
around the entropy maximum (R is the gas constant). Along
the same lines, one can show that the lateral compressibility
k
T
is given by

d A
2

ARTk
T
; (2)
i.e., it is proportional to the uctuations in area A. Heimburg
(27) and Ebel et al. (28) have shown that changes in heat, in
volume, and in area in lipid transitions are proportional func-
tions. This leads to the relation
Dk
A
T

g
2
T
A
Dc
p
; (3)
meaning that the lateral compressibility changes are propor-
tional to the excess heat capacity (g is a material constant
of ~1 m
2
/J (27)). This further implies that it does not matter
whether one considers a perturbation in enthalpy (after
a temperature jump) or a perturbation in volume or area.
The uctuations in volume and area have the same lifetimes
as those of the heat.
Pore formation in absence of channel-forming proteins
occurs because of thermal area uctuations. According to
Nagle and Scott (29), the work to create a pore in the
membrane is proportional to its lateral compressibility,
which is proportional to the excess heat capacity (Eq. 3).
As a consequence, pore formation is facilitated in the melting
regime. Membranes are expected to be more permeable at the
heat capacity maximum (for details, see(19)). Equating the
uctuation lifetime with the lifetime of pores, this implies
that the pore opening times are proportional to the heat
capacity. This relation has already been discussed in Seeger
et al. (23), and in this article we will demonstrate that this
relation leads to reasonable predictions. Both permeability
and mean pore open time are at maximum in the chain
melting transition. Experimental values for pore lifetimes
are close to those estimated from Eq. 1.
Permeability changes during lipid membrane
phase transition
To study the temperature dependence of lipid bilayers,
a BLM was prepared at 40

C well above the phase transition

of the lipid mixture used (5:95 DOPC/D15PC). Before the
BLM experiments, the heat capacity of the lipid mixture
has been measured (inset of Fig. 1), revealing a melting tran-
sition at T
m
z30

C. To assure bilayer formation, the electric

capacitance of the membrane lm has been monitored. The
bilayer was then left to equilibrate for 15 min. After lm
formation, the stability of the bilayer was tested by a stepwise
increase of the clamp voltage until signicant current uctu-
ation occurred (i.e., threshold voltage). Subsequently, the
applied voltage V
M
was decreased until the current uctua-
tions disappeared, leaving the system close to the conducting
state. After 10 min waiting for equilibration, the temperature
was lowered at a rate of 0.2

C/min at constant clamp voltage.

Fig. 1 shows the overall transmembrane conductivity (aver-
aged over 60 s) at V
M
400 mV as a function of tempera-
ture. At T 36

C the transmembrane conductivity begins

to rapidly rise to reach a maximum at T z31.5

C. Clearly,
ion permeability and heat capacity follow the same qualita-
tive temperature dependence with a full-width at half height
Biophysical Journal 96(11) 45924597
Membrane Current Fluctuations 4593
of 36 K. The slight shift of I
tot
(T) toward higher tempera-
tures is probably due to the different sample preparations.
After sonication, small unilamellar vesicles are believed
to be under some tension, whereas BLMs are rather relaxed.
Tension, however, tends to increase the membrane area,
thereby supporting a lowering in T
m
. Finally, uncontrollable
lipid accumulation around the septum might be an additional
source of changes in T
m
. We were able to observe the same
correlation between heat capacity and ion permeability for
D15PC/DMPC mixtures (see Supporting Material). How-
ever, the introduction of DOPC in our lipid mixtures signif-
icantly increased the stability of the planar lipid membrane.
This can also be seen in Fig. 2 b, where the bilayer resists
membrane potentials as high as 1000 mV.
One possible origin of the increase in overall permeability
is the increasing number of lattice defects at the gel/uid
phase boundary. This was suggested by several theoretical
ndings (30) and is supported by the work of Papahadjopou-
los et al. (6), who found a maximum leakage of radioactive
labeled sodium ions during lipid phase transition. An alterna-
tive approach to explain the increased permeability in the
phase transition region was given by Kaufmann et al. (31)
as well as by Nagel and Scott (29) and Blicher et al. (19),
who address the changes in lateral compressibility k
T
and
their relation to the uctuations in area hvA
2
i (Eq. 2).
Considering such area uctuations as a source of defects
excellently agrees with the correlation between heat capacity
and conductivity.
Current characteristics strongly depend
on lipid phase state
To unravel the origin of the transient conductivity behavior,
we prepared BLMs from a DOPC/D15PC (5:95) mixture
both in the uid phase (40

C) and in the phase transition

region (27

C). After a stepwise increase in V

M
until values
were reached at which current uctuations were observed,
the voltage V
M
was again slightly reduced, resulting in stable
current uctuations over several minutes.
In the uid phase (Fig. 2 a, T 33

C, V
M
100 mV),
spikelike events were observed and did not change their char-
acteristics at even higher temperatures. The current uctua-
tions in the phase transition regime (Fig. 2 b, T 31,5

C,
V
M
1000 mV), however, appear rather steplike and quan-
tized, with longer opening times. Again, the bilayer exhibited
very high stability (the relation between applied voltage and
initial resistance will be addressed in a separate article). For
the DMPC/D15PC mixture, for example, strong current uc-
tuations were found at ~500 mV and below (see Supporting
Material). An analysis of the current amplitudes results in
a rather broad distribution with two small maxima at ~12 pS
and 24 pS in the uid state (Fig. 3 a). In the phase transition
FIGURE 1 Relative conductance of a D15PC/DOPC (95:5) mixture as
a function of temperature at V
M
400 mV. A maximum is observed
between 35

C and 25

C. The current trace clearly correlates with the heat

capacity prole (see inset). The maximum corresponds to a conductivity
of 2 x 10
7
S/cm
2
. Conductivity measurements outside the transition region
in the uid (squares) and gel phase (triangles) have been performed on
individual BLMs and are further described in the Supporting Material.
FIGURE 2 Typical current traces of a D15PC/DOPC (95:5) (top panel) in
the uid phase (33

C, V
M
100 ~ mV) and (bottom panel) in the phase tran-
sition regime (31.5

C, V
M
1000 mV). In the uid phase, spikelike current
uctuations on short timescales are observed. In the phase transition regime,
however, quantized uctuations appear at longer timescales. Note that the
typical timescales are strongly increased as compared with the top panel.
Biophysical Journal 96(11) 45924597
4594 Wunderlich et al.
regime, these maxima become very pronounced (Fig. 3 b). In
addition, the gel state exhibited some current uctuations,
which, however either slowly disappeared when waiting for
extended times or led to membrane rupture (see Supporting
Material). Distinct quantized current uctuations in lipid
membranes have been reported before (2,1218,29), ranging
between ~1 pAand ~1 nAclose to the phase transition temper-
ature. However, so far, no clear relation among current ampli-
tudes, distribution, and uctuations for the different thermo-
dynamic states of the lipid membrane has been given.
The existence of current uctuations in both states
suggests that the nature of the underlying mechanism for
these current uctuations is the same, but the specic charac-
teristics seem to be determined by the physical properties of
the membrane. In agreement with our results, we believe,
that uctuations in area hvA
2
i are the origin of lattice defect
formation and hence permeating ions. The origin of the step-
like shape of the current uctuations remains unclear, yet is
probably related to the physical properties of the liquid
crystal. In principle, and based on our experimental results,
one could model the pore size necessary for an ion to pass
the membrane. For a one-step uctuation (Fig. 2 b), this
suggests a single pore diameter of ~1 nm, using a cylindrical
pore approximation assuming free instead of surface-bound
water within the pore.
Timescales of current uctuation increase
during phase transition
To unravel the relationship between thermodynamic
behavior and the observed uctuations, we analyzed the
typical timescales of the opening times. Peaks were identi-
ed and characterized with a threshold criterion on the
same set of data used to describe the conductance character-
istics above (Fig. 3). In Fig. 4, we show the resulting data for
the uid phase (circles) and the phase transition (triangles).
The logarithmic plot already reveals that the opening times
are signicantly increased within the phase transition regime.
In the uid phase, we found a mean average opening time of
~3 ms (exponential t). The behavior alters signicantly
when the phase transition region is entered. The mean
average timescales shift by almost an order of magnitude
to ~20 ms, including long-lasting states of up to ~100 ms.
We also observed a broad variety of events including short
events in the transition region and long uctuations in the
uid state during measurements over extended periods of
times. This is also reproduced by the broad distributions
FIGURE 3 Conductivity histogram of current uctuations in (top panel)
the uid phase (T 33

C, V
M
100 mV) and (bottom panel) the phase tran-
sitionregime (T31.5

C, V
M
1000mV). The peakat G0pScorresponds
to the baseline. In the uid phase, no signicant peaks appear. In the phase
transition regime, distinct maxima around G 12 pS and G 24 pS appear,
with some additional substructure around G 20 and G 27 pS. Data were
collected from three individual bilayers by analyzing a 200 s long trace. The
observed maxima agree in all measurements within a range of 20%.
FIGURE 4 Logarithmic plot of the timescales of the current uctuation in
the uid phase (triangles) and the phase transition regime (circles). The
transmembrane currents have been measured under constant membrane
potential V
m
. The timescales of the uid phase follow an exponential decay,
whereas those in the transition can only be approximated by a double-
exponential decay. The average lifetimes t center around t 3 ms in the
uid and t 20 ms in the phase transition. This is in good agreement
with our theoretical prediction.
Biophysical Journal 96(11) 45924597
Membrane Current Fluctuations 4595
shown in Fig. 3, and reects uncontrollable variations in the
lipid membrane structure and dynamics like, for example,
the pool of lipids accumulating around the septum or the
degree of asymmetry. However, the tendency toward
increased timescales during lipid phase transitions is obvious
and reproducible in every experiment. Importantly, these
enhanced timescales are in line with the increasing relaxation
times of the membrane in this regime (20) and originate most
likely from the small restoring forces of the at thermody-
namic potential around T
m
. Experimentally, increased relax-
ation times during the main phase transition of articial
membranes have been reported before by Tsong (32) as
well as by Blume and Hillmann (33). Since the relaxation
times reect the reorganization of lipids in the membrane,
one would expect that pore nucleation, pore opening, and
closing, respectively, follow the same time evolution as lipid
relaxation.
Taking the phenomenological coefcient L for DMPC
MLV (as given above) and numbers for c
p
from Fig. 1
(at ~31

C), Eq. 1 yields lifetimes of 165 ms, whereas using

the LfromDMPC LUVyields lifetimes of 73 ms. Considering
the transition half-width of only 2

C, this is very close to the

experimentally found average lifetime of 20 ms (Fig. 4).
CONCLUSION
The behavior of current uctuations in an unmodied phos-
pholipid membrane under a constant voltage was found to be
strongly sensitive to the phase state. Whereas uctuations in
the uid phase reveal an unordered pattern on short time-
scales, in the phase-transition region, quantized uctuations
on much longer timescales occur. Using the uctuation dissi-
pation theorem and the linear relationship between heat
capacity and area compressibility, we were able to predict
the correct timescales and identify weak restoring forces of
the at thermodynamic potential as the origin of the extended
timescales during lipid phase transition. Therefore, this
article demonstrates the thermodynamic potential of lipid
membranes to be the physical origin of lipid membrane
current uctuations.
SUPPORTING MATERIAL
Five gures are available at http://www.biophysj.org/biophysj/supplemental/
S0006-3495(09)00663-8.
We thank Dr. K. Kaufmann (Gottingen) for very helpful discussions and
draw the readers attention to his earlier work (31). M.F.S. personally thanks
K. Kaufmann, who inspired him to work in this eld, described the funda-
mental difculty of transport across lipid membranes, and outlined the
solution and its thermodynamic origin in his book in 1989 (31).
Financial support by the Deutsche Forschungsgemeinschaft (No. SPP 1313)
and the German Excellence Initiative via the Nanosystems Initiative
Munich (NIM) is gratefully acknowledged. C.L. thanks the Bavarian
Science Foundation for nancial support.
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