a. PORE FORMATION IN PLANAR LIPID BILAYER
MEMBRANES
Gianluigi Monticelli
Istituto di Fisiologia generale e Chimica biologica, Università degli Studi, Via Saldini
50, 20133 Milano - ITALY
INTRODUCTION
Porins constitute a class of proteins located in the outer membranes of mitochondria and
of gram-negative bacteria. These proteins form large water filled pores through the
hydrophobic core of the outer membrane (1-4). This channel system acts as a molecular
filter with a defined exclusion limit for hydrophilic substances (3, 5, 6). Sugars and other
hydrophilic solutes up to a molecular weight of 500-700 (7-10) permeate the outer
membrane of gram-negative bacteria, such as Escherichia coli, Salmonella
thyphimurium, Proteus mirabilis.
In some cases it seems that porin forms solute-specific channels as protein P of
Pseudomonas aeruginosa (11) and maltoporin of Escherichia coli (12, 13).
The outer mitochondrial membrane is freely permeable to various small solutes, too, but
not to molecules of large weight (14-18).
Porin, this pore forming protein, was identified in the outer mitochondrial membrane of
a variety of eukariotic cells (17, 19-24). It was named mitochondrial porin in analogy to
the bacterial porins and it is also known as voltage-dependent anion-selective channel
(VDAC) (19, 20). Mitochondrial porins are polypeptides with molecular weights
between 30000 and 35000 (18, 21-26).
In gram-negative bacteria proteins giving this passive permeability were variously
called: protein I (27), protein 1 (28), matrix protein (29) and porins (1). These proteins
range in molecular weight from 28000 to 50000 and common features were determined
examining more than 40 different porins (30).
Mitochondrial porin formed pores share some similarities with the bacterial porins
located in the bacterial outer membranes (24). The formed diffusion pores have a
diameter of 1.3 - 2.2 nm in bacteria and 1.7 - 2 nm in mitochondria approximatively (23,
24, 31).
It has been proposed that porins from bacteria usually exist in the membrane as trimers
and the pore appears to be formed by β-sheet rather than α-helices (32); moreover it has
been demonstrated that three channels on the outer surface of the cell merge into a single
channel at the periplasmic face (33).
The outer mitochondrial membrane contains only one type of porin (21) and the pore
permeability seems to be controlled by a transmembrane potential or an intrinsic
membrane potential; many studied porins are voltage-dependent and switch to substates
when transmembrane potential is higher than 10-20 mV (19-24). Open pores present an
higher permeability and a different ionic selectivity than the substates (19, 23, 24, 34).
Pore forming properties can be studied in reconstitution experiments with planar lipid
bilayers and liposomes.
Incorporation of lipid components from bacteria
vesicles rendered the vesicle membrane permeable
and it has been demonstrated that incorporation of
coli into phospholipid/lipopolysaccharide vesicles
solutes up to molecular weight of about 550 (1).
outer membrane into phospholipid
to small sugar molecules (1, 35, 36)
the matrix protein from Escherichia
induced permeability to hidrophilic
Planar bilayers which separate two well defined aqueous phases offer the possibility of
directly measuring electrical parameters as well as to follow the kinetics of porin
incorporation.
Many papers have been published on the determination of ion permeability through the
channels made of porins from outer membrane of bacteria (31, 37-40) and of
mitochondria (18-21, 23, 41-44).
PORIN CHANNELS
The addition of small amounts of porin to the solutions bathing a lipid bilayer membrane
having small surface area allowed the resolution of step increases of conductance.
The average conductance increment, as determined by measuring a large number of
events, for different lipid membranes and porins are reported in Table I with the
reference source.
TABLE I
c
Λ
Λ/λ
[M]
[nS]
[Å]
Egg lecithin/n-decane (pH 6.0-7.0: Vm=50 mV: t=25 ºC)
Pseudomonas aeruginosa protein F: l nM
NaC1
0.10
0.480
5.10
1.00
4.500
5.40
KC1
1.00
5.600
5.10
λ
[mS/cm]
r=l/λ
[Ωcm]
d
[nm]
Ref.
n.
9.4
83.3
109.8
106.250
12.000
9.107
2.21
2.27
2.21
31
31
31
Asolectin (L-α-phosphatidylcholine)/n-decane (pH 6.0: Vm=5 mV: t=25 ºC)
Rat liver porin l µg/ml
KC1
0.01
0.050
3.60
1.4
720.000
0.03
0.150
3.80
3.9
253.333
0.10
0.480
3.70
13.0
77.083
0.30
1.300
3.60
36.1
27.692
1.00
4.400
4.00
110.0
9.091
NaC1
1.00
3.800
4.50
84.4
11.842
1.85
1.90
1.88
1.85
1.95
2.07
21
21
21
21
21
21
Salt
LiC1
RbC1
MgC12
K2S04
Tris+Hepes- (pH 8.0)
N(C2H5)+Hepes-
1.00
1.00
0.50
0.50
0.50
0.50
3.400
4.200
2.700
2.400
0.230
0.170
4.80
3.50
4.20
3.20
3.20
3.50
14.118
8.333
15.556
13.333
139.130
205.882
2.14
1.83
2.00
1.75
1.75
1.83
21
21
21
21
21
21
Diphytanoylphosphatidylcholine/n-decane (pH 6.0-7.0: Vm=10 mV: t=25 ºC)
Rat brain porin 1.2 ng/ml
KC1
0.01
0.050
4.20
1.2
84.000
0.05
0.270
4.50
6.0
166.667
0.10
0.400
4.20
9.5
105.000
0.30
1.400
4.10
34.1
29.286
1.00
4.000
3.60
111.1
9.000
3.00
11.500
4.60
250.0
4.000
NaC1
1.00
4.000
4.70
85.1
11.750
RbC1
1.00
5.000
4.30
116.3
8.600
CH3COOK
1.00
2.500
4.00
62.5
16.000
LiC1
1.00
3.500
4.90
71.4
14.000
MgC12
0.50
3.000
4.70
63.8
15.667
Na2SO4
0.50
2.500
4.60
54.3
18.400
Tris-HC1
0.50
1.500
5.00
30.0
33.333
2.00
2.07
2.00
1.98
1.85
2.10
2.12
2.03
1.95
2.16
2.12
2.10
2.19
41
41
41
41
41
41
41
41
41
41
41
41
41
c
Λ
Λ/λ
[M]
[nS]
[Å]
Oxidized cholesterol/n-decane (pH 6.0-7.0: Vm=50 mV)
E. coli porin 0.5 ng/ml
KC1
1.00
1.900
1.70
NaC1
1.00
1.200
1.43
RbC1
1.00
2.100
1.88
LiC1
1.00
0.720
1.01
CsC1
1.00
2.000
1.74
NH4C1
1.00
2.000
1.79
MgC12
0.50
0.430
0.67
CaC12
0.50
0.440
0.56
BaC12
0.50
0.430
0.55
K2S04
0.50
0.960
1.26
MgS04
0.50
0.240
0.73
Na+Hepes- (pH 9.0)
0.50
0.240
1.33
+
Tris C1
0.50
0.300
1.00
Tris+Hepes- (pH 8.0)
0.50
0.064
0.89
+
Glucosamine C1 (pH
1.00
0.460
1.02
3.0)
N(CH3)4+C1–
1.00
0.670
0.94
+
N(CH3)4 Hepes (pH 8.5) 0.50
0.087
0.58
N(C2H5)+Hepes0.50
0.032
0.67
Salt
Salmonella typhimurium SH5551 (40K) 1 pM trimers
LiC1
1.00
0.900
1.30
NaC1
1.00
1.800
2.10
70.8
120.0
64.3
75.0
7.2
4.9
λ
[mS/cm]
r=l/λ
[Ωcm]
d
[nm]
Ref.
n.
111.8
83.9
111.7
71.3
114.9
111.7
64.2
78.6
78.2
76.2
32.9
18.0
30.0
7.2
45.1
8.947
11.917
8.952
14.028
8.700
8.950
15.581
12.727
12.791
13.125
30.417
55.417
33.333
139.063
22.174
1.27
1.17
1.34
0.98
1.29
1.31
0.80
0.73
0.72
1.10
0.83
1.13
0.98
0.92
0.99
37
37
37
37
38
37
38
38
38
38
38
38
38
38
38
71.3
15.0
4.8
14.030
66.667
209.375
0.95
0.74
0.80
38
38
38
69.2
85.7
14.444
11.667
1.11
1.42
39
39
KC1
NH4C1
RbC1
CsC1
MgC12
CaC12
K2S04
MgS04
Na+Hepes- (pH 9.0)
Tris+C1Tris+Hepes- (pH 8.0)
Glucosamine+C1- (pH
3.0)
N(CH3)4+C1N(CH3)4+Hepes- (pH 8.5)
N(C2H5)+Hepes-
1.00
1.00
1.00
1.00
0.50
0.50
0.50
0.50
0.50
0.50
0.50
1.00
2.400
2.600
2.200
2.300
0.620
0.730
1.400
0.230
0.390
0.380
0.088
0.600
2.10
2.30
1.90
2.00
1.00
0.94
1.80
1.00
2.20
1.30
1.20
1.30
114.3
113.0
115.8
115.0
62.0
77.7
77.8
32.0
17.7
29.2
7.3
46.2
8.750
8.846
8.636
8.696
16.129
12.877
12.857
31.250
56.410
34.211
136.364
21.667
1.42
1.48
1.35
1.38
0.98
0.95
1.31
0.98
1.45
1.11
1.07
1.11
39
39
39
39
39
39
39
39
39
39
39
39
1.00
0.50
0.50
0.820
0.120
0.045
1.20
0.80
0.94
68.3
15.0
4.8
14.634
66.667
208.889
1.07
0.87
0.95
39
39
39
Pseudomonas aeruginosa protein F 1 nM
NaC1
0.01
0.052
0.10
0.450
1.00
4.300
KC1
0.10
0.570
1.00
5.900
MgC12
0.50
2.900
CaC12
0.50
2.700
K2S04
0.50
3.600
Na+Hepes- (pH 9.0)
0.50
0.910
Tris+C10.50
1.400
+
Tris Hepes- (pH 8.0)
0.50
0.350
N(CH3)4+C10.50
3.100
+
N(CH3)4 Hepes (pH 8.5) 0.50
0.680
+
N(C2H5) Hepes
0.50
0.220
4.70
4.80
5.10
4.10
5.40
4.50
3.50
4.70
5.10
4.70
4.90
4.40
4.50
4.60
1.1
9.4
84.3
13.9
109.3
64.4
77.1
76.6
17.8
29.8
7.1
70.5
15.1
4.8
903.846
106.667
11.860
71.930
9.153
15.517
12.963
13.056
56.044
33.571
140.000
14.194
66.176
209.091
2.12
2.14
2.21
1.98
2.27
2.07
1.83
2.12
2.21
2.12
2.16
2.05
2.07
2.10
31
31
31
31
31
31
31
31
31
31
31
31
31
31
In this table in addition to the specific conductance (Λ), the specific conductance λ of
the corresponding aqueous solution, the ratio Λ/λ and the effective pore diameter d are
given.
The pore diameter d was calculated assuming a membrane tickness l=7.5 nm and
considering that the pore is a cylinder with a circular cross section and is filled with an
aqueous solution of the same conductance as the external bulk phase.
With these assumptions it is:
Λ = λπr2/l
-1-
and the pore diameter (2r) can be calculated.
The analysis of Table I shows that: 1) pores of different diameter can be formed
depending on the porin origin and on the bilayer composition; 2) pore conductance is a
linear function of the specific conductance of the aqueous phase; 3) large organic cations
and anions are able to permeate porin channels practically without interactions with the
pore walls.
SIMPLE GEOMETRICAL CONSIDERATIONS
From a geometrical point of view the pore formation into a planar lipid bilayer
membrane can be represented as schematized in Fig. 1.
After addition of porin to bulk phases, as time passes, there is an increase of the number
of pores. If Sp and So are the single pore area and the total area occupied by lipids at t=0
respectively, the membrane lipid area at time t, when n(t) aqueous channels have been
formed, is:
S(t) = So - n(t) Sp
-2–
This evolution of the membrane incorporating protein channels can be schematized
electrically as in Fig. 2. Before porin addition the BLM equivalent circuit is constituted
by a parallel combination of the resistance Rmo and capacitance Cmo. As pores are
formed the surface area of membrane lipid part decreases so that the relative resistance
and capacitance are time dependent: Rlm(t) and Cm(t). Single channel resistances (Rsc)
are connected in parallel constituting pathways for ion transport.
At time t the equivalent electrical circuit can be semplified as in Fig. 3 where Rlm [n(t)]
is the resistance of lipid part of the membrane, Rp[n(t)] is the total electrical resistance of
pores and Cm[n(t)] is the membrane capacitance.
In this picture pores are characterized by the conductance only and moreover it has been
neglected the thickness increase of the lipid part because of the aqueous holes (pores)
into the membrane.
Increasing the number of channels Rp[n(t)] decreases as Rsc/n(t).
Due to the pore formation the resistance of the membrane lipid part increases according
to:
Rlm(t) = Rmo {1/[1-Sp n(t)/So]}
-3-
For the same reason the capacitance of membrane lipid part decreases following
Cm(t) = Cmo [1-Sp n(t)/So]
-4-
and the capacitative reactance Xc = 1/ω Cm(t) (ω = 2πf, where f is the frequency)
increases with the number of pores.
With this simple picture of the system and with the restrictions introduced by the
simplifying hypothesis (constant tickness during porin incorporation and homogeneity
of the lipid film) it is possible to calculate the different electrical characteristics of the
membrane if it is known the resistance and the diameter of a single channel.
From the experimental point of view one problem can be constituted by the membrane
capacitance as it has been well shown by White and Thompson (45, 46). In the
normalization of capacitance with respect to area the bilayer film area must be precisely
determined.
Knowing the kinetics of pore formation, n(t), the dependence of calculated parameters
on the number of formed pores can be transformed in the dependence on time.
On the other hand current measurements during channel formation give informations on
the number of protein complex functionning as pores.
ACKNOWLEDGEMENTS
I wish to thank Prof.s E. Gallucci and S. Micelli as well as Dr. J. Hladyszowski for the
useful discussions and criticism. Work supported in part by a MPI 60% grant.
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