Ionchannelactivityofbrainabundant protein
BASP1 inplanarlipid bilayers
Olga S. Ostroumova
1
, Ludmila V. Schagina
1
, Mark I. Mosevitsky
2
and Vladislav V. Zakharov
2
1 Laboratory of Ionic Channels of Cell Membranes, Institute of Cytology of RAS, St Petersburg, Russia
2 Division of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute of RAS, Gatchina, Leningrad District, Russia
Introduction
Under the current concept, many neurodegenerative
diseases (e.g. Alzheimer’s, Parkinson’s, Huntington’s
diseases, etc.) are associated with the abnormal aggre-
gation of amyloid proteins. Soluble oligomers of these
proteins are now considered to be the main neurotoxic
species that contribute to disease-associated neurode-
generation. The precise mechanism of amyloid protein
toxicity is not well understood. A large body of evi-
dence suggests that amyloid protein oligomers can
exert their toxicity by indirect mechanisms, through
binding to various receptors, existing ion channels
or other cellular proteins (as described for amyloid
b-protein [1,2]). Alternatively, the amyloid oligomers
can cause membrane permeabilization, which directly
Keywords
amyloid proteins; BASP1; ion channels;
lipid bilayer; protein oligomers
Correspondence
V. V. Zakharov, Division of Molecular and
Radiation Biophysics, Petersburg Nuclear
Physics Institute of Russian Academy of
Sciences, 188300 Gatchina, Leningrad
District, Russia
Fax: +7 813 7132303
Tel: +7 812 3282802
E-mail: vlad.v.zakharov@mail.ru
(Received 23 September 2010, revised 14
November 2010, accepted 18 November
2010)
doi:10.1111/j.1742-4658.2010.07967.x
BASP1 (also known as CAP-23 and NAP-22) is a brainabundant myri-
stoylated protein localized at the inner surface of the presynaptic plasma
membrane. Emerging evidence suggests that BASP1 is critically involved in
various cellular processes, in particular, in the accumulation of phosphati-
dylinositol-4,5-diphosphate (PIP
2
) inlipid raft microdomains. We have
recently shown that BASP1 forms heterogeneously-sized oligomers and
higher aggregates with an outward similarity to oligomers and protofibrils
of amyloid proteins. However, BASP1 is not known to be related to any
amyloid disease. In the present study, we show that BASP1 induces single
channel currents across negatively-charged planarlipidbilayers (containing
phosphatidylserine or PIP
2
) bathed in 0.1–0.2 M KCl (pH 7.5). By their
characteristics, BASP1 channels are similar to amyloid protein channels.
BASP1 channels exhibit multiple conductance levels, in the range
10–3000 pS, with the most frequently observed conductance state of
approximately 50 pS. The channels demonstrate a linear current–voltage
relationship and voltage-independent kinetics of opening and closing. Their
K
+
to Cl
)
permeability ratio is approximately 14, indicating that BASP1
channels are cation-selective. The ionchannelactivityofBASP1 is in
accordance with the pore-like structure ofBASP1 oligomers observed by
electron microscopy on a lipid monolayer. Neuronal protein GAP-43,
which is functionally related to BASP1 and also forms oligomers, elicited
no ionchannel currents under the conditions used in the present study.
Elucidation of the physiological or pathological roles ofionchannel activ-
ity of membrane-bound BASP1 oligomers will help to define the precise
mechanism of amyloid protein toxicity.
Abbreviations
BASP1, brain acid-soluble protein-1; GAP-43, axonal growth-associated protein-43; PC, phosphatidylcholine; PE, phosphatidylethanolamine;
PIP
2
, phosphatidylinositol-4,5-diphosphate; PS, phosphatidylserine; TEM, transmission electron microscopy.
FEBS Journal 278 (2011) 461–469 ª 2010 The Authors Journal compilation ª 2010 FEBS 461
perturbs cellular calcium ion homeostasis. This fact
was demonstrated both inlipid bilayer and cell mem-
brane experiments [3,4]. Many amyloid proteins, such
as amyloid b-protein, islet amyloid polypeptide, poly-
glutamine and a-synuclein, have been reported to form
ion channels inlipid membranes [5–9]. It has been also
suggested that amyloid oligomers can increase mem-
brane permeability by channel-independent perturba-
tion oflipid bilayers, allowing nonspecific membrane
leakage of larger molecules [10–13]. In support of the
channel hypothesis, atomic force microscopy of amy-
loid oligomers on supported lipidbilayers demon-
strates the presence of pore-like (annular) structures
[8,14–16]. In addition, electron microscopy of amyloid
protein preparations has also revealed the presence of
heterogeneous annular oligomers [17–20]. The size
diversity of amyloid oligomers probably contributes to
the observed heterogeneity of amyloid protein channels
with respect to their kinetic behaviour, conductance
and ion selectivity [21,22].
BASP1 (brain acid-soluble protein-1; also known as
cortical cytoskeleton-associated protein CAP-23 and
neuronal tissue-enriched acidic protein NAP-22) is an
abundant 22–25 kDa proteinin neuronal axon termi-
nals, where it resides at the inner surface of the
plasma membrane, predominantly inlipid rafts
[23,24]. With respect to certain biochemical and func-
tional properties, it is similar to axonal growth-
associated protein GAP-43 [25,26]. Although GAP-43
is almost exclusively neuron-specific, BASP1 was also
detected in considerable amounts in other cell types
and tissues [23,27]. Knocking out the BASP1 gene in
mice results in severe abnormalities in the nervous
system and a high incidence (approximately 90%) of
early postnatal lethality, indicating that the protein is
critically involved in normal physiological processes
[25]. Like GAP-43, BASP1 participates in neurite
growth, axon guidance and regeneration [25,28,29].
The molecular mechanism ofBASP1activity presum-
ably involves the regulation of actin cytoskeleton
dynamics through either direct interaction with actin-
binding proteins [30] or modulation of the plasmalemmal
distribution of phosphatidylinositol-4,5-diphosphate
(PIP
2
) [31,32]. Recently obtained data indicate that
BASP1 may also act as a potential tumor suppressor
in non-neuronal cells, where it inhibits transcriptional
activity of WT1 and Myc proteins [33,34].
BASP1 and GAP-43 are not related to any amyloid
disease. However, recently, we have shown that GAP-
43 and particularly BASP1 form oligomers resembling
the oligomers of amyloid proteins by their annular
appearance in electron micrographs [35]. In addition,
BASP1 can also form linear rod-like aggregates
reminiscent of protofibrils of amyloid proteins. In the
present study, taking into account an outward similar-
ity ofBASP1 to amyloid oligomers, we explored
whether BASP1 oligomers can induce ionic currents
across planarlipid bilayers.
Results
BASP1 exhibits single channel-like activity on
planar lipid bilayers
The activityofBASP1 and GAP-43 proteins in nega-
tively-charged lipidbilayers under near-physiological
conditions (0.1–0.2 m KCl, pH 7.5) was examined using
the electrophysiological recording technique. The addi-
tion ofBASP1 to the solution in the cis-compartment
resulted in the spontaneous incorporation of BASP1
molecules into the planar bilayer, which was accom-
panied by the appearance of single channel-like
currents (Fig. 1A). Figure 2 shows representative exam-
ples of current fluctuations of the single BASP1 chan-
nels in phosphatidylcholine (PC) ⁄ phosphatidylserine
(PS) (50 : 50 mol %), PC ⁄ PIP
2
(90 : 10 mol %) and
phosphatidylethanolamine (PE) ⁄ PS (50 : 50 mol %)
bilayers at the most frequently observed conductance
level. The burst activity is a prominent characteristic of
this conductance level, regardless of the bilayer compo-
sition, whereas the dwell time distributions of BASP1
channels were slightly different in the bilayers used
(Fig. 2). The channels demonstrate slower kinetics
in PC ⁄ PIP
2
(90 : 10 mol %) membranes (mean life time
is approximately 2.5-fold larger) compared to
PC ⁄ PS (50 : 50 mol %) and PE ⁄ PS (50 : 50 mol %)
membranes.
A
B
Fig. 1. Study ofionchannelactivityofBASP1 and GAP-43 in the
PC ⁄ PS (50 : 50 mol %) bilayer. (A) BASP1 induces discrete single-
channel current fluctuations. (B) GAP-43 has no channel-like activ-
ity. Membrane-bathing solutions were 0.2
M KCl, 10 mM Hepes-
KOH (pH 7.5) in both compartments. The current records are
obtained at 50 mV transmembrane potential. The start of the
records corresponds to 50 min after the addition ofprotein to the
cis-compartment solution.
Ion channelactivityofBASP1 O. S. Ostroumova et al.
462 FEBS Journal 278 (2011) 461–469 ª 2010 The Authors Journal compilation ª 2010 FEBS
At the most frequently observed conductance
level (shown in Fig. 2), BASP1 channels generate a
linear current–voltage relationship with a slope of
50 ± 3 pS, irrespective of the bilayer composition
(Fig. 3).
The frequency ofchannel appearance was signifi-
cantly dependent on the bilayer composition. BASP1
ion channelactivity was only rarely observed in PE ⁄ PS
bilayers (two of eight trials), whereas 100% of trials
with PC ⁄ PS and PC⁄ PIP
2
bilayers (27 and 13 trials,
respectively) resulted in appearance ofionchannel cur-
rents. A trial was considered negative if no discrete
membrane conductance changes were observed within
180 min (starting from the protein addition). The
latency time before the appearance ofion channel
activity was highly variable (in the range 30–120 min)
and decreased with increasing protein concentration.
Less latency time (approximately 20 min) was observed
when BASP1 was pre-incubated (for 30 min) with
PC ⁄ PS (50 : 50 mol %) liposomes, which were used as
0.2
0.4
0.6
Relative frequency
Time (s)
Time (s)
Time (s)
t
t
= 0.29 ± 0.03 s
t
t
= 0.82 ± 0.24 s
t
t
= 0.33 ± 0.03 s
0.2
0.4
0.6
Relative frequency
0.2
0.4
0.6
Relative frequency
0.0
0.1
0.2
Relative frequency
Current (pA)
Current (pA)
Current (pA)
I = –4.6 ± 0.5 pA
I = –1.9 ± 0.3 pA
I = –5.2 ± 0.6 pA
0.0
0.2
0.4
Relative frequency
5 pA
5 s
–100 mV
4 pA
A
B
C
5 s
–75 mV
2 pA
5 s
–50 mV
0.0 0.5 1.0 1.5
0246
0123
–6 –5 –4 –3
–7 –6 –5 –4 –3
–3 –2 –1
0.0
0.1
0.2
Relative frequency
Fig. 2. Single ionchannelactivityofBASP1inplanarlipidbilayersof different composition: (A) PC ⁄ PS (50 : 50 mol %); (B) PC ⁄ PIP
2
(90 : 10 mol %); (C) PE ⁄ PS (50 : 50 mol %). Experimental conditions were the same as described in Fig. 1. The applied transmembrane
potential is indicated above the current records. The dotted line corresponds to the closed state of the channel (0 pA). Current amplitude his-
tograms (central panels) and channel dwell time histograms (right panels) for the corresponding bilayer compositions are shown. The
mean ± SD current, I, and dwell time, s, were evaluated by Gaussian and single-exponential fit, respectively (with significance at P < 0.05
by the chi-square test). The total numbers of events used for the analysis were 50–150 and 50–400, respectively.
–200 –100 100 200
–10
–5
5
10
V (mV)
I (pA)
Fig. 3. Amplitude of the BASP1-induced single-channel currents,
I, plotted as a function of transmembrane potential, V. Bilayer
compositions were: squares, PC ⁄ PS (50 : 50 mol %); triangles,
PC ⁄ PIP
2
(90 : 10 mol %); circles, PE ⁄ PS (50 : 50 mol %). Experi-
mental conditions were the same as described in Fig. 1. Each point
of the I–V plot represents the mean ± SD of at least ten current
readings. The line shows a linear regression fit of the data points.
O. S. Ostroumova et al. Ionchannelactivityof BASP1
FEBS Journal 278 (2011) 461–469 ª 2010 The Authors Journal compilation ª 2010 FEBS 463
carriers to incorporate the protein into PC ⁄ PS
(50 : 50 mol %) bilayer. Under these conditions, a ser-
ies of stepwise current changes was observed, which
probably corresponds to successive fusions of lipo-
somes (containing active BASP1 channels) with
the bilayer (Fig. 4A). In three of three trials, the ion
channel activityofBASP1in PC ⁄ cholesterol ⁄ PIP
2
(63 : 32 : 5 mol %) bilayers was similar to that
observed in the absence of cholesterol. The inclusion
of cholesterol into the bilayer did not significantly
affect either the conductance ofBASP1 channels or
the latency time of their appearance (30–50 min).
By contrast to BASP1, GAP-43 did not affected
bilayer conductance under the con ditions used (Fig. 1B).
Channel-like current fluctuations were not observed in
either PC ⁄ PS (50 : 50 mol %), PE ⁄ PS (50 : 50 mol %),
PC ⁄ PIP
2
(90 : 10 mol %) or PC ⁄ cholesterol⁄ PIP
2
(63 : 32 : 5 mol %) bilayers, regardless of the protein
concentration and method of incorporation (addition
of soluble GAP-43 or GAP-43-containing liposomes).
Multiple conductance states ofBASP1 channels
Besides the most frequently observed conductance level
of approximately 50 pS, BASP1 often induced the
formation ofion channels with higher conductance.
Figure 5 shows the spontaneous transitions of BASP1
channels between multiple conductance levels, when
the protein was incorporated into the bilayer directly
from the membrane-bathing solution. The conductance
transitions were observed within the picosiemens
range. As a rule, the higher conductance states were
more long-lived than the 50 pS conductance state
(Fig. 5). Their usual life time was from several seconds
up to several minutes (Fig. 1A). Rarely, conductance
levels of less than 50 pS or more than 1000 pS were
also observed.
Multichannel activityofBASP1 was studied by
liposome-mediated protein delivery to the bilayer.
Multichannel measurements produced linear current–
A
B
100 pA
10 s
0 pA
0 pA
50 pA
20 s
–50 mV
–25 mV
–100 –50 50 100
–900
–600
–300
300
600
900
I (pA)
V (mV)
Fig. 4. Multichannel activity induced by BASP1 incorporated into
PC ⁄ PS (50 : 50 mol %) lipid bilayer by fusion of BASP1-containing
liposomes. Membrane-bathing solutions were 0.1
M KCl, 1 mM
CaCl
2
,5mM Hepes-KOH (pH 7.5). (A) Current records obtained at
the transmembrane potential of )50 mV (upper) and )25 mV
(lower). (B) I–V plots corresponded to different time intervals after
liposome addition: squares, 30 min; circles, 45 min; triangles,
145 min. The lines show linear regression fits of three data series
with a slope (conductance) of approximately 200 pS, 1 nS and
10 nS, respectively. The larger conductance corresponds to a larger
number ofBASP1 channels incorporated by liposome fusion. The
data in each series were measured within 20 s.
100 mV
5 pA
ABC
5 s
~ 500 pS
~ 100 pS
15 pA
3 s
50 mV
~ 50 pS
~ 300 pS
200 mV
~ 50 pS
~ 200 pS
10 pA
5 s
Fig. 5. Multiple conductance levels induced by direct incorporation ofBASP1 into planarlipidbilayersof different composition: (A) PC ⁄ PS
(50 : 50 mol %); (B) PC ⁄ PIP
2
(90 : 10 mol %); (C) PE ⁄ PS (50 : 50 mol %). Experimental conditions were the same as described in Fig. 1.
Applied transmembrane potential is indicated above the current records. The dotted line corresponds to the closed state of the channel
(0 pA). Conductance values are indicated adjacent to the corresponding current jumps.
Ion channelactivityofBASP1 O. S. Ostroumova et al.
464 FEBS Journal 278 (2011) 461–469 ª 2010 The Authors Journal compilation ª 2010 FEBS
voltage relationships, indicating that all conductance
levels exhibit ohmic behaviour (Fig. 4B).
Cation-selective behaviour ofBASP1 channels
To determine the charge selectivity ofBASP1 channels,
we studied the protein-induced currents in the asym-
metrical 0.5 ⁄ 0.05 m KCl solution system (Fig. 6A). At
zero membrane potential, a net negative current
(approximately ) 1.3 pA) was measured (Fig. 6B),
which corresponded to a positive charge moving
from the trans- to the cis-compartment. This result
demonstrates that the bulk of the current is carried by
potassium ions. The reversal potential (V-intercept in
Fig. 6B) was estimated to be approximately 50 mV.
Similar values of the reversal potential (45 ± 5 mV)
were obtained for seven bilayers, with the overall con-
ductance in the range 10–100 pS. Using the Goldman–
Hodgkin–Katz equation [36], we calculated that the
permeability ratio P
K
⁄ P
Cl
is approximately 14, whereas
the transport number for K
+
ions (i.e. the fraction of
current carried by K
+
ions) is equal to approximately
0.93. These results indicate that BASP1 channels are
cation-selective.
BASP1 channels are voltage-independent
Using multichannel measurements, we investigated the
effect of transmembrane voltage on gating of BASP1
channels. Ionchannel voltage gating may be character-
ized by an effective gating charge [37]. Switching the
applied voltage polarity (± 50 mV) did not signifi-
cantly change the absolute value of BASP1-induced
steady-state transmembrane current. A two-fold reduc-
tion of the applied voltage (from )50 to )25 mV)
decreased the current in the same manner (Fig. 7A).
Using the steady-state current measurements, we deter-
mined the effective gating charge ofBASP1 channels
as being approximately 0 (Fig. 7B). Consequently, the
channels are not voltage-gated. Taking into account
the linear current–voltage relationships of BASP1
channels (Fig. 4B), these data clearly indicate that the
channels are voltage-independent.
Electron microscopy ofBASP1 oligomers on
a lipid monolayer
Using transmission electron microscopy (TEM), we pre-
viously demonstrated that BASP1 oligomers adsorbed
on the nitrocellulose support film predominantly have
an annular structure with a central pore (Fig. 8A) [35].
The size of annular BASP1 oligomers was confirmed
as being very heterogeneous, with a diameter in the
range 10–25 nm. To examine the structure of BASP1
oligomers under conditions close to those employed in
the present study, we used the lipid monolayer tech-
nique. Figure 8B shows that BASP1 oligomers formed
on PC ⁄ PS (50 : 50 mol %) monolayer patches have a
similar pore-like appearance. The mean ± SD diame-
ter of the oligomers was 17.2 ± 3.8 nm (n = 52).
Discussion
In the present study, we have shown for the first time
that the brainabundantproteinBASP1 forms ion
channels in a manner similar to that observed for amy-
loid proteins. In the present experiments, we used neg-
atively-charged lipid bilayers, which were previously
found to induce the formation ofBASP1 and GAP-43
oligomers [35] and stimulate the activityof amyloid
protein channels [5–7,9,38]. The BASP1-induced
A
B
–300 –200 –100 100 200
–9
–6
–3
3
6
V (mV)
I (pA)
–55 mV
50 mV
170 mV
–280 mV
45 mV
55 mV
20 mV
95 mV
0 pA
3 pA
10 s
Fig. 6. Cation-selectivity ofBASP1 channels. (A) BASP1-induced
current recorded in asymmetric system at different transmembrane
potentials. Lipid bilayer composition was PC ⁄ cholesterol ⁄ PIP
2
(63 : 32 : 5 mol %). Membrane-bathing solutions: in the cis-
compartment, 0.5
M KCl, 5 mM Hepes-KOH (pH 7.5); in the trans-
compartment, 0.05
M KCl, 5 mM Hepes-KOH (pH 7.5). BASP1 was
added to the solution in the cis-compartment. Values of transmem-
brane potential are indicated. (B) I–V plot corresponded to the
current record shown in (A).
O. S. Ostroumova et al. Ionchannelactivityof BASP1
FEBS Journal 278 (2011) 461–469 ª 2010 The Authors Journal compilation ª 2010 FEBS 465
increase in membrane conductance occurred in the
form of discrete unitary conductance changes with evi-
dence of open and closed states that are characteristic
of ion channels. It is notable that cholesterol, which is
a main component oflipid rafts, had no noticeable
effect on the BASP1channel activity. Similarly,
BASP1 oligomerization induced by negatively-charged
liposomes was also unaffected by the addition of
cholesterol [35]. BASP1 channels show cation selectiv-
ity, generate linear voltage–current relationships and
appear to be voltage-independent. Similar characteris-
tics were reported for ion channels formed by amyloid
b-protein and prion protein fragment 106–126
[5,21,39,40]. In addition, similar to most amyloid pro-
tein channels, BASP1 channels exhibit spontaneous
transitions between multiple conductance states (chan-
nel subtypes), including those with sufficiently high
conductance (up to several thousands of picosiemens)
[7,9,21,39–41]. The conductance levels of BASP1
channels are highly heterogeneous. Some levels are
characterized by burst activity (such as the 50 pS
conductance level), whereas others are long-lived.
Similar to amyloid protein channels, BASP1 channels
have irregular latency of appearance [21]. The latency
may be explained by the time-consuming step of BASP1
oligomerization into an active channel structure. In sup-
port of this suggestion, liposome-mediated incorpora-
tion of preformed BASP1 oligomers into the lipid
bilayer resulted in a smaller latency time. We have
shown previously that BASP1 is predominantly mono-
meric in solution but forms oligomers in the presence of
liposomes composed of anionic lipids [35]. The forma-
tion of an ion-conducting channel probably proceeds
through the association of several BASP1 monomers on
the surface of the anionic lipid bilayer. The BASP1
molecule has a net negative charge, while its N-terminus
is positively charged. Electrostatic binding of the
N-terminal domains to anionic lipids probably provides
the proper conformation and orientation of the protein
molecules allowing them to self-associate.
Using electron microscopy, we demonstrated that
BASP1 oligomers predominantly have an annular
structure with a central pore. This was confirmed for
oligomers formed on the nitrocellulose support film, as
well as on the PC ⁄ PS lipid monolayer. We suggest that
the pore-like structure ofBASP1 oligomers is responsi-
ble for the generation of ion-permeable channels in
lipid bilayers. The pore-like BASP1 oligomers are very
heterogeneous in diameter (10–25 nm), which can
account for the multiple conductance states of BASP1
channels. Different conductance levels are probably
related to different numbers of the protein monomers
in the oligomeric channel structure. A similar explana-
tion was proposed for multiple single-channel conduc-
tances of amyloid protein channels [14,16,21,42]. In the
presence of GAP-43, no ion channels were observed
under the conditions used. It should be noted that, in
contrast to BASP1, GAP-43 preferably forms discoid
rather than annular oligomers [35].
The most intriguing question concerns the physio-
logical or pathological role ofBASP1 channels. First,
–25 mV
–50 mV
0 pA
100 pA
5 min
50 mV
A
B
–2 0 2
4
8
12
VF/RT
ln (G, pS)
Fig. 7. Voltage-independent behavior ofBASP1 channels. (A)
Records of BASP1-induced multichannel activity at different trans-
membrane potentials. Experimental conditions were the same as
described in Fig. 4. Arrows indicate voltage steps. (B) A semiloga-
rithmic plot of BASP1-induced membrane conductance, G,asa
function of the applied voltage, V, at steady-state conditions for
two independent experiments. Taking into account the linear cur-
rent–voltage relationships ofBASP1 channels (Figs 3 and 4B), the
average number of open channels under steady-state conditions,
N
ch
, is proportional to steady-state membrane conductance, G. The
effective gating charge, Q, that characterizes the effect of electric
field on the conformational equilibrium of the channels can be cal-
culated by the formula: Q = d(lnN
ch
) ⁄ d(VF ⁄ RT ) [46], where R, T
and F represent the universal gas constant, the absolute tempera-
ture and the Faraday constant, respectively. The linear regression
fit of the plotted data gives Q = 0.081 ± 0.037 (squares) and
Q = 0.004 ± 0.046 (circles).
Ion channelactivityofBASP1 O. S. Ostroumova et al.
466 FEBS Journal 278 (2011) 461–469 ª 2010 The Authors Journal compilation ª 2010 FEBS
BASP1 oligomer formation may be related to a certain
pathology at which the oligomers exhibit toxicity as a
result of their ionchannel activity. However, BASP1 is
not known to be associated with any amyloid disease.
Recent data have indicated that, in neurons, BASP1
forms large oligomeric complexes, which can be
revealed by glutaraldehyde cross-linking of proteins in
plasma membrane rafts (V. V. Zakharov & O. S. Vity-
uk, unpublished results). This suggests that the mem-
brane-bound oligomers probably represent a functional
form of BASP1. Recently, the nonpathological protein
stefin B (in amyloid-like aggregates and in the native
form) was shown to also form ion channels in planar
lipid bilayers [43]. It has been suggested that the ion
channel activityof stefin B may be a physiological
function of the protein. Whether or not raft-associated
BASP1 oligomers act as ion channels in vivo (e.g. as a
kind of leak channels) remains unclear. Further
experiments are needed to address this possibility. The
studies ofionchannelactivityof nonpathological
amyloid-like proteins probably will help to define the
precise mechanism of amyloid protein toxicity.
Materials and methods
BASP1 and GAP-43 proteins were isolated from bovine
brain as described previously [44]. The proteins were
purified to homogeneity using a combination of preparative
polyacrylamide gel electrophoresis and reversed-phase
HPLC [35]. Protein stock solutions (0.5–3.5 mgÆmL
)1
)
in water were stored at )20 °C in aliquots. Water was
distilled twice and deionized. PC (1,2-dioleoyl-sn-glycero-
3-phosphocholine), PS (1,2-dioleoyl-sn-glycero-3-phospho-
serine), PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)
and porcine brain PIP
2
were purchased from Avanti Polar
Lipids (Alabaster, AL, USA). Cholesterol was obtained
from Sigma-Aldrich (St Louis, MO, USA).
Solvent-free planarlipidbilayers were formed according
to the lipid monolayer opposition technique [45] on a
50 lm diameter aperture in the 10 lm thick Teflon film
separating two compartments (cis and trans) of the Teflon
chamber. The aperture was pre-treated with hexadecane.
Under symmetric conditions, an electric potential was
applied to the bilayer with a pair of Ag ⁄ AgCl electrodes
via 2 m KCl, 1.5% agarose bridges. Under asymmetric con-
ditions (0.5 ⁄ 0.05 m KCl), Ag ⁄ AgCl electrodes were used. In
the latter case, the difference in the two half-cell potentials
(measured to be approximately 53 mV) was subtracted
from the measured potential to obtain the transmembrane
potential. The trans-compartment was held at virtual
ground. Negative current represents the flow of cations
from trans-tocis-side. Proteins were added to the mem-
brane-bathing solution in the cis-compartment to a final
concentration of 2–10 lm. In some experiments, BASP1
and GAP-43 were preliminary incorporated into PC ⁄ PS
(50 : 50 mol %) liposomes (protein ⁄ lipid molar ratio of
1 : 50, lipid concentration of 2 mm) by a method described
previously [35]. 5 lL of the protein ⁄ liposome preparation
was added to the membrane-bathing solution in the cis-
compartment (1.5 mL). To facilitate fusion of the liposomes
with the bilayer, CaCl
2
was added to the solutions in both
compartments up to 1 mm. Transmembrane currents were
recorded with a custom made amplifier and digitized with a
pclamp-compatible board. Data collection was performed
with a 2 kHz sampling frequency and low-pass filtering at
200 Hz. Data were analyzed using pclamp, version 9.0
(Axon Instruments, Foster City, CA, USA) and Origin,
version 7.0 (OriginLab Corp., Northampton, MA, USA).
All experiments were carried out at room temperature
(21 ± 2 °C).
TEM specimens were prepared using the lipid monolayer
technique. 1 lL of 0.1 mgÆmL
)1
lipid solution (PC ⁄ PS,
50 : 50 mol %) in chloroform ⁄ methanol (19 : 1) was spread
on 60 lL of the buffer (10 mm sodium phosphate, pH 7.3,
0.1 m NaCl) in a Teflon well (4 mm in diameter) and
allowed to incubate in a humid chamber for 1 h at room
temperature. Formvar ⁄ carbon-coated copper grid (200
mesh) (SPI Supplies, West Chester, PA, USA) was placed
onto the top of the buffer in the well for 1 min. Then the
grid with adsorbed lipid monolayer was transferred onto a
10 lL drop ofBASP1 solution (0.1 mgÆmL
)1
) in the same
AB
Fig. 8. TEM micrographs ofBASP1 oligomers stained with uranyl acetate. (A) BASP1 oligomers adsorbed on the nitrocellulose support film.
(B) BASP1 oligomers adsorbed on the lipid monolayer composed of PC and PS (50 : 50 mol %). Dark areas correspond to the lipid mono-
layer patches on the Formvar ⁄ carbon support film. Black objects represent electron-dense material. Scale bars = 50 nm.
O. S. Ostroumova et al. Ionchannelactivityof BASP1
FEBS Journal 278 (2011) 461–469 ª 2010 The Authors Journal compilation ª 2010 FEBS 467
buffer and exposed for 20 min. Subsequently, the grid was
successively rinsed on three drops of water (for 30 s on
each) and stained with 1% uranyl acetate for 30 s. The
excess fluid was drained off with filter paper, and the grid
was air dried. The specimens ofBASP1 oligomers on the
nitrocellulose support film were prepared as described pre-
viously [35].
Acknowledgements
This work was supported by the Russian Foundation
for Basic Research (grants 08-04-00432, 09-04-00883),
the Molecular and Cell Biology Program of the Presid-
ium of the Russian Academy of Sciences, the President
of Russian Federation (grant SS-3796.2010.4) and the
Russian State Contract P1372 (MES, FTP ‘‘SSEPIR’’).
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. Ion channel activity of brain abundant protein BASP1 in planar lipid bilayers Olga S. Ostroumova 1 , Ludmila V. Schagina 1 , Mark I. Mosevitsky 2 and Vladislav V. Zakharov 2 1 Laboratory of Ionic. bilayers. Results BASP1 exhibits single channel- like activity on planar lipid bilayers The activity of BASP1 and GAP-43 proteins in nega- tively-charged lipid bilayers under near-physiological conditions. regeneration [25,28,29]. The molecular mechanism of BASP1 activity presum- ably involves the regulation of actin cytoskeleton dynamics through either direct interaction with actin- binding proteins