Puroindoline-a and a1-purothionin form ion channels
in giant liposomes but exert different toxic actions
on murine cells
Paola Llanos
1
, Mauricio Henriquez
1
, Jasmina Minic
2
, Khalil Elmorjani
3
, Didier Marion
3
,
Gloria Riquelme
1
, Jordi Molgo
´
2
and Evelyne Benoit
2
1 Instituto de Ciencias Biome
´
dicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
2 Laboratoire de Neurobiologie Cellulaire et Mole
´
culaire, UPR 9040, Centre National de la Recherche Scientifique, Gif-sur-Yvette cedex,
France
3 Biopolyme
`
res Interactions Assemblages, Institut National de la Recherche Agronomique, Nantes, France

genic rice plants expressing genes encoding PINs (pinA
and ⁄ or pinB) reduce in vitro the growth of rice fungal
Keywords
a1-purothionin; giant liposomes; ion
channels; neuromuscular transmission;
puroindoline-a
Correspondence
E. Benoit, Laboratoire de Neurobiologie
Cellulaire et Mole
´
culaire, UPR 9040, Centre
National de la Recherche Scientifique, ba
ˆ
t.
32–33, 91198 Gif-sur-Yvette cedex, France
Fax: +33 169 82 41 41
Tel: +33 169 82 36 52
E-mail:
(Received 20 July 2005, revised 13 February
2006, accepted 17 February 2006)
doi:10.1111/j.1742-4658.2006.05185.x
Puroindoline-a (PIN-a) and a1-purothionin (a1-PTH), isolated from wheat
endosperm of Triticum aestivum sp., have been suggested to play a role in
plant defence mechanisms against phytopathogenic organisms. We investi-
gated their ability to form pores when incorporated into giant liposomes
using the patch-clamp technique. PIN-a formed cationic channels
( 15 pS) with the following selectivity K
+
>Na
+

ion concentration gradients essential for the mainten-
ance of cellular homoeostasis [10–13]. Also, b-PTH
extracted from wheat flour has been shown to form
cation-selective ion channels in artificial lipid bilayer
membranes and in the plasmalemma of rat hippocam-
pal neurons [14]. PIN-a and a1-PTH have also been
reported to swell the nodes of Ranvier of frog myeli-
nated axons, and pore formation in the nodal mem-
brane has been suggested to be responsible for these
effects [15]. In addition, PINs have also been shown to
be cytotoxic to Xenopus oocytes [16]. However, the
mechanisms involved in the toxicity of PTHs and PINs
to mammalian cells remain poorly understood. There-
fore, as a first step toward understanding these mecha-
nisms, we characterized (a) the pore-forming activity
of PIN-a and a1-PTH in giant liposomes and (b) their
toxicity to mammalian phrenic nerve ⁄ hemidiaphragm
muscle preparations and cultured neuroblastoma
(NG108-15) cells. A preliminary account of part of this
work has been published in abstract form [17].
Results
Molecular masses of purified PIN-a and a1-PTH
A typical electrospray mass spectrum of purified wheat
PIN-a (Fig. 1A) reveals that its apparent heterogeneity
is related to complex post-translational proteolytic
maturation which leads to two major forms (M
r
12 750 and 12 919) and three minor ones (M
r
12 634.7, 12 803.5 and 13 083.6). However, as reported

tionship (Fig. 2C).
4,700
4,800
4,900 5,000 5,100 5,200
α1-PTH
4,921
B
12,60012,200 13,000 13,400 13,800
DVA-(PIN)
-GTIG
12,919
DVA-(PIN)
-GTIGY
13,083.6
VA-(PIN)
-GT
12,634.7
VA-(PIN)
-GTIG
12,803.05
DVA-(PIN)
-GT
12,750
A
Fig. 1. MALDI-TOF mass spectrum of PIN-a and a1-PTH. Deconvo-
luted and reconstructed electrospray mass spectra from multi-
charged ion spectra of the purified PIN-a (A) and a1-PTH (B). Note
the homogeneity of the protein preparations.
P. Llanos et al. Toxic actions of puroindoline-a and a1-purothionin
FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS 1711

⁄ P
Cl
) was  13 (n ¼ 5). To
determine the K
+
to Na
+
permeability ratio
(P
K
⁄ P
Na
), we replaced the bath NaCl with KCl. When
140 mm KCl was perfused in the bath solution, the
current recorded in response to a potential ramp
showed an almost linear current–voltage relation-
ship, and it had a reversal potential of )9.2 ± 0.8 mV
(n ¼ 8). Under these conditions, the permeability ratio
was 1.43 ± 0.04 (n ¼ 8). These results indicate that
PIN-a forms a cationic channel, the permeability
sequence of which is K
+
>Na
+
 Cl
–
.
a1-PTH forms ionic channels in giant liposomes
Giant liposomes containing a1-PTH also produced
excised patches with seals of high resistance. Channel

) was  7, which indicates that the high-
500 ms
1 pA
500 ms
1 pA
5 s
1 pA
AB
V (mV)
-100 -50 50 100
-1.5
-1.0
-0.5
0.5
1.0
1.5
C
I (pA)
100-100
-50
50
-100
-50
50
100
V (mV)
D
I (pA)
I (pA)
%emiT

conductance channel formed by a1-PTH is an anionic
channel. The Ca
2+
selectivity of the channels was nil.
Indeed, when the concentration of CaCl
2
was increased
from 2.6 mm to 10 or 20 mm in the bath solution, no
significant effect on current amplitude or reversal
potential values was detected in response to potential
ramps (data not shown).
Currents through low-conductance channels formed
by a1-PTH were recorded at holding potentials varying
from 0 to ± 80 mV in steps of 40 mV (Fig. 4A). Unit-
ary conductances of 46 ± 5 and 34 ± 2 pS were cal-
culated from current potential relationships at holding
potentials of +100 and )100 mV, respectively
(Fig. 4B), observed during 21 experiments using sym-
metrical NaCl (140 mm, n ¼ 18) or sodium gluconate
(140 mm, n ¼ 3) concentrations. When the NaCl
concentration in the bath solution was decreased from
140 to 40 mm, the reversal potential of the current
recorded in response to potential ramps shifted from 0
to +23 mV (Fig. 4C), which is close to the Na
+
equi-
librium potential (+ 27.8 mV). A P
Na
⁄ P
Cl

-10
-5
5
10
15
20
25
-20 mV
I (pA)
Fig. 3. High-conductance channel activities exhibited by giant lipo-
somes containing a1-PTH. (A) Unitary current traces recorded at
the indicated holding potentials. (B) Current–voltage relationships in
the presence of either 140 m
M NaCl (s)or40mM NaCl (d) in the
bathing solution. Under these conditions, the voltages correspond-
ing to zero current were 0 and )20 mV (arrow), respectively.
A
-20
0
20
1 s
80 mV
40 mV
0 mV
-80 mV
-40 mV
B
V (mV)
I (pA)
-150 -100 -50 50 100 150

)2.5 ± 1.1 mV (n ¼ 4), brought about by replacing
NaCl (140 mm) with KCl (140 mm) in the bath solu-
tion. These results indicate that the low-conductance
channel formed by a1-PTH is a cationic channel.
The selectivity of the low-conductance channel to
bivalent cations was studied by changing the CaCl
2
concentration in the bath solution. Figure 5A shows
unitary currents, recorded at a holding potential of
0 mV, in the presence of 2.6 mm (control conditions)
and 20 m m CaCl
2
. In response to potential ramps,
the reversal potential shifted from 0 (Fig. 5B) to
)8.0 ± 0.8 mV (n ¼ 4, Fig. 5C) when the CaCl
2
con-
centration was increased from 2.6 to 10 mm, and it
was )13.3 ± 0.4 mV (n ¼ 5) when the CaCl
2
concen-
tration was 20 mm. Under these conditions, the expec-
ted equilibrium potential calculated for Ca
2+
was
)17.3 mV and )26.2 mV for 10 and 20 mm CaCl
2
,
respectively. A Ca
2+

ade was not reversed after extensive washing with the
standard physiological solution. Similar concentrations
of a1-PTH also blocked twitches evoked by direct
electric stimulation of the muscle (Fig. 6A,B). Thus,
a1-PTH is toxic to isolated mouse phrenic nerve ⁄ hemi-
diaphragm muscle preparations. In contrast, when we
examined the ability of PIN-a (0.01–1 lm) to alter
muscle twitches and tetanic responses evoked by nerve
stimulation at 0.2 and 40 Hz, respectively, no signifi-
cant changes were detected in the contraction ampli-
tude (Fig. 6B).
Membrane permeability changes caused by the pore-
forming ability of a1-PTH may explain the above
effects. Therefore, we performed intracellular record-
ings to measure the effect of a1-PTH and PIN-a on
the resting membrane potential of mouse hemidia-
phragm muscle fibres. When added to the standard
medium, a1-PTH (0.05–1 lm) caused dose-dependent
membrane depolarization (Fig. 7A). A representative
recording of the time course of 1 lm a1-PTH-induced
depolarization of skeletal muscle fibres is shown in
Fig. 7B. The time required by a1-PTH to exert half-
010 30 40 50 60
4 pA
0 pA
5 s
4 pA
0 pA
Control
CaCl

was increased from 2.6 mM (control) to 20 mM
(arrow). The dotted lines indicate the zero current level. The arrows
show in an expanded time basis unitary currents. (B,C) Representa-
tive currents recorded during the same experiment in response to
potential ramps from )100 mV to +100 mV in the presence of
either 2.6 m
M (B) or 10 mM (C) CaCl
2
in the bathing solution. Under
these conditions, the voltages corresponding to zero current were
0 (B) and )9 mV (C, arrow).
Toxic actions of puroindoline-a and a1-purothionin P. Llanos et al.
1714 FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS
maximal depolarization was 3.5 ± 0.8 min (n ¼ 4).
The magnitude of the depolarization was independent
of the external CaCl
2
concentration between 0 and
2mm (Fig. 7C). However, when a1-PTH was added to
the standard medium in which the CaCl
2
concentration
was raised from 2 mm to 5 or 10 mm, no significant
change was detected in the resting membrane potential
of the muscle fibres (Fig. 7B,C). In contrast with the
marked effect of a1-PTH, PIN-a (0.05–1 lm) did not
significantly alter the resting membrane potential of
the muscle fibres (Fig. 7A). However, at a higher con-
centration (10 lm), the protein hyperpolarized the
muscle membrane by 21 ± 2.5 mV within about 5 min

(filled symbols), and directly elicited muscle twitch (s). The twitch
tension is expressed with respect to controls and as means ± SEM
for n experiments (numbers beside data points). Note the complete
blockade of the twitch response in the presence of 1 l
M a1-PTH,
and the quasi-absence of effect of similar concentrations of PIN-a.
Protein concentration
α1-PTH (2 mM CaCl
2
)
PIN-a (2 m
M CaCl
2
)
)Vm(laitnetopenarbmemgnitseR
-90
-80
-70
-60
-50
-40
-30
-20
-10
0
A
)Vm(laitnetop
enarbmemgn
itseR
Control

)
1 µ
M α1-PTH (10 mM CaCl
2
)
B
//
//
//
Fig. 7. Effects of a1-PTH and PIN-a on the resting membrane
potential of skeletal muscle fibres (A), and the influence of extracell-
ular Ca
2+
concentration on the effect of a1-PTH (B and C). Note
that in (A) only a1-PTH produces membrane depolarization, and in
(B) and (C) increasing extracellular Ca
2+
concentration (from 2 to
10 m
M) markedly reduces a1-PTH-induced muscle depolarization,
whereas decreasing extracellular Ca
2+
concentration (from 2 to
0m
M) has no significant effect. In (A) and (C), each column repre-
sents the mean ± SEM obtained from 3 to 31 fibres. In (B), the
points represent the membrane potential of single muscle fibres as
a function of time after addition of a1-PTH to the medium.
P. Llanos et al. Toxic actions of puroindoline-a and a1-purothionin
FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS 1715

the size of the cells. A similar increase in both fluores-
cence intensity and 3D projected area occurred when
the cells were exposed to 10 lm a1-PTH in a CaCl
2
-
free medium. However, under these conditions, bleb
formation was not observed (Fig. 9Ad). In contrast,
exposure to 10 lm PIN-a had no detectable effect on
the morphology of NG108-15 cells (Fig. 9Bc),
although higher concentrations (50 and 100 lm) pro-
duced a 10–15% decrease in the cells’ 3D projected
area (Fig. 9Bb,c).
Effects of a1-PTH and PIN-a on the actin network
of NG108-15 cells
The significant membrane blebbing observed in the
presence of a1-PTH, but not in the presence of PIN-
a, prompted us to determine whether the two pro-
teins affect the cytoskeleton of NG108-15 cells. Thus,
immunofluorescence studies were performed to detect
eventual changes in the actin network organization
after exposure to a1-PTH (10 lm) or PIN-a (10 lm)
for 2–4 h. In comparison with control cells, and with
cells treated with PIN-a, significant changes in fila-
mentous actin immunolabelling distribution were
observed in cells treated with a1-PTH (Fig. 9C). Dis-
organization and disarray of actin were reflected by
disruption and clumping of actin filaments, which
was depicted as a punctuate pattern throughout the
cytoplasm (Fig. 9Cb). Similar results were observed
in NG108-15 cells after AlexaFluor-594-conjugated

and 1 l
M (right trace) a1-PTH. The arrow indicates the stimulation
artefact of the phrenic nerve. (B) Average of 30 sequential MEPPs
recorded before (left trace) and after 20 min exposure to 0.5 l
M
(middle trace) and 1 lM a1-PTH (right trace). (C) Average of 30
sequential MEPPs recorded before (left trace) and after 20 min
exposure to 1 l
M PIN-a (right trace). Note the subthreshold EPP (A,
middle trace), the reduction and complete block of averaged
MEPPs induced by a1-PTH (B, right trace), and the absence of
effect of PIN-a on the amplitude of averaged MEPPs (C, right
trace). Note the different scales in A, B and C.
Toxic actions of puroindoline-a and a1-purothionin P. Llanos et al.
1716 FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS
forms ion channels in biological and artificial mem-
branes. In addition, we found that PIN-a forms a cat-
ion-selective channel with a 15 pS conductance. This
channel is 13 times more permeable to a univalent cat-
ion (Na
+
) than to Cl
–
and 1.4-fold more permeable to
K
+
than to Na
+
. These results considerably extend
those previously obtained on voltage-clamped Xenopus

for this channel is the following: Ca
2+
>Na
+
 K
+
.
These results are consistent with and extend previ-
ous observations suggesting that PTHs form cation-
selective ion channels, and in particular with data
obtained with b-PTH showing the formation of cation-
selective ion channels in artificial lipid bilayer mem-
branes and in the plasmalemma of rat hippocampal
neurons [14].
Although a concentration-dependent study of a1-
PTH has not been performed, it is possible that the
two different channel behaviours detected are the con-
sequence of protein–protein interactions in the recon-
stituted system, leading to channel clustering or similar
processes. Thus, it is likely that progressive recruitment
of additional monomers will contribute to increase the
pore size. The formation of transmembrane chan-
nels ⁄ pores by bundles of amphipathic a-helices of
a1-PTH and PIN-a polypeptides may occur via a ‘bar-
rel-stave’ mechanism [19], in such a manner that their
hydrophobic surfaces interact with the lipid core of the
membrane and their hydrophilic surfaces point inward,
producing an aqueous pore. According to this model,
10 µm
a

1GN fo ae
ra evitaleR
12
*
*
20
* *
16
* *
16
* *
34
* *
* *
21
20
Concentration (time of application)
10 µ
M
(90 min)
50 µ
M
(30min)
50 µ
M
(60 min)
50 µ
M
(120 min)
100 µ

2+
-con-
taining medium. Note the marked increase in fluorescence intensity
in the cells’ cytosol, the large membrane blebs (arrows), and the
increase in projected area of the cells. Cells imaged before (Ac) and
after (Ad) exposure to 10 l
M a1-PTH in a Ca
2+
-free medium. Note
the absence of blebbing, but a similar increase in cells’ cytosol
fluorescence intensity. (B) Cells were imaged before (Ba) and after
(Bb) 50 l
M PIN-a exposure to a Ca
2+
-containing medium. In (Bc),
the bars indicate the relative projected area of the cells as a funct-
ion of PIN-a concentration and time of exposure. (C) Immunostain-
ing of actin under control conditions (Ca) and after exposure of the
cells to either 10 l
M a1-PTH (Cb) or 10 lM PIN-a. In (Cb), note the
distinct distribution and clumping of the immunolabelling.
P. Llanos et al. Toxic actions of puroindoline-a and a1-purothionin
FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS 1717
progressive recruitment of monomers would increase
the pore size. Under our conditions, once the cationic
pore is formed, the aggregation of further monomers
would not only augment the pore size, but also, by
exposing some amino-acid residues, create a new ani-
onic-selectivity filter. Although this may explain the
different channel behaviours detected with a1-PTH,

meability mainly to K
+
ions and that the reversal
potential for K
+
ions is more negative than the resting
membrane potential of muscle fibres. Therefore, the
increased permeability induced by PIN-a will result in
K
+
outflux and, as a consequence, in membrane
hyperpolarization.
The consequences of the pore-forming ability of
a1-PTH and PIN-a were also evaluated in NG108-15
cells stained with the styryl dye FM1-43. This vital dye
partitions into the plasma membrane and does not
ordinarily ‘flip-flop’ across it [20]. During exposure of
cells to a1-PTH action, the fluorescent staining of the
cell’s membrane by the FM1-43 dye was particularly
useful for delineating membrane blebbing and follow-
ing the 3D projected area of the cells. The develop-
ment of blebs in the presence of a1-PTH is probably
related to the membrane permeability changes it indu-
ces, as, in other neuronal cells, blebbing has been asso-
ciated with raised intracellular Na
+
concentration [21].
Also, a1-PTH-treated NG108-15 cells exhibited an
increase in FM1-43 fluorescence intensity, similar to
that previously observed at the nodes of Ranvier of

NaCl, 5 mm EDTA and 5% Triton X-114 (pH 7.8). After
stirring (12 h, 4 °C) and centrifugation (8000 g, 30 min),
the supernatants were heated at 30 °C to allow phase par-
titioning, and the upper, detergent-poor phase was discar-
ded. The lower, detergent-rich phase was diluted with
5 vol. water and loaded on a column packed with a cation
exchanger (SP Biobeads; Pharmacia, Montigny-le-Breton-
neux, France). Proteins were eluted by applying a gradient
from 0.02 to 0.7 m NaCl in Tris buffer without Triton
X-114. Analysis of the collected fractions by SDS ⁄ PAGE
indicated that the PTHs were eluted as a single peak just
after the PINs. Separate pools of the PTH-containing and
PIN-containing crude fractions were dialyzed against de-
ionized water and freeze-dried, and a1-PTH, a2-PTH and
b-PTH were separated (at room temperature) by semipre-
parative RP-HPLC. The HPLC column was packed with
Nucleosil C18 (5 lm, 300 A
˚
), the PTHs were eluted with
an acetonitrile gradient (0.1% trifluoroacetic acid in deion-
ized water to 0.1% trifluoroacetic acid in acetonitrile), and
the fractions containing a1-PTH were pooled and freeze-
dried after dilution with deionized water. PIN-a was puri-
fied from the crude, freeze-dried, PIN-containing fraction
by cation-exchange chromatography on a 6 mL Resource
Toxic actions of puroindoline-a and a1-purothionin P. Llanos et al.
1718 FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS
S column (Pharmacia), as previously described [26]. The
homogeneity of the purified a1-PTH and PIN-a prepara-
tions was monitored by MS, as detailed by Elmorjani

pipette
), and the junction poten-
tial was compensated for when necessary. To study the
ionic selectivity of the protein-induced channels, we deter-
mined the relative ionic permeabilities from the reversal
potentials of the currents recorded in solutions of various
compositions, in response to potential ramps [ +150 to
)150 mV (60 mVÆs
)1
) or +100 to )100 mV (40 mVÆs
)1
)].
They were calculated from changes in reversal potentials,
brought about by ion replacement based on the Goldman–
Hodgkin–Katz flux equation [30,31]. The reversal potential
of a cationic current as a function of the concentration or
activity and the permeability of each ion species was calcu-
lated as previously described [32,33].
Patch-clamp data were analyzed off-line with TAC soft-
ware (Bruxton Corporation, Seattle, WA, USA) and Pulse
Fit (Heka Elektronic) software. All measurements were
made at  25 °C, and the pipette and bath solutions usu-
ally had the following composition:140 mm NaCl, 2.6 mm
CaCl
2
, 1.3 mm MgCl
2
, and 10 mm Hepes (adjusted to
pH 7.4 with NaOH). In some experiments, NaCl was
replaced by either KCl or sodium gluconate. All reagents

(8–18 MW resistance), using conventional techniques and
an Axoclamp-2A system (Axon Instruments, Union City,
CA, USA). Recordings were made continuously from the
same endplate before and during treatment with the pro-
teins being tested. Electrical signals after amplification were
collected and digitized, at a sampling rate of 25 kHz, with
the aid of a computer equipped with an analogue-to-digital
interface board (DT 2821; Data Translation, Marlboro,
MA, USA). Computerized data acquisition and analysis
were performed with a program kindly provided by
J. Dempster (University of Strathclyde, Scotland, UK).
For twitch tension measurements, one of the tendons of
the hemidiaphragm muscle was tied with silk thread, via an
adjustable stainless steel hook, to an FT03 isometric transdu-
cer (Grass Instruments, West Warwick, RI, USA), and the
other tendon was pinned to the Rhodorsil-lined chamber.
Twitches were evoked either by stimulating the motor nerve
of isolated neuromuscular preparations via a suction micro-
electrode adapted to the diameter of the nerve, or by direct
muscle stimulation via an electrode assembly placed along
the length of the fibres. Pulses were supplied by a S-44 stimu-
lator (Grass Instruments) at frequencies of 0.2–40 Hz. For
each preparation investigated, the resting tension was
adjusted to obtain maximal contractile responses. Signals
from the isometric transducer were amplified, collected, and
digitized with the aid of a computer equipped with a DT
2821 analogue-to-digital interface board (Data Translation).
Cultured neuroblastoma cells
Rodent neuroblastoma (NG108-15) cells were grown in
monolayer cultures on glass coverslips using Dulbecco’s

incubated for 2–4 h at 37 °C. Then, they were rinsed with
phosphate-buffered saline (NaCl ⁄ P
i
) and fixed with either
4% paraformaldehyde in NaCl ⁄ P
i
(15 min, 37 °C) or with
100% methanol (4 min, )20 °C). After being washed three
times with NaCl ⁄ P
i
to remove excess fixative, the cells were
permeabilized and blocked (l h, room temperature) with
NaCl ⁄ P
i
containing 0.1% Triton X-100 and 3% BSA
(blocking buffer). Subsequently, cells fixed by either method
were incubated with the primary JLA-20 antibodies (1 : 100
dilution; Jackson ImmunoResearch Laboratories, Inc, West
Grove, PA, USA), washed with NaCl ⁄ P
i
, and incubated
(1 h, room temperature) with Texas-red-conjugated secon-
dary antibodies (1 : 100 dilution; Molecular Probes). Cover-
slips were mounted on to glass slides with Vectashield
antifading mounting medium (Vector Laboratories, Inc,
Burlingame, CA, USA). In some experiments, AlexaFluor-
594-conjugated phalloidin (Molecular Probes) was used to
visualize F-actin in fixed cells.
Confocal laser scanning microscopy
Time-lapsed imaging of cells was performed using a Saras-

dicale. We thank Dr M. Malo and Dr B. Rouzaire-
Dubois for providing the NG108-15 cells used in this
study, and Dr H. Rogniaux for performing MS.
Confocal microscopy studies were performed on the
Plate-forme Imagerie et Biologie Cellulaire of the
Gif-sur-Yvette Campus.
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