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 p
Trang 1in giant liposomes but exert different toxic actions
on murine cells
Paola Llanos1, Mauricio Henriquez1, Jasmina Minic2, Khalil Elmorjani3, Didier Marion3,
Gloria Riquelme1, Jordi Molgo´2 and Evelyne Benoit2
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
Various cationic lipid-binding proteins, the folding of
which is stabilized by four or five disulfide bonds, have
been isolated from wheat endosperm They include
lipid-transfer proteins (LTPs), puroindolines (PINs)
and a⁄ b-purothionins (PTHs) [1] PINs are restricted
to Triticae and Avenae species [2,3], whereas LTPs and
PTHs are ubiquitous plant proteins found in most
plant organs [1,4,5] PIN-a and PIN-b, two isoforms
sharing 60% sequence homology, have been purified
from wheat seeds PIN-a displays a unique
trypto-phan-rich domain (WRWKWWK), which is slightly
truncated in PIN-b (WPTKWWK) The 3D structures
of LTPs and PINs are closely related and rich in
a-helices, as suggested by their cysteine pairing and
secondary-structure characterization [1,6], and they
dif-fer from the structure of the PTHs [5] Furthermore, in
contrast with LTPs, PINs and PTHs can be isolated
by Triton X-114 phase partitioning [7], an observation that is in agreement with differences in their lipid-binding properties Indeed, whereas LTPs bind lipid monomers in a hydrophobic cavity, PINs and PTHs interact with lipid aggregates, e.g micelles and lipid bilayers [1]
Because of their toxic activity against fungi, yeast and bacteria, PTHs have been suggested to play a role
in plant defence against microbial pathogens [4,5] PINs are also thought to have a role in plant defence because of their antifungal properties in vitro, and especially because they enhance the antimicrobial effects of PTHs [8] In addition, leaf extracts of trans-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, baˆt.
32–33, 91198 Gif-sur-Yvette cedex, France
Fax: +33 169 82 41 41
Tel: +33 169 82 36 52
E-mail: benoit@nbcm.cnrs-gif.fr
(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+ Cl– Also, a1-PTH formed channels of 46 pS and 125 pS at +100 mV, the selectivity of which was Ca2+> Na+ K+ Cl– and Cl– Na+, respectively In isolated mouse neuromuscular preparations, a1-PTH induced muscle mem-brane depolarization, leading to blockade of synaptic transmission and directly elicited muscle twitches Also, a1-PTH caused swelling of differen-tiated neuroblastoma NG108-15 cells, membrane bleb formation, and dis-organization of F-actin In contrast, similar concentrations of PIN-a had
no detectable effects The cytotoxic actions of a1-PTH on mammalian cells may be explained by its ability to induce cationic-selective channels
Abbreviations
EPP, endplate potential; LTP, lipid-transfer protein; MEPP, miniature endplate potential; PIN, puroindoline; PTH, purothionin.
Trang 2pathogens [9] The generalized toxicity of PTHs may
be due to their ability to form ion channels in the
membrane of target cells, resulting in dissipation of
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 (Mr
12 750 and 12 919) and three minor ones (Mr
12 634.7, 12 803.5 and 13 083.6) However, as reported
by Blochet et al [18], all these polypeptides originate
from a unique polypeptide template with different
extensions at both the N-terminus and C-terminus
(Fig 1A) The mass spectrum of a1-PTH is depicted in
Fig 1B All the masses reported here fit very well with
the expected calculated molecular masses for native
PIN-a and a1-PTH
PIN-a forms ionic channels in giant liposomes
Seals of high-resistance and excised patches in an
‘inside out’ configuration were obtained from 19
prep-arations of giant liposomes containing PIN-a
Forty-five of 72 patches studied ( 60%) exhibited channel
activity Usually, multiple current levels corresponding
to a similar conductance were observed, and at least
two distinct levels were detected in 35 of the 45
pat-ches ( 78%) The unitary level was difficult to
observe in isolation, which suggested the presence of
substrates or clustering of channels in the patch How-ever, because it was not possible to distinguish between these two possibilities, we assume that each level above the baseline corresponds to a single channel, which opens and closes independently Unitary currents recorded at a holding potential of +100 mV are shown in Fig 2A, and the corresponding current amplitude distribution is depicted in Fig 2B Six chan-nels were present in the patch, as judged by the num-ber of simultaneous unitary current steps and histogram peaks A unitary conductance of 14.8 ± 0.6 pS (n¼ 17) was determined, between )80 and +80 mV, from the slope of the current–voltage rela-tionship (Fig 2C)
4,700 4,800 4,900 5,000 5,100 5,200
α1-PTH
4,921
B
12,600
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.
Trang 3The selectivity of the channels for Na+ vs Cl– was
determined by increasing or decreasing the NaCl
con-centration in the bath solution In the presence of a
high NaCl concentration (440 mm instead of 140 mm)
in the bath, the reversal potential of the current
recor-ded in response to potential ramps shifted from 0 to
)25 ± 1 mV (n ¼ 3, Fig 2D), which is close to the
)28 mV theoretical equilibrium potential for Na+(the
equilibrium potential for Cl–was +29.6 mV under this
condition) Similarly, in the presence of a low NaCl
concentration (40 mm instead of 140 mm) in the bath,
the reversal potential of the current shifted from 0 to
+24 mV (data not shown), which is close to the
+30 mV equilibrium potential for Na+ The Na+ to
Cl– permeability ratio (PNa⁄ PCl) was 13 (n ¼ 5) To
determine the K+ to Na+ permeability ratio
(PK⁄ PNa), 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
(n¼ 8) Under these conditions, the permeability ratio
was 1.43 ± 0.04 (n¼ 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 activity was found in 31 ( 41%) of 75 recorded pat-ches Single channels with a high unitary conductance and single channels with a low unitary conductance were detected in 32% (n¼ 10) and 68% (n ¼ 21) of the recordings, respectively In two independent experi-ments, low-conductance and high-conductance open-ings were detected simultaneously in the same patch, but these data have not been included in this study because of difficulties with their analysis
Typical current recordings through high-conduct-ance channels formed by a1-PTH are shown in Fig 3A The unitary conductance was 125 and 100 pS
at holding potentials of +100 and )100 mV, respect-ively Figure 3B depicts the current vs voltage plot at different holding potentials in the presence of 140 mm and 40 mm NaCl in the bath solution When the bath concentration of NaCl was decreased, the reversal potential of the current shifted from 0 to )20 mV, which is close to the )28 mV equilibrium potential for Cl– The calculated Cl– to Na+permeability ratio (PCl⁄ PNa) was 7, which indicates that the
high-500 ms
1 pA
500 ms
1 pA
5 s
1 pA
V (mV)
-1.5
-1.0
-0.5
0.5
1.0
1.5
C
I (pA)
100
-100 -50
50 100
V (mV)
D
I (pA)
I (pA)
1.5
Fig 2 Ion-channel activities exhibited by giant liposomes containing PIN-a (A) Unitary current traces recorded at a holding potent-ial of +100 mV The zero current level is indicated by the dotted line, and channel openings are indicated by upward deflect-ions The arrows show in an expanded time base the corresponding unitary currents (B) Time distribution of current amplitude corresponding to the recordings shown in (A) The time was expressed as a percent-age of the total recording time (C) Current– voltage relationship obtained from current amplitude distributions at various holding potentials A unitary conductance of 14.8 ± 0.6 pS (n ¼ 17) was determined from the slope of the relationship by linear regression between )80 and +80 mV (D) Representative currents recorded during potential ramps from )100 to +100 mV in the presence of either 140 or 440 m M NaCl
in the bathing solution Under these condit-ions, the voltages corresponding to zero current were 0 and )24.3 mV (arrow), respectively.
Trang 4conductance channel formed by a1-PTH is an anionic
channel The Ca2+ selectivity of the channels was nil
Indeed, when the concentration of CaCl2was 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, n¼ 18) or sodium gluconate
(140 mm, n¼ 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 PNa⁄ PCl of 11 was calculated The low-conductance channels were almost equally selective to Na+ and K+ The PK⁄ PNa was
A
-60
-30
0
30
60
1 s
150 mV
50 mV
0 mV
-150 mV -50 mV
B
I ( pA)
V (mV)
-20 -15 -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) or 40 m M 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)
-4 -2
2 4 6 8
23 mV
V (mV)
I (pA)
-100 -50
50
100
C
Fig 4 Low-conductance channel activities exhibited by giant lipo-somes containing a1-PTH (A) Unitary current traces recorded in response to holding potentials varying from 0 to ± 80 in steps of
40 mV (B) Representative current–voltage relationship The calcula-ted unitary conductance was 51 and 35 pS at holding potentials of +100 mV and )100 mV, respectively (C) Representative currents recorded during potential ramps from )100 mV to +100 mV in the presence of either 140 or 40 m M NaCl in the bathing solution Under these conditions, the voltages corresponding to zero current were 0 and +23 mV (arrow), respectively.
Trang 51.10 ± 0.04 (n¼ 4), as calculated from changes in
the current reversal potential, e.g from 0 to
)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 CaCl2
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 mm CaCl2 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 CaCl2
con-centration was increased from 2.6 to 10 mm, and it
was )13.3 ± 0.4 mV (n ¼ 5) when the CaCl2
concen-tration was 20 mm Under these conditions, the
expec-ted equilibrium potential calculaexpec-ted for Ca2+ was
)17.3 mV and )26.2 mV for 10 and 20 mm CaCl2,
respectively A Ca2+ to Na+ permeability ratio
(PCa⁄ PNa) of 5 was calculated from the changes in the
measured reversal potential Thus, the relative ionic
permeability sequence for the cationic channel formed
by a1-PTH is Ca2+> Na+ K+ Cl–
Effects of a1-PTH and PIN-a on isolated mouse
neuromuscular preparations
The addition of a1-PTH (0.01–1 lm) to the
physiologi-cal medium bathing isolated preparations produced a
concentration-dependent decrease in muscle twitches
and tetanic responses evoked by nerve stimulation at
0.2 and 40 Hz, respectively (Fig 6A) The
concentra-tion of a1-PTH that reduced the contracconcentra-tion amplitude
by 50% was 0.16 lm (Fig 6B) Complete blockade of
nerve-evoked muscle twitches and tetanic responses
occurred with 1 lm a1-PTH (n¼ 10), and the
block-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
4 pA
0 pA
5 s
4 pA
0 pA
Control CaCl 2 (20mM in bath)
0 mV
min
A
V (mV)
I (pA)
-100 -50
50 100
-100 -50
50
100
I (pA)
V (mV)
B
C
Fig 5 Low-conductance channel selectivity for Ca2+ (A) Current traces recorded at a holding potential of 0 mV when the bath con-centration of CaCl2was increased from 2.6 m M (control) to 20 m M (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 m M (C) CaCl2in the bathing solution Under these conditions, the voltages corresponding to zero current were
0 (B) and )9 mV (C, arrow).
Trang 6maximal depolarization was 3.5 ± 0.8 min (n¼ 4).
The magnitude of the depolarization was independent
of the external CaCl2 concentration between 0 and
2 mm (Fig 7C) However, when a1-PTH was added to
the standard medium in which the CaCl2concentration
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
(n¼ 4)
Analysis of synaptic transmission at single
neuro-muscular junctions revealed that a1-PTH (0.05–1 lm)
produced a dose-dependent decrease in endplate
poten-tial (EPP) amplitude Thus, in the presence of 0.25 lm
a1-PTH, EPPs had a subthreshold amplitude, being
unable to reach the threshold for action potential
gen-eration in muscle fibres, and were almost completely
blocked by 1 lm a1-PTH (Fig 8A) Parallel recordings
of spontaneous miniature endplate potentials (MEPPs) showed that 0.5 lm a1-PTH increased the frequency of MEPPs Thus, MEPP frequency was 0.7 ± 0.2 s-1 (n¼ 12) in control conditions and 6.7 ± 0.4 s1(n¼ 6) after the addition of 0.5 lm a1-PTH In addition, a1-PTH caused a marked decrease in MEPP ampli-tude, which attained the basal noise level with 1 lm a1-PTH (Fig 8B) This precluded the recording of
A
B
Fig 6 Effects of a1-PTH and PIN-a on nerve-evoked and directly
elicited muscle twitches (A) Superimposed tracings of muscle
twit-ches evoked by nerve stimulation (left panel, 0.2 Hz), tetanic nerve
stimulation (middle panel, 40 Hz), and direct muscle stimulation
(right panel) Tracings were recorded before and after 20 min
expo-sure to a1-PTH (0.05–1 l M ) (B) Dose–response curves of the
effects of a1-PTH (circles) and PIN-a (squares) on nerve-evoked
(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 m M CaCl2) PIN-a (2 m M CaCl2)
R -90
-80 -70 -60 -50 -40 -30 -20 -10 0
A
Control α1- PTH (1 µ M ) -90
-80 -70 -60 -50 -40 -30 -20 -10 0
C CaCl2 concentration
0 -10 -20 -30 -40 -50 -60 -70 -80
0 1 2 3 4 5 6 7 29 30 31 32
Time (min)
1 µ M α1-PTH (2 m M CaCl2)
1 µ M α1-PTH (10 m M CaCl2)
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
0 m 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.
Trang 7MEPPs with concentrations higher than 0.5 lm
a1-PTH The above results indicate that a1-PTH,
within the range of concentrations studied, causes
per-meability changes in the presynaptic and postsynaptic
membranes of the neuromuscular junction In contrast,
MEPPs (Fig 8C) and EPPs were not significantly
affected by 1 lm PIN-a
Cytotoxic effects of a1-PTH and PIN-a on
neuroblastoma (NG108-15) cells
The effects of a1-PTH and PIN-a were studied on the
morphology of NG108-15 cells stained with the
fluor-escent dye FM1-43 and imaged with a confocal laser
scanning microscope (see Experimental procedures)
Images from each experiment were processed
identic-ally, and the effects were quantified using the same
cells examined before and during the action of the
proteins The 3D projected area of cells was measured
as an index of cell volume The addition of 10 lm a1-PTH to the standard mammalian physiological solution (e.g containing 2 mm CaCl2) produced, after
a latent period of about 15 min, a marked increase in the fluorescence intensity and a slight but significant (P¼ 0.0001) swelling of the cells (Fig 9Ab) Within 30–45 min, the 3D projected area of the cells reached a maximum increase of 128 ± 17% (n¼ 14), when com-pared with control values In addition, large membrane bleb formation followed by bleb dilation was consis-tently observed, and, in most cases, the blebs attained 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 CaCl2 -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 phalloidin staining to visualize F-actin (data not shown)
Discussion
In this work, the ion channels formed by PIN-a and a1-PTH reconstituted into giant liposomes were characterized, and their toxicity examined in murine isolated neuromuscular preparations and cultured NG108-15 cells To our knowledge, this is the first study to demonstrate that highly purified a1-PTH
0.5 mV
1.00 µ M
5 ms
C
0.25 µ M
0.00 µ M
20 mV
1.00 µ M α1-PTH
5 ms
A
B 0.00 µ M
0.5 mV
5 ms
Fig 8 Effects of a1-PTH and PIN-a on nerve-evoked action
potent-ial, EPPs and MEPPs recorded from isolated hemidiaphragm
mus-cles (A) Single action potential recorded before (left trace), and
EPPs recorded after, 20 min exposure to 0.25 l M (middle trace)
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 l M 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.
Trang 8forms 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
oocytes [16]
In the case of a1-PTH, two types of channels with
different conductance and selectivity were detected
One of them is an anion channel with a conductance
larger than 100 pS (high-conductance channel), which
is 7 times more permeable to Cl– than to Na+ Our
results constitute the first description of single-channel
anion currents induced by a1-PTH The
low-conduct-ance channel formed by a1-PTH is a cationic channel
with single unitary conductance of 30–45 pS, which is
11 times more permeable to Na+than to Cl–, and has
a permeability ratio between Na+and K+ close to 1
However, this channel is 5 times more permeable to
Ca2+ than to Na+ Thus, the cation selectivity order
for this channel is the following: Ca2+> 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
10 µm
10 µ M α1-PTH (0 m M CaCl2)
Control (0 m M CaCl2)
Control 10 µ M α1-PTH 10 µ M PIN-a
* P > 0.02
* * P< 0.002
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 M
(15 min)
100 µ M
(30 min)
100 µ M
(90 min) 0.0
0.2 0.4 0.6 0.8 1.0 1.2 c
B A
5 µm
Fig 9 Effects of a1-PTH and PIN-a on NG108-15 cells In (A) and
(B), the cells were stained with 2 l M FM1-43 dye for 30 min, and
thereafter abundantly washed with dye-free solution before imaging
and the addition of a1-PTH or PIN-a to the medium Cells imaged
before (Aa) and after (Ab) exposure to 10 l M a1-PTH in a Ca 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 concentratfunct-ion 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 l M PIN-a In (Cb), note the
distinct distribution and clumping of the immunolabelling.
Trang 9progressive 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,
further experiments are needed to support this model
In isolated mouse hemidiaphragm preparations,
a1-PTH completely blocked directly and indirectly
electrical muscle twitches As a1-PTH caused
mem-brane depolarization, voltage-dependent sodium
chan-nels must be inactivated and unable to generate action
potentials in muscle fibres a1-PTH effects can be
rela-ted to its predominant ability to form low-conductance
cationic-selective channels, as reported in liposomes
The fact that increasing external Ca2+prevented
mus-cle depolarization by a1-PTH is not surprising because
pore formation induced by PTHs and PINs is inhibited
by high Ca2+concentrations [14,16] Also, a1-PTH
eli-cited permeability changes that may depolarize nerve
terminals supplying the neuromuscular junction Such
an action would explain the decrease in evoked
trans-mitter release (revealed by the decrease in EPP
ampli-tudes) and the increased spontaneous quantal release
(manifested by an enhanced MEPP frequency) Thus,
both presynaptic and postsynaptic permeability
chan-ges co-operate to block neuromuscular transmission
and muscle contraction evoked by nerve stimulation
In contrast, no changes were detected with PIN-a,
except for hyperpolarization of the muscle membrane
when high concentrations were used This is expected
if one considers that PIN-a increases membrane
per-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 myelinated axons [15] This may be due to dye entry into cells via a1-PTH-formed channels, as previously reported for mechanotransducer channels [22] Another possibility is that the increased fluorescence intensity reflects changes in membrane potential, as FM1-43 has also been used as a voltage-sensitive dye [20] In this case, a 3.3% fluorescence increase per 100 mV poten-tial change would be expected This value is too low to account for the marked increase in FM1-43 fluores-cence intensity observed in NG108-15 cells FM1-43 has also been reported to be a useful probe for monit-oring phospholipid scrambling [23] Taking into account that one of the earliest detectable events in cells undergoing apoptosis is phospholipid scrambling [24], the FM1-43 fluorescence increase detected during a1-PTH action, together with membrane blebbing and disorganization and disarray of cytoskeletal actin, may represent apoptotic events involved in a1-PTH cyto-toxicity
Experimental procedures
Extraction and purification of PIN-a and a1-PTH
PIN-a (Mr 12 920) and a1-PTH (Mr 4921.89) were puri-fied from wheat seeds of Triticum aestivum sp., using a modification of previously described procedures [18,25] Briefly, 4 kg wheat endosperm flour was extracted with a 10-L solution containing 100 mm Tris buffer, 100 mm NaCl, 5 mm EDTA and 5% Triton X-114 (pH 7.8) After stirring (12 h, 4C) and centrifugation (8000 g, 30 min),
the supernatants were heated at 30C 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
Trang 10S 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
et al [27]
Reconstitution of PIN-a and a1-PTH into giant
liposomes
Giant liposomes were prepared by subjecting a mixture
(2 mL) of the protein (100 lg either PIN-a or a1-PTH) and
asolectin lipid vesicles (13 mm, in terms of lipid
phospho-rus) to a partial dehydration⁄ rehydration cycle, as reported
previously [28] After the partial dehydration⁄ rehydration
cycle, the diameter of the resulting giant multilamellar
lipo-somes ranged from 5 to 100 lm
Patch-clamp measurements
Aliquots (3–15 lL) of giant liposome preparations, in Petri
dishes (3.5 cm diameter), were mixed with 1 mL of the
buf-fer of choice (the bath solution) for electrical recording,
and unitary current recordings were performed using the
patch-clamp technique in an excised patch ‘inside out’
con-figuration, as previously described [29] Giga seals were
formed on giant liposomes with glass microelectrodes of 5–
10 MW resistance After sealing, withdrawal of the pipette
from the liposome surface resulted in an excised patch
Current was recorded with an EPC-9 patch-clamp amplifier
(Heka Elektronic, Lambrecht⁄ Pfalzt, Germany) at a gain of
50–100 mVÆpA)1and a filter setting of 10 kHz The holding
potential was applied to the interior of the patch pipette
The bath potential was maintained at virtual ground via an
agar bridge (V¼ Vbath) Vpipette), 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
CaCl2, 1.3 mm MgCl2, 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
and chemicals were purchased from Sigma Biochemical Co
(St Louis, MO, USA) and Merck (Darmstadt, Germany)
Electrophysiological and mechanical recordings
in isolated mouse hemidiaphragm muscles
Left and right hemidiaphragm muscles with their associated phrenic nerves were isolated from adult Swiss-Webster mice (20–25 g) killed by cervical vertebrae dislocation followed by immediate exsanguination Phrenic nerve⁄ hemidiaphragm muscle preparations were mounted in a Rhodorsil-lined (Rhoˆne-Poulenc, St Fons, France) Plexiglas chamber (2-mL
or 4-mL capacity) and bathed in a standard physiological solution gassed with pure O2 and composed of 154 mm NaCl, 5 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 11 mm glucose, and 5 mm Hepes (adjusted to pH 7.4 with NaOH) Lyophilized PIN-a and a1-PTH were dissolved in 100 mm Hepes buffer and stored as 1-mm stock solutions at)18 C The stock solutions were diluted with the standard physiolo-gical solution before experiments were performed at room temperature All experiments on mice were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC), regarding the ethical use of animals for experimental procedures
Membrane potentials and synaptic potentials were recor-ded with intracellular microelectrodes filled with 3 m KCl (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