Puroindoline-a and a1-purothionin form ion channels in giant liposomes but exert different toxic actions on murine cells Paola Llanos1, Mauricio Henriquez1, Jasmina Minic2, Khalil Elmorjani3, Didier Marion3, ´ Gloria Riquelme1, Jordi Molgo2 and Evelyne Benoit2 ´ Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile ´ Laboratoire de Neurobiologie Cellulaire et Moleculaire, UPR 9040, Centre National de la Recherche Scientifique, Gif-sur-Yvette cedex, France ` Biopolymeres Interactions Assemblages, Institut National de la Recherche Agronomique, Nantes, France Keywords a1-purothionin; giant liposomes; ion channels; neuromuscular transmission; puroindoline-a Correspondence E Benoit, Laboratoire de Neurobiologie ´ Cellulaire et Moleculaire, UPR 9040, Centre ˆ National de la Recherche Scientifique, bat 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) 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 investigated 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 membrane depolarization, leading to blockade of synaptic transmission and directly elicited muscle twitches Also, a1-PTH caused swelling of differentiated neuroblastoma NG108-15 cells, membrane bleb formation, and disorganization 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 doi:10.1111/j.1742-4658.2006.05185.x 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 tryptophan-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 differ 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 lipidbinding 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 transgenic rice plants expressing genes encoding PINs (pinA and ⁄ or pinB) reduce in vitro the growth of rice fungal Abbreviations EPP, endplate potential; LTP, lipid-transfer protein; MEPP, miniature endplate potential; PIN, puroindoline; PTH, purothionin 1710 FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS P Llanos et al pathogens [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 maintenance 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 hippocampal neurons [14] PIN-a and a1-PTH have also been reported to swell the nodes of Ranvier of frog myelinated axons, and pore formation in the nodal membrane 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 Therefore, as a first step toward understanding these mechanisms, 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] Toxic actions of puroindoline-a and a1-purothionin DVA-(PIN)-GT 12,750 DVA-(PIN)-GTIG 12,919 A VA-(PIN)-GTIG 12,803.05 DVA-(PIN)-GTIGY 13,083.6 VA-(PIN)-GT 12,634.7 12,200 12,600 13,000 13,400 13,800 B α1-PTH 4,921 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 preparations of giant liposomes containing PIN-a Fortyfive 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 patches ( 78%) The unitary level was difficult to observe in isolation, which suggested the presence of 4,700 4,800 4,900 5,000 5,100 5,200 Fig MALDI-TOF mass spectrum of PIN-a and a1-PTH Deconvoluted and reconstructed electrospray mass spectra from multicharged ion spectra of the purified PIN-a (A) and a1-PTH (B) Note the homogeneity of the protein preparations substrates or clustering of channels in the patch However, 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 channels were present in the patch, as judged by the number of simultaneous unitary current steps and histogram peaks A unitary conductance of 14.8 ± 0.6 pS (n ¼ 17) was determined, between )80 and +80 mV, from the slope of the current–voltage relationship (Fig 2C) FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS 1711 Toxic actions of puroindoline-a and a1-purothionin B 1.5 Time % A P Llanos et al pA 5s pA 500 ms I (pA) pA 500 ms D 1.5 C 100 I (pA) I (pA) 1.0 50 0.5 -100 -50 50 -0.5 100 -100 50 -50 V (mV) -50 -1.0 -100 -1.5 The selectivity of the channels for Na+ vs Cl– was determined by increasing or decreasing the NaCl concentration 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 recorded in response to potential ramps shifted from to )25 ± 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 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 relationship, 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– 1712 100 V (mV) Fig Ion-channel activities exhibited by giant liposomes containing PIN-a (A) Unitary current traces recorded at a holding potential of +100 mV The zero current level is indicated by the dotted line, and channel openings are indicated by upward deflections 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 percentage 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 mM NaCl in the bathing solution Under these conditions, the voltages corresponding to zero current were and )24.3 mV (arrow), respectively 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 patches Single channels with a high unitary conductance and single channels with a low unitary conductance were detected in 32% (n ¼ 10) and 68% (n ¼ 21) of the recordings, respectively In two independent experiments, low-conductance and high-conductance openings 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-conductance 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, respectively 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 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- FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS P Llanos et al Toxic actions of puroindoline-a and a1-purothionin A A 150 mV 60 80 mV 20 40 mV 30 mV 50 mV -40 mV mV -50 mV -80 mV -20 -150 mV -30 1s B -60 I ( pA) 1s I (pA) 25 B I ( pA) 20 15 -150 10 -100 -50 -150 -100 100 150 V (mV) -4 50 -5 50 -2 -20 mV 100 150 C 100 V (mV) I (pA) -10 50 -15 -20 -100 Fig High-conductance channel activities exhibited by giant liposomes containing a1-PTH (A) Unitary current traces recorded at the indicated holding potentials (B) Current–voltage relationships in the presence of either 140 mM NaCl (s) or 40 mM NaCl (d) in the bathing solution Under these conditions, the voltages corresponding to zero current were and )20 mV (arrow), respectively conductance channel formed by a1-PTH is an anionic channel The Ca2+ selectivity of the channels was nil Indeed, when the concentration of CaCl2 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 to ± 80 mV in steps of 40 mV (Fig 4A) Unitary conductances of 46 ± and 34 ± pS were calculated from current potential relationships at holding potentials of +100 and )100 mV, respectively (Fig 4B), observed during 21 experiments using symmetrical NaCl (140 mm, n ¼ 18) or sodium gluconate (140 mm, n ¼ 3) concentrations When the NaCl -50 50 100 V (mV) -50 23 mV -100 Fig Low-conductance channel activities exhibited by giant liposomes containing a1-PTH (A) Unitary current traces recorded in response to holding potentials varying from to ± 80 in steps of 40 mV (B) Representative current–voltage relationship The calculated 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 mM NaCl in the bathing solution Under these conditions, the voltages corresponding to zero current were and +23 mV (arrow), respectively 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 to +23 mV (Fig 4C), which is close to the Na+ equilibrium 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 FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS 1713 Toxic actions of puroindoline-a and a1-purothionin P Llanos et al 1.10 ± 0.04 (n ¼ 4), as calculated from changes in the current reversal potential, e.g from to )2.5 ± 1.1 mV (n ¼ 4), brought about by replacing NaCl (140 mm) with KCl (140 mm) in the bath solution 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 mV, in the presence of 2.6 mm (control conditions) and 20 mm CaCl2 In response to potential ramps, the reversal potential shifted from (Fig 5B) to )8.0 ± 0.8 mV (n ¼ 4, Fig 5C) when the CaCl2 concentration was increased from 2.6 to 10 mm, and it was )13.3 ± 0.4 mV (n ¼ 5) when the CaCl2 concentration was 20 mm Under these conditions, the expected equilibrium potential calculated 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 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– A Control CaCl2 (20mM in bath) 10 30 60 pA mV 5s pA pA 100 B I (pA) 50 -100 -50 50 100 V (mV) -50 -100 100 Effects of a1-PTH and PIN-a on isolated mouse neuromuscular preparations 1714 50 pA C The addition of a1-PTH (0.01–1 lm) to the physiological 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 concentration of a1-PTH that reduced the contraction amplitude by 50% was 0.16 lm (Fig 6B) Complete blockade of nerve-evoked muscle twitches and tetanic responses occurred with lm a1-PTH (n ¼ 10), and the blockade 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 ⁄ hemidiaphragm 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 significant changes were detected in the contraction amplitude (Fig 6B) Membrane permeability changes caused by the poreforming ability of a1-PTH may explain the above effects Therefore, we performed intracellular recordings to measure the effect of a1-PTH and PIN-a on the resting membrane potential of mouse hemidia- 40 I (pA) 50 -100 50 100 V (mV) -50 CaCl2 (10mM in bath) -100 Fig Low-conductance channel selectivity for Ca2+ (A) Current traces recorded at a holding potential of mV when the bath concentration of CaCl2 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) Representative currents recorded during the same experiment in response to potential ramps from )100 mV to +100 mV in the presence of either 2.6 mM (B) or 10 mM (C) CaCl2 in the bathing solution Under these conditions, the voltages corresponding to zero current were (B) and )9 mV (C, arrow) 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 lm a1-PTH-induced depolarization of skeletal muscle fibres is shown in Fig 7B The time required by a1-PTH to exert half- FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS Toxic actions of puroindoline-a and a1-purothionin A B Protein concentration A Resting membrane potential (mV) P Llanos et al -10 -20 -30 -40 -50 -60 -70 -80 -90 maximal depolarization was 3.5 ± 0.8 (n ¼ 4) The magnitude of the depolarization was independent of the external CaCl2 concentration between and mm (Fig 7C) However, when a1-PTH was added to the standard medium in which the CaCl2 concentration was raised from mm to 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 concentration (10 lm), the protein hyperpolarized the muscle membrane by 21 ± 2.5 mV within about (n ¼ 4) Analysis of synaptic transmission at single neuromuscular junctions revealed that a1-PTH (0.05–1 lm) produced a dose-dependent decrease in endplate potential (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 generation in muscle fibres, and were almost completely -10 -20 -30 -40 -50 -60 -70 -80 Time (min) // 29 30 31 32 // // µM α1-PTH (2 mM CaCl2) µM α1-PTH (10 mM CaCl2) CaCl2 concentration C Resting membrane potential (mV) Fig Effects of a1-PTH and PIN-a on nerve-evoked and directly elicited muscle twitches (A) Superimposed tracings of muscle twitches 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 exposure to a1-PTH (0.05–1 lM) (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 lM a1-PTH, and the quasi-absence of effect of similar concentrations of PIN-a Resting membrane potential (mV) B α1-PTH (2 mM CaCl 2) PIN-a (2 mM CaCl 2) -10 -20 -30 -40 -50 -60 -70 -80 -90 Control α1- PTH (1 µM) Fig Effects of a1-PTH and PIN-a on the resting membrane potential of skeletal muscle fibres (A), and the influence of extracellular Ca2+ 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 Ca2+ concentration (from to 10 mM) markedly reduces a1-PTH-induced muscle depolarization, whereas decreasing extracellular Ca2+ concentration (from to mM) has no significant effect In (A) and (C), each column represents the mean ± SEM obtained from 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 blocked by 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 (n ¼ 12) in control conditions and 6.7 ± 0.4 s (n ¼ 6) after the addition of 0.5 lm a1-PTH In addition, a1-PTH caused a marked decrease in MEPP amplitude, which attained the basal noise level with lm a1-PTH (Fig 8B) This precluded the recording of FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS 1715 Toxic actions of puroindoline-a and a1-purothionin A 0.00 µM 0.25 µM 1.00 µM P Llanos et al α1-PTH 20 mV ms B 0.00 µM 0.50 µM 1.00 µM α1-PTH 0.5 mV ms C 0.00 µM 1.00 µM PIN-a 0.5 mV ms Fig Effects of a1-PTH and PIN-a on nerve-evoked action potential, EPPs and MEPPs recorded from isolated hemidiaphragm muscles (A) Single action potential recorded before (left trace), and EPPs recorded after, 20 exposure to 0.25 lM (middle trace) and lM (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 exposure to 0.5 lM (middle trace) and lM a1-PTH (right trace) (C) Average of 30 sequential MEPPs recorded before (left trace) and after 20 exposure to lM 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 MEPPs with concentrations higher than 0.5 lm a1-PTH The above results indicate that a1-PTH, within the range of concentrations studied, causes permeability changes in the presynaptic and postsynaptic membranes of the neuromuscular junction In contrast, MEPPs (Fig 8C) and EPPs were not significantly affected by 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 fluorescent dye FM1-43 and imaged with a confocal laser scanning microscope (see Experimental procedures) Images from each experiment were processed identically, and the effects were quantified using the same cells examined before and during the action of the 1716 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 mm CaCl2) produced, after a latent period of about 15 min, a marked increase in the fluorescence intensity and a slight but significant (P ¼ 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% (n ¼ 14), when compared with control values In addition, large membrane bleb formation followed by bleb dilation was consistently observed, and, in most cases, the blebs attained the size of the cells A similar increase in both fluorescence intensity and 3D projected area occurred when the cells were exposed to 10 lm a1-PTH in a CaCl2free 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) produced 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 PINa, prompted us to determine whether the two proteins 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 filamentous actin immunolabelling distribution were observed in cells treated with a1-PTH (Fig 9C) Disorganization 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 FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS P Llanos et al Fig Effects of a1-PTH and PIN-a on NG108-15 cells In (A) and (B), the cells were stained with lM 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 lM a1-PTH in a Ca2+-containing 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 lM a1-PTH in a Ca2+-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 lM PIN-a exposure to a Ca2+-containing medium In (Bc), the bars indicate the relative projected area of the cells as a function of PIN-a concentration and time of exposure (C) Immunostaining of actin under control conditions (Ca) and after exposure of the cells to either 10 lM a1-PTH (Cb) or 10 lM PIN-a In (Cb), note the distinct distribution and clumping of the immunolabelling Toxic actions of puroindoline-a and a1-purothionin A a b c d 10 µm 10 µM α1-PTH (0 mM CaCl 2) Control (0 mM CaCl2) B a b 10 µm Control 50 µM PIN-a * P > 0.02 * * P< 0.002 c Relative area of NG108-15 cells forms ion channels in biological and artificial membranes In addition, we found that PIN-a forms a cation-selective channel with a 15 pS conductance This channel is 13 times more permeable to a univalent cation (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 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-conductance 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 However, this channel is 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 previous observations suggesting that PTHs form cationselective ion channels, and in particular with data obtained with b-PTH showing the formation of cationselective ion channels in artificial lipid bilayer membranes and in the plasmalemma of rat hippocampal neurons [14] Although a concentration-dependent study of a1PTH has not been performed, it is possible that the two different channel behaviours detected are the consequence of protein–protein interactions in the reconstituted system, leading to channel clustering or similar processes Thus, it is likely that progressive recruitment 1.2 1.0 0.8 12 20 * * 16 16 ** ** 20 34 ** ** 0.6 21 ** 0.4 0.2 0.0 50 µM 100 µM 100 µM 100 µM 10 µM 50 µM 50 µM (90 min) (30min) (60 min) (120 min) (15 min) (30 min) (90 min) Concentration (time of application) C a c b µm Control 10 µM α1-PTH 10 µM PIN-a of additional monomers will contribute to increase the pore size The formation of transmembrane channels ⁄ pores by bundles of amphipathic a-helices of a1-PTH and PIN-a polypeptides may occur via a ‘barrel-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, FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS 1717 Toxic actions of puroindoline-a and a1-purothionin P Llanos et al 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 anionic-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 membrane depolarization, voltage-dependent sodium channels must be inactivated and unable to generate action potentials in muscle fibres a1-PTH effects can be related to its predominant ability to form low-conductance cationic-selective channels, as reported in liposomes The fact that increasing external Ca2+ prevented muscle 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 elicited permeability changes that may depolarize nerve terminals supplying the neuromuscular junction Such an action would explain the decrease in evoked transmitter release (revealed by the decrease in EPP amplitudes) and the increased spontaneous quantal release (manifested by an enhanced MEPP frequency) Thus, both presynaptic and postsynaptic permeability changes 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 permeability 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 following the 3D projected area of the cells The development of blebs in the presence of a1-PTH is probably related to the membrane permeability changes it induces, as, in other neuronal cells, blebbing has been associated with raised intracellular Na+ concentration [21] Also, a1-PTH-treated NG108-15 cells exhibited an 1718 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 potential change would be expected This value is too low to account for the marked increase in FM1-43 fluorescence intensity observed in NG108-15 cells FM1-43 has also been reported to be a useful probe for monitoring 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 cytotoxicity Experimental procedures Extraction and purification of PIN-a and a1-PTH PIN-a (Mr 12 920) and a1-PTH (Mr 4921.89) were purified from wheat seeds of Triticum aestivum sp., using a modification of previously described procedures [18,25] Briefly, kg wheat endosperm flour was extracted with a 10-L solution containing 100 mm Tris buffer, 100 mm NaCl, mm EDTA and 5% Triton X-114 (pH 7.8) After stirring (12 h, °C) and centrifugation (8000 g, 30 min), the supernatants were heated at 30 °C to allow phase partitioning, and the upper, detergent-poor phase was discarded The lower, detergent-rich phase was diluted with vol water and loaded on a column packed with a cation exchanger (SP Biobeads; Pharmacia, Montigny-le-Bretonneux, 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 deionized water and freeze-dried, and a1-PTH, a2-PTH and b-PTH were separated (at room temperature) by semipreparative 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 deionized water to 0.1% trifluoroacetic acid in acetonitrile), and the fractions containing a1-PTH were pooled and freezedried after dilution with deionized water PIN-a was purified from the crude, freeze-dried, PIN-containing fraction by cation-exchange chromatography on a mL Resource FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS P Llanos et al S column (Pharmacia), as previously described [26] The homogeneity of the purified a1-PTH and PIN-a preparations 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 phosphorus) to a partial dehydration ⁄ rehydration cycle, as reported previously [28] After the partial dehydration ⁄ rehydration cycle, the diameter of the resulting giant multilamellar liposomes ranged from to 100 lm Patch-clamp measurements Aliquots (3–15 lL) of giant liposome preparations, in Petri dishes (3.5 cm diameter), were mixed with mL of the buffer 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’ configuration, 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 mVỈpA)1 and 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 (V ¼ Vbath ) Vpipette), and the junction potential was compensated for when necessary To study the ionic selectivity of the protein-induced channels, we determined 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 mVỈs)1) or +100 to )100 mV (40 mVỈs)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 calculated as previously described [32,33] Patch-clamp data were analyzed off-line with TAC software (Bruxton Corporation, Seattle, WA, USA) and Pulse Fit (Heka Elektronic) software All measurements were made at  25 °C, and the pipette and bath solutions usually 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) Toxic actions of puroindoline-a and a1-purothionin 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 RhodorsilÒ-lined (Rhone-Poulenc, St Fons, France) Plexiglas chamber (2-mL ˆ or 4-mL capacity) and bathed in a standard physiological solution gassed with pure O2 and composed of 154 mm NaCl, mm KCl, mm CaCl2, mm MgCl2, 11 mm glucose, and 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 physiological 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 recorded with intracellular microelectrodes filled with 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 proteins 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 transducer (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 microelectrode 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 stimulator (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 FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS 1719 Toxic actions of puroindoline-a and a1-purothionin P Llanos et al modified Eagle’s medium supplemented with 5% fetal bovine serum, 100 lm hypoxanthine, 0.4 lm aminopterin, 16 lm thymidine, mm glutamine, and lm glycine, as described previously [34] Three days before the experiments, cells were differentiated by adding 0.5 mm dibutyryl-cAMP to the medium and reducing the serum concentration to 1% Culture reagents were purchased from Sigma-Aldrich Chimie (Saint Quentin-Fallavier, France) and Invitrogen (Cergy Pontoise, France) The cultures were maintained at 37 °C in a humidified atmosphere containing 95% air ⁄ 5% CO2 Plasma membrane staining of neuroblastoma cells Before imaging, NG108-15 cells were rinsed free of the culture medium, and their plasma membrane was stained (30 min, room temperature) with the styryl dye N-[3-(triethylammonium)propyl]-4-(4-dibutylaminostyryl pyridinium) dibromide (FM1-43; Molecular Probes Europe Bv, Leiden, the Netherlands; lm) dissolved in standard physiological solution Thereafter, cells were rinsed with dye-free solution The dry extracts of the two wheat endosperm proteins were dissolved as described above, and added to the bathing solution during the experiments Actin immunostaining of neuroblastoma cells The culture medium of NG108-15 cells was added with either PIN-a (10 lm) or a1-PTH (10 lm), and cells were incubated for 2–4 h at 37 °C Then, they were rinsed with phosphate-buffered saline (NaCl ⁄ Pi) and fixed with either 4% paraformaldehyde in NaCl ⁄ Pi (15 min, 37 °C) or with 100% methanol (4 min, )20 °C) After being washed three times with NaCl ⁄ Pi to remove excess fixative, the cells were permeabilized and blocked (l h, room temperature) with NaCl ⁄ Pi 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 ⁄ Pi, and incubated (1 h, room temperature) with Texas-red-conjugated secondary antibodies (1 : 100 dilution; Molecular Probes) Coverslips were mounted on to glass slides with Vectashield antifading mounting medium (Vector Laboratories, Inc, Burlingame, CA, USA) In some experiments, AlexaFluor594-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 Sarastro-2000 confocal system (Molecular Dynamics, Sunnyvale, CA, USA) mounted on a Nikon Optiphot-2 upright 1720 microscope The system was controlled with imagespace 3.10 software and a Silicon Graphics Personal Iris 4D ⁄ 35G workstation (Mountain View, CA, USA) The 488 and 514 nm lines of an argon-ion laser were used for fluorescence excitation of FM1-43 and Texas-red or AlexaFluor-594, respectively Images were collected at room temperature using a water-immersion lens (· 40; numerical aperture ¼ 0.75), and the aperture setting of the confocal pinhole was 100 lm A 3% neutral density transmission filter was used in all experiments, and the photomultiplier gain was kept constant during experiments Series of optical sections were collected using a standard scanning mode format of 512 · 512 pixels, and ‘look through’ projections were constructed from the sections Statistical analysis Values in the text are expressed as means ± SEM of n experiments Statistical analysis of data was performed using Student’s t test (two-tailed) Data were considered significant at P < 0.05 Acknowledgements This work was made possible by the ECOS Sud-CONICYT (C03S02) exchange program, and was supported in part by Fondecyt-Chile (grant No 1040546), the Centre National de la Recherche Scientifique and the Institut National de la Recherche Agronomique J.M was supported by the Fondation pour la Recherche ´ Medicale We thank Dr M Malo and Dr B RouzaireDubois 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 References Douliez JP, Michon T, Elmorjani K & Marion D (2000) Structure, biological and technological functions of lipid transfer proteins and indolines, the major lipid binding proteins from cereal kernels J Cereal Sci 32, 1–20 Tanchak MA, Schernthaner JP, 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puroindoline-a and a1-purothionin P Llanos et al progressive recruitment of monomers would increase the pore size Under our conditions, once the cationic... 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 preparations of giant liposomes