Báo cáo khoa học: Puroindoline-a and a1-purothionin form ion channels in giant liposomes but exert different toxic actions on murine cells

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 1 Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile 2 Laboratoire de Neurobiologie Cellulaire et Moleculaire, UPR 9040, Centre National de la Recherche Scientifique, Gif-sur-Yvette cedex, France 3 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 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) 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. 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 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- lipid-transfer proteins (LTPs), puroindolines (PINs) binding properties. Indeed, whereas LTPs bind lipid 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 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 of LTPs and PINs are closely related and rich in especially because they enhance the antimicrobial 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 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 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. Toxic actions of puroindoline-a and a1-purothionin 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 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- A DVA-(PIN)-GT 12,750 DVA-(PIN)-GTIG 12,919 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- VA-(PIN)-GTIG 12,803.05 VA-(PIN)-GT 12,634.7 DVA-(PIN)-GTIGY 13,083.6 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]. 12,200 12,600 13,000 13,400 13,800 B a1-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 4,700 4,800 4,900 5,000 5,100 5,200 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. 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 Seals of high-resistance and excised patches in an recorded at a holding potential of +100 mV are ‘inside out’ configuration were obtained from 19 prep- shown in Fig. 2A, and the corresponding current arations of giant liposomes containing PIN-a. Forty-five of 72 patches studied ( 60%) exhibited channel amplitude distribution is depicted in Fig. 2B. Six chan-nels were present in the patch, as judged by the num- activity. Usually, multiple current levels corresponding ber of simultaneous unitary current steps and to a similar conductance were observed, and at least two distinct levels were detected in 35 of the 45 pat- histogram peaks. A unitary conductance of 14.8 ± 0.6 pS (n ¼ 17) was determined, between )80 and ches ( 78%). The unitary level was difficult to +80 mV, from the slope of the current–voltage rela-observe in isolation, which suggested the presence of tionship (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 P. Llanos et al. A B 1.5 1 pA 5 s 1 pA 500 ms 0 2 4 I (pA) 6 1 pA 500 ms 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 C 1.5 D 100 I (pA) I (pA) 1.0 50 0.5 (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) -100 -50 50 100 V (mV) -0.5 -1.0 -100 -50 -50 50 100 Representative currents recorded during V (mV) potential ramps from )100 to +100 mV in the presence of either 140 or 440 mM NaCl in the bathing solution. Under these condit- ions, the voltages corresponding to zero current were 0 and )24.3 mV (arrow), -1.5 -100 respectively. The 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 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% (n ¼ 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 current recorded in response to a potential ramp different holding potentials in the presence of 140 mm showed an almost linear current–voltage relation- and 40 mm NaCl in the bath solution. When the bath 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–. 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- 1712 FEBS Journal 273 (2006) 1710–1722 ª 2006 The Authors Journal compilation ª 2006 FEBS P. Llanos et al. A 60 A 150 mV 20 Toxic actions of puroindoline-a and a1-purothionin 80 mV 40 mV 30 50 mV 0 0 mV 0 0 mV -40 mV -50 mV -20 -80 mV -30 -150 mV 1 s -60 I (pA) B B 1 s I (pA) 25 I (pA) 20 15 8 6 4 2 -20 mV 10 5 -150 -100 -50 -2 -4 50 100 150 V (mV) -150 -100 -5 -10 -15 50 100 150 C 100 V (mV) I (pA) 50 -20 Fig. 3. High-conductance channel activities exhibited by giant lipo- -100 -50 50 100 somes containing a1-PTH. (A) Unitary current traces recorded at V (mV) the indicated holding potentials. (B) Current–voltage relationships in -50 the presence of either 140 mM NaCl (s) or 40 mM NaCl (d) in the 23 mV bathing solution. Under these conditions, the voltages correspond- ing to zero current were 0 and )20 mV (arrow), respectively. -100 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 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 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 mM NaCl in the bathing solution. Under these conditions, the voltages corresponding to zero current were 0 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 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 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 0 to )2.5 ± 1.1 mV (n ¼ 4), brought about by replacing A Control 0 10 CaCl2 (20mM in bath) 30 40 50 60 min 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 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 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 4 pA 0 mV 0 pA 5 s 4 pA 0 pA B 100 I (pA) 50 -100 -50 50 100 V (mV) -50 -100 C 100 I (pA) 50 concentration-dependent decrease in muscle twitches -100 50 100 and tetanic responses evoked by nerve stimulation at V (mV) 0.2 and 40 Hz, respectively (Fig. 6A). The concentra- -50 tion 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 CaCl (10mM in bath) -100 occurred with 1 lm a1-PTH (n ¼ 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). ... - tailieumienphi.vn