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Cation channel formed at lipid bilayer by Cinnamomin, a new type II ribosome-inactivating protein
PERGAMON
Toxicon 37 (1999) 1313±1322
Cation channel formed at lipid bilayer by Cinnamomin, a new type II ribosomeinactivating protein Guang-ping Zhang a, Yu-liang Shi a, *, Wen-ping Wang a, Wang-yi Liu b a
Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China b Shanghai Institute of Biochemistry, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China
Received 14 September 1998; accepted 16 December 1998 Abstract Cinnamomin, a new type II ribosome-inactivating protein, puri®ed from the seeds of Cinnamonum camphora is reconstituted into the membranes of planar lipid bilayer and giant liposome. The channel-forming activity of the cinnamomin is found and cation permeability of the channel is characterized by patch clamp. In an asymmetric solution system, bath 150/ pipette 100 mM KCl, the unit conductance is 140 2 7 pS and the reversal potential is 10.4 2 0.6 mV, very close to the theoretical value of the K + electrode. The results oer an interpretation for internalization of the RIP and the cytotoxicity dierence between single and two chain RIP. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Ribosome-inactivating protein; Cation channel; Cinnamomin; Lipid bilayer; Giant liposome
Abbreviations: RIP, ribosome-inactivating protein, CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Hepes, (N-[2-hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid]), NMDG, Nmethyl-D-glucamine. * Corresponding author. Unit of Bioactive Substances and Ion Channels, Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China. Tel.: +86-21-6437-0080; Fax: +86-21-6433-2445; E-mail: [email protected] 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 0 7 8 - 1
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1. Introduction Plant ribosome-inactivating proteins (RIPs) are a group of ribotoxins. They are RNA N-glycosidases which arrest protein synthesis by selectively hydrolyzing the N-glycosidic bond of a conserved adenosine in the largest ribosomal RNA (Stirpe, 1982; Stirpe and Barbieri, 1986; Stirpe et al., 1992; Barbieri et al., 1993). According to chemical structure, RIPs are divided into two groups. Type I is composed of a single chain with the molecular weight between 11,000±30,000 Da, while type II has a molecular weight of around 60,000 Da and consists of two chains (A- and B-chain) (Stirpe, 1982; Barbieri et al., 1993). There are considerable dierences between the two types of RIPs in the toxicity to both integral cell and cell-free translation systems. Type I markedly inhibits protein synthesis in cell-free translation systems and seldom aects on integral cell. Type II has strong cytotoxicity to the integral cell and a low toxic eect on the cell-free translation system (Stirpe et al., 1983; Bolognesi et al., 1990). The A-chain of type II acts on ribosome in the same way as type I RIP does. The B-chains are a lectin, binding to the receptors on the cell surface and allowing entry of the ribotoxin (Barbieri et al., 1993). Cinnamomin (cin) is a new type II RIP puri®ed from the seeds of Cinnamomum camphora. Its A-chain has the RNA N-glycosidase activity and the intact cin expresses the inhibitory eects in culture carcinoma cells (Ling et al., 1995; Ling and Liu, 1996; Li et al., 1997). In this work, in order to elucidate its transmembrane mechanism, Cinnamomin was reconstituted into the membranes of the planar lipid bilayer and giant liposome. Its channel-forming activity is observed by the patch clamp technique. The results oer an interpretation for the internalization of type II RIP and the toxicity dierences between two types of RIPs.
2. Materials and methods 2.1. Fusing Cinnamomin into planar lipid bilayer In the initial experiment, the interaction between Cinnamomin and the bilayer membrane was determined by using a two-compartment system (Finkelstein, 1974). As described previously, the lipid bilayer was formed by painting an Ndecane suspension of lecithin and cholesterol (20 and 5 mg/ml, respectively) over a 500 mm ori®ce in a Te¯on partition separating the two 3 ml solution-®lled compartments (cis and trans compartments) (Shi et al., 1992; Zhang and Shi, 1994; Ma and Shi, 1997; Chan et al., 1997). Only the stable bilayers, no channellike noise in 30 min under holding potentials of 2100 mV, were used for experiments. The reconstitution was achieved by adding 2±3 ml Cinnamomin to the cis-compartment (®nal protein concentration 1 mg/ml) and stirring for a minute. The salt concentration between the two compartments excised an osmotic
G. Zhang et al. / Toxicon 37 (1999) 1313±1322
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gradient (cis 200/trans 100 mM KCl), which was used to promote the protein fusion, the signals were picked up by a pair of calomel electrodes connecting the two compartments to the inputs of the patch clamp ampli®er (CEZ-2300, Nihon Kohden, Tokyo, Japan) in voltage-clamping model. The output signals were monitored on an oscilloscope, displayed on a chart recorder and simultaneously recorded on videotape with a video cassette recorder (VCR-N85, NEC, Tokyo, Japan) via an audio pulse code modulator (PCM) processor (Modi®ed 501 ES, Sony, Osaka, Japan) for further analysis. The current was de®ned as being positive when cations ¯owed into the trans side. All measurements were made at room temperature (0228C). 2.2. Reconstituting Cinnamomin into a giant liposome The formation procedure of the giant liposome used in this study was a modi®cation of that previously described by Keller et al. (1988). Brie¯y, 100 mg lecithin from soybean (type 2-S, Sigma) was suspended in 1 ml of distilled water by sonication in Branson soni®er at 40 W for 5±10 min under nitrogen. The lipid suspension was further diluted 10-fold with dialysis solution (in mM: 10 Hepes; 100 NaCl, pH 7.4), then the Cinnamomin and CHAPS were added at ®nal concentrations of 10 ng/ml and 10 mM, respectively. The mixture sample was dialyzed against 600 volumes of dialysis solution for 70 h. The dialyzed sample (02.5 ml) was ultra-centrifuged at 40,000 rpm for 1 h and the pellet was resuspended with 200 ml Hepes buer containing 5% ethylene glycol, pH 7.4. The resuspension sample was deposited to a clean glass slide in 15 ml per aliquot and was submitted to partial dehydration (3±6 h) in a desiccator containing anhydrous CaCl2, then stored in a refrigerator. Before use, the sample was rehydrated by dropping 15 ml of 100 mM KCl solution to cover the dehydrated sample on slide and leaving the slide in a closed petri dish with a wet paper pad on its bottom overnight. Next day, the giant liposome could be observed. The preparation of the control giant liposome was the same as mentioned above, but only without adding Cinnamomin. All the operations were performed at 48C. 2.3. Patch clamping giant liposome and recording channel activity An aliquot of the giant liposome solution (010 ml) was mixed with 200 ml of a DEAE-Sephedex A-50 suspension (3 mg/ml in the dialysis solution) in a small dish and incubated at room temperature for 15±30 min. This treatment anchored the liposome to the gel beads and to the bottom of the dish, making it easier to get a giga-seal (Riquelme et al., 1990). After gigaohm seal (10±100 GO) formation between the giant liposome and the patch pipette, the pipette was withdrawn from the liposome surface. This resulted in an excised patch. Single channel recordings were obtained by using the patch-clamp technique as described by Hamill et al. (1981). The current was de®ned as positive when cation ¯owed out of the pipette into the bath. The signal was ampli®ed through an EPC-7 patch clamp ampli®er
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(List Medical Electronics), then recorded and stored on videotape via the audio PCM processor. The current signal was monitored simultaneously on an oscilloscope. The stored records were transferred, using an analog-to-digital interface (Digidata 1200, Axon Instrument Co.) and the pClamp 6.02 software (Axon Instrument Co.) to a computer for analysis. AxoScope1.01, SigmaPlot 2.0 and CorelDraw 6.0 were used to plot ®gures. The results were presented as mean 2 SD. 2.4. Reagents The Cinnamomin was extracted and puri®ed from the seeds of Cinnamonum camphora by the method previously described (Li et al., 1997). The purity was characterized as a homogeneous band on SDS-polyacrylamide gel. CHAPS, Hepes, NMDG and lecithin were purchased from Sigma, N-decane and cholesterone from Avanti Polar and Merck-Schuchardt, respectively. All other reagents were of analytical grade.
3. Results 3.1. Cinnamomin-induced conductance in planar lipid bilayer After bilayer formation and stabilization, no channel-like noise at voltage 2100 mV in 30 min, in the cis 200/trans 100 mM KCl solution system, Cinnamomin is added to the cis side solution in a ®nal concentration of 1 mg/ml at +30 mV. In 11 successful experiments of more than 50 experiments, the channel-like ¯uctuations of the membrane current are usually observed within 10 min, then an increase in steady state membrane conductance can be observed. In an asymmetrical solution system, cis 200/trans 100 mM KCl, the current±voltage relationship (I±V curve) obtained by plotting the steady state current versus the holding potential is a straight line. At its intersection with the zero current line, the reverse potential is about 20 mV (n = 3) closing to the predicated value for a perfect Nernst K + electrode. These preliminary results indicate that Cinnamomin has been incorporated into the bilayer and the formed channels are cationselective. 3.2. Unit conductance To characterize the channel properties in detail, Cinnamomin was reconstituted into giant liposomes by a de-rehydration cycle (see Section 2) and the channel activity studied by the standard patch clamp technique. After gigaohm seal formation between the giant liposome and pipette, the patch was excised and the single channel current recorded. In this case, giga-seal formation was very easy and stable recordings can be maintained for several hours. The success rates of
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giga-seal and excised patch formation are 86% (104/120) and 80% (95/120), respectively. A gigaohm seal was also obtained easily from the giant liposome which was prepared by the same procedure except for addition of Cinnamomin, but no channel activity was found (n = 8). All the results below are obtained from excised patches of giant liposomes. If the concentration of Cinnamomin was above 50 ng/ml in the process of reconstitution, sometimes, multi-channel activity with the same conductance was recorded from one patch (as shown in Fig. 1A). When the concentration of Cinnamomin was decreased to 10 ng/ml, usually only a single channel in a patch was obtained (Fig. 1B). The current±voltage relationship obtained by plotting the unit current against the holding potential is a straight line (Fig. 2). In symmetrical 100 mM KCl solution, the unit conductance was 90 25 pS (n = 12). In an asymmetrical solution, bath 150/pipette 100 mM KCl or bath 300/pipette 100 mM KCl, the unit conductance is 1402 7 pS (n = 18) and 155 pS (n = 3), respectively. 3.3. Cation selectivity In order to estimate the ion selectivity of this conductance, a single channel current is recorded in asymmetrical solutions at dierent holding potentials. The reversal potential is obtained from the current±voltage curve (Fig. 2). In bath 150/ pipette 100 mM KCl and bath 300/pipette 100 mM KCl, the reversal potentials are 10.4 20.6 mV (n = 18) and 26.5 2 0.5 mV (n = 3), respectively (Fig. 2), very close to the theoretical values of K + electrodes (10.6 and 27 mV, respectively). These are consistent with the results obtained from the planar lipid bilayer experiment. Whether Na+ can pass through the Cinnamomin channel is tested by replacement of K+ in the pipette solution with Na+ (bath 100 KCl/pipette 100 mM NaCl). In the Na+/K+ bi-ionic condition, the unit conductance does not change much (90 2 6.1 pS, n = 27), the reversal potential only has a 1±3 mV positive shift (n = 10) relative to the K+/K+ condition (Fig. 2). However, an obvious decrease in the channel open probability (Po) is encountered in all the patches. All of the Po are near 0.8 in the symmetric 100 mM KCl solution, but lower than 0.2 in the bath 100 mM KCl/pipette 100 mM NaCl solution system, at a voltage of 2100 mV, as shown in Fig. 4(C) as an example. No obvious voltagedependence of the Cinnamomin channel is observed in all patches of this work at a potential ranging from ÿ150 to +150 mV in the KCl solution system. In sixteen other experiments, when cations are completely replaced with NMDG at both sides of the bilayer, no channel activity can be found in a voltage range of 2100 mV (data not shown). In an asymmetric solution system, bath 100 KCl/ pipette 100 mM NMDG±Cl, in which all of the cations in the pipette are replaced by impermeable NMDG, the channel events can be observed only at a negative potential (n = 10, Fig. 3 as an example). These results demonstrate further the cation selectivity of the Cinnamomin channel.
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Fig. 1. Recordings of Cinnamomin-formed channels in bilayer membranes of giant liposomes. (A) There are three Cinnamomin-channels in one patch at least; (B) single channel recordings at various holding potentials from an excised patch of a giant liposome reconstituted with Cinnamomin. Solution system (mM): bath 150/pipette 100 KCl, solutions buered with 10 mM Hepes, pH 7.4, the number in the right of each trace: holding potential (mV); ®lter frequency, 3 kHz.
3.4. Two gating modes It is found that the transition rates between open and close are obviously dierent at dierent patches. In most patches (72/85), as shown in Fig. 1 and Fig. 4(A), the channel activity displayed a slow transition between open and closed states, however, in some patches (13/85, Fig. 4B), the transition of two
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Fig. 2. Current±voltage relationship obtained by plotting the unitary current against the holding potential (HP) in dierent solution systems (mM) buered with 10 mM Hepes, pH 7.4. The reversal potentials are very close to the theoretical values of K + electrode. The data in each curve is from one patch.
states is fast. Moreover, the transfer between the slow and the fast mode has never been observed in the same channel, even in a 1 h period of recording time.
4. Discussion In this work, Cinnamomin-formed channels in bilayer membranes are characterized by patch clamp recording. This is the ®rst report that a ribosome-
Fig. 3. Recording trace in an asymmetrical solution system (mM) with bath 100 KCl/pipette 100 NMDG-Cl, buered with 10 mM Hepes, pH 7.4, ®lter frequency 3 kHz.
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Fig. 4. (A and B) Slow and fast kinetics of Cinnamomin channels. There are dierent transition rates between open and closed states in the same solution system (mM): bath 100 KCl/pipette 100 KCl, buered with 10 Hepes, pH 7.4, ®lter frequency 3 kHz. (C) The eect of Na + on open probability of the channel. Po under each voltage is estimated from a recording period of 30±50 s.
inactivating protein can incorporate into a bilayer and form transmembrane channels. The results oer a reasonable explanation for the transmembrane mechanism of type II RIP and the toxicity dierence between type I and type II RIP. For two types of ribosome-inactivating protein, type II RIP is called cytotoxin because of its toxicity on the integral cell, type I RIP is hemitoxin because of its lower toxicity on the integral cell. The B-chain of type II RIP, a monovalence lectin, functions in binding the membrane receptor on the cell surface and allowing the entry of the A-chain (Barbieri et al., 1993). The results reported here indicate that Cinnamomin can form a cation channel in the bilayer. The viewpoint that the B-chain of type II may be the channel-forming chain is supported by the following data: (1) channel-forming properties of lectin in the lipid bilayer have been reported (Shi et al., 1992; Zhang and Shi, 1994); (2) some toxins containing two chains similar to type II RIP such as diphtheria, tetanus toxin, botulinum toxin or leukotoxin can form channels in the bilayer via their B-chain (Donovan et al., 1981; Boquet and Du¯ot, 1982; Sandvig and Olsnes, 1988; Schmid et al., 1994; Lear et al., 1995; Gouaux, 1997; Lanzrein et al., 1997); (3) trichosanthin, a type I RIP, has no channel-forming ability. However, ricin, a type II RIP, can form a channel in the lipid bilayer (Shi et al., unpublished data). The results that there are no signi®cant dierences at the reversal potential and conductance of the channel in both K+/K+ and K+/Na+ conditions indicate that the Cinnamonin channel passes Na+ and K+ with a similar permeability. It is unclear why Na+ aects the Po of the Cinnamomin channel. However, it is interesting that the Po of the Na+-dependent K+ channel from guinea-pig ventricular myocytes is [Na+]-dependent, reducing Po when decreasing [Na+]i (Mistry et al., 1997).
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In summary, the present results are the ®rst to demonstrate the channelformation of a type II ribosome-inactivating protein in a bilayer membrane and suggest that the B-chain of the protein may be responsible for the action.
Acknowledgements We thank Mr. R.G. Hu for his assistance in preparation of the Cinnamomin. This work was supported by the Natural Science Foundation of China.
References Barbieri, L., Battelli, M.G., Stirpe, F., 1993. Ribosome-inactivating proteins from plants. Biochim. Biophys. Acta 1154, 237±282. Bolognesi, A., Barbieri, L., Abbondanza, A., Falasca, A.I., Carnicelli, D., Battelli, M.G., Stirpe, F., 1990. Puri®cation and properties of new ribosome-inactivating proteins with RNA N-glycosidase activity. Biochim. Biophys. Acta 1087, 293±302. Boquet, P., Du¯ot, E., 1982. Tetanus toxin fragment forms channels in lipid vescles at low pH. Proc. Natl. Acad. Sci. USA 79, 7614±7618. Chan, H.C., Zhou, T.S., Fu, W.O., Wang, W.P., Shi, Y.L., Wong, P.Y.D., 1997. Cation and anion channels in fat and human spermatozoa. Biochim. Biophys. Acta 1323, 117±129. Donovan, J.J., Simon, M.I., Draper, R.K., Montal, M., 1981. Diphtheria toxin forms transmembrane channels in planar lipid bilayers. Proc. Natl. Acad. Sci. USA 78, 172±176. Finkelstein, A., 1974. Bilayers: formation, measurements and incorporation of components. In: Fleischer, S., Pacher, L. (Eds.), Methods Enzymol. 32(part B), 489±501. Gouaux, E., 1997. Channel-forming toxins: tales of transformation. Curr. Opin. Struct. Biol. 7, 566±573. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp technique for high-resolution current recording from cells and cell-free membrane patches. P¯uegers Arch. 391, 85±100. Keller, B., Hedrich, R., Vaz, W.L.C., Criado, M., 1988. Single channel recordings of reconstituted ion channel protein: an improved technique. P¯uegers Arch. 411, 94±100. Lanzrein, M., Falnes, P.O., Sand, O., Olsnes, S., 1997. Structure±function relationship of the ion channel formed by diphtheria toxin in vero cell membranes. J. Membrane Biol. 156, 141±148. Lear, J.D., Furblur, U.G., Lally, E.T., Tanaka, J.T., 1995. Actinobacillus itans leukotoxin forms large conductance, voltage-gated ion channels when incorporated into planar lipid bilayers. Biochim. Biophys. Acta 1238, 34±41. Li, X.D., Chen, W.F., Liu, W.Y., Wang, G.H., 1997. Large-scale preparation of two new ribosome-inactivating proteins, Cinnamomin and camphorin, from the seeds of Cinnamomin camphora. Protein Express. Purif. 10, 15±22. Ling, J., Liu, W.Y., 1996. Cytotoxicity of two new ribosome-inactivating proteins, cinnamomin and camphorin, to carcinoma cells. Cell Biochem. Funct. 14, 157±161. Ling, J., Liu, W.Y., Wang, T.P., 1995. Simultaneous existence of the seeds of Cinnamomun camphora: characterization of the enzymatic activaties of these cytotoxic proteins. Biochim. Biophys. Acta 1252, 15±22. Ma, X.H., Shi, Y.L., 1997. Eects of ADP, DTT and Mg2+ on the ion-conductive property of chloroplast H + -ATPase (CF0±CF1) reconstituted into bilayer membrane. Biochem. Biophys. Res. Commun. 232, 461±463.
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Mistry, D.K., Tripathi, O., Chapman, R.A., 1997. Kinetic properties of unitary Na + -dependent K + channels in inside-out patches from isolated guinea-pig ventricular myocytes. J. Physiol. (London) 500, 39±50. Riquelme, G., Lopez, E., Garcia-Segura, L.M., Ferragut, J.A., Gonzalez-Ros, J.M., 1990. Giant liposome: a model system in which to obtain patch-clamp recordings of ionic channels. Biochemistry 29, 11215± 11222. Sandvig, K., Olsnes, S., 1988. Diphtheria toxin-induced channels in vero cells selective for monovalent cations. J. Biochem. Chem. 263, 12352±12359. Schmid, A., Benz, R., Just, I., Aktories, K., 1994. Interaction of Clostridium botulinum C2 toxin with lipid bilayer membranes-Formation of cation-selective channels and inhibition of channel function by chloroquine. J. Biol. Chem. 269, 16706±16711. Shi, Y.L., Wang, W.P., Zhang, H., Wang, K.Y., Guo, M., 1992. Cation channels formed in lipid bilayer by pinellia ternata lectin. Acta Physiol. Sin. 44, 142±148. Stirpe, F., 1982. On the action of ribosome-inactivating protein: are plant ribosomes species-speci®c?. Biochem. J. 202, 279±280. Stirpe, F., Barbieri, L., 1986. Ribosome-inactivating proteins up to data. FEBS Lett. 195, 1±8. Stirpe, F., Barbieri, L., Battelli, M.G., Soria, M., Lappi, D.A., 1992. Ribosome-inactivating proteins from plants: present status and future prospects. Biotech. 10, 405±412. Stirpe, F., Gasperi-Campani, A., Barbieri, L., Falasca, A.I., Abbondanza, A., Stevens, W.A., 1983. Ribosome-inactivating proteins from the seeds of Saponaria ocinalis L. (soapwort), of Agrostenma githago L. (corn codkle) and Asparagus ocinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree). Biochem. J. 216, 617±625. Zhang, H., Shi, Y.L., 1994. Cation selectivity of channels formed at plannar lipid bilayer by pinella lectin. Sci. China (Ser. B) 37, 547±556.
Toxicon 37 (1999) 1313±1322
Cation channel formed at lipid bilayer by Cinnamomin, a new type II ribosomeinactivating protein Guang-ping Zhang a, Yu-liang Shi a, *, Wen-ping Wang a, Wang-yi Liu b a
Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China b Shanghai Institute of Biochemistry, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China
Received 14 September 1998; accepted 16 December 1998 Abstract Cinnamomin, a new type II ribosome-inactivating protein, puri®ed from the seeds of Cinnamonum camphora is reconstituted into the membranes of planar lipid bilayer and giant liposome. The channel-forming activity of the cinnamomin is found and cation permeability of the channel is characterized by patch clamp. In an asymmetric solution system, bath 150/ pipette 100 mM KCl, the unit conductance is 140 2 7 pS and the reversal potential is 10.4 2 0.6 mV, very close to the theoretical value of the K + electrode. The results oer an interpretation for internalization of the RIP and the cytotoxicity dierence between single and two chain RIP. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Ribosome-inactivating protein; Cation channel; Cinnamomin; Lipid bilayer; Giant liposome
Abbreviations: RIP, ribosome-inactivating protein, CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Hepes, (N-[2-hydroxyethyl]piperazine-N 0 -[2-ethanesulfonic acid]), NMDG, Nmethyl-D-glucamine. * Corresponding author. Unit of Bioactive Substances and Ion Channels, Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China. Tel.: +86-21-6437-0080; Fax: +86-21-6433-2445; E-mail: [email protected] 0041-0101/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 0 7 8 - 1
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1. Introduction Plant ribosome-inactivating proteins (RIPs) are a group of ribotoxins. They are RNA N-glycosidases which arrest protein synthesis by selectively hydrolyzing the N-glycosidic bond of a conserved adenosine in the largest ribosomal RNA (Stirpe, 1982; Stirpe and Barbieri, 1986; Stirpe et al., 1992; Barbieri et al., 1993). According to chemical structure, RIPs are divided into two groups. Type I is composed of a single chain with the molecular weight between 11,000±30,000 Da, while type II has a molecular weight of around 60,000 Da and consists of two chains (A- and B-chain) (Stirpe, 1982; Barbieri et al., 1993). There are considerable dierences between the two types of RIPs in the toxicity to both integral cell and cell-free translation systems. Type I markedly inhibits protein synthesis in cell-free translation systems and seldom aects on integral cell. Type II has strong cytotoxicity to the integral cell and a low toxic eect on the cell-free translation system (Stirpe et al., 1983; Bolognesi et al., 1990). The A-chain of type II acts on ribosome in the same way as type I RIP does. The B-chains are a lectin, binding to the receptors on the cell surface and allowing entry of the ribotoxin (Barbieri et al., 1993). Cinnamomin (cin) is a new type II RIP puri®ed from the seeds of Cinnamomum camphora. Its A-chain has the RNA N-glycosidase activity and the intact cin expresses the inhibitory eects in culture carcinoma cells (Ling et al., 1995; Ling and Liu, 1996; Li et al., 1997). In this work, in order to elucidate its transmembrane mechanism, Cinnamomin was reconstituted into the membranes of the planar lipid bilayer and giant liposome. Its channel-forming activity is observed by the patch clamp technique. The results oer an interpretation for the internalization of type II RIP and the toxicity dierences between two types of RIPs.
2. Materials and methods 2.1. Fusing Cinnamomin into planar lipid bilayer In the initial experiment, the interaction between Cinnamomin and the bilayer membrane was determined by using a two-compartment system (Finkelstein, 1974). As described previously, the lipid bilayer was formed by painting an Ndecane suspension of lecithin and cholesterol (20 and 5 mg/ml, respectively) over a 500 mm ori®ce in a Te¯on partition separating the two 3 ml solution-®lled compartments (cis and trans compartments) (Shi et al., 1992; Zhang and Shi, 1994; Ma and Shi, 1997; Chan et al., 1997). Only the stable bilayers, no channellike noise in 30 min under holding potentials of 2100 mV, were used for experiments. The reconstitution was achieved by adding 2±3 ml Cinnamomin to the cis-compartment (®nal protein concentration 1 mg/ml) and stirring for a minute. The salt concentration between the two compartments excised an osmotic
G. Zhang et al. / Toxicon 37 (1999) 1313±1322
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gradient (cis 200/trans 100 mM KCl), which was used to promote the protein fusion, the signals were picked up by a pair of calomel electrodes connecting the two compartments to the inputs of the patch clamp ampli®er (CEZ-2300, Nihon Kohden, Tokyo, Japan) in voltage-clamping model. The output signals were monitored on an oscilloscope, displayed on a chart recorder and simultaneously recorded on videotape with a video cassette recorder (VCR-N85, NEC, Tokyo, Japan) via an audio pulse code modulator (PCM) processor (Modi®ed 501 ES, Sony, Osaka, Japan) for further analysis. The current was de®ned as being positive when cations ¯owed into the trans side. All measurements were made at room temperature (0228C). 2.2. Reconstituting Cinnamomin into a giant liposome The formation procedure of the giant liposome used in this study was a modi®cation of that previously described by Keller et al. (1988). Brie¯y, 100 mg lecithin from soybean (type 2-S, Sigma) was suspended in 1 ml of distilled water by sonication in Branson soni®er at 40 W for 5±10 min under nitrogen. The lipid suspension was further diluted 10-fold with dialysis solution (in mM: 10 Hepes; 100 NaCl, pH 7.4), then the Cinnamomin and CHAPS were added at ®nal concentrations of 10 ng/ml and 10 mM, respectively. The mixture sample was dialyzed against 600 volumes of dialysis solution for 70 h. The dialyzed sample (02.5 ml) was ultra-centrifuged at 40,000 rpm for 1 h and the pellet was resuspended with 200 ml Hepes buer containing 5% ethylene glycol, pH 7.4. The resuspension sample was deposited to a clean glass slide in 15 ml per aliquot and was submitted to partial dehydration (3±6 h) in a desiccator containing anhydrous CaCl2, then stored in a refrigerator. Before use, the sample was rehydrated by dropping 15 ml of 100 mM KCl solution to cover the dehydrated sample on slide and leaving the slide in a closed petri dish with a wet paper pad on its bottom overnight. Next day, the giant liposome could be observed. The preparation of the control giant liposome was the same as mentioned above, but only without adding Cinnamomin. All the operations were performed at 48C. 2.3. Patch clamping giant liposome and recording channel activity An aliquot of the giant liposome solution (010 ml) was mixed with 200 ml of a DEAE-Sephedex A-50 suspension (3 mg/ml in the dialysis solution) in a small dish and incubated at room temperature for 15±30 min. This treatment anchored the liposome to the gel beads and to the bottom of the dish, making it easier to get a giga-seal (Riquelme et al., 1990). After gigaohm seal (10±100 GO) formation between the giant liposome and the patch pipette, the pipette was withdrawn from the liposome surface. This resulted in an excised patch. Single channel recordings were obtained by using the patch-clamp technique as described by Hamill et al. (1981). The current was de®ned as positive when cation ¯owed out of the pipette into the bath. The signal was ampli®ed through an EPC-7 patch clamp ampli®er
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(List Medical Electronics), then recorded and stored on videotape via the audio PCM processor. The current signal was monitored simultaneously on an oscilloscope. The stored records were transferred, using an analog-to-digital interface (Digidata 1200, Axon Instrument Co.) and the pClamp 6.02 software (Axon Instrument Co.) to a computer for analysis. AxoScope1.01, SigmaPlot 2.0 and CorelDraw 6.0 were used to plot ®gures. The results were presented as mean 2 SD. 2.4. Reagents The Cinnamomin was extracted and puri®ed from the seeds of Cinnamonum camphora by the method previously described (Li et al., 1997). The purity was characterized as a homogeneous band on SDS-polyacrylamide gel. CHAPS, Hepes, NMDG and lecithin were purchased from Sigma, N-decane and cholesterone from Avanti Polar and Merck-Schuchardt, respectively. All other reagents were of analytical grade.
3. Results 3.1. Cinnamomin-induced conductance in planar lipid bilayer After bilayer formation and stabilization, no channel-like noise at voltage 2100 mV in 30 min, in the cis 200/trans 100 mM KCl solution system, Cinnamomin is added to the cis side solution in a ®nal concentration of 1 mg/ml at +30 mV. In 11 successful experiments of more than 50 experiments, the channel-like ¯uctuations of the membrane current are usually observed within 10 min, then an increase in steady state membrane conductance can be observed. In an asymmetrical solution system, cis 200/trans 100 mM KCl, the current±voltage relationship (I±V curve) obtained by plotting the steady state current versus the holding potential is a straight line. At its intersection with the zero current line, the reverse potential is about 20 mV (n = 3) closing to the predicated value for a perfect Nernst K + electrode. These preliminary results indicate that Cinnamomin has been incorporated into the bilayer and the formed channels are cationselective. 3.2. Unit conductance To characterize the channel properties in detail, Cinnamomin was reconstituted into giant liposomes by a de-rehydration cycle (see Section 2) and the channel activity studied by the standard patch clamp technique. After gigaohm seal formation between the giant liposome and pipette, the patch was excised and the single channel current recorded. In this case, giga-seal formation was very easy and stable recordings can be maintained for several hours. The success rates of
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giga-seal and excised patch formation are 86% (104/120) and 80% (95/120), respectively. A gigaohm seal was also obtained easily from the giant liposome which was prepared by the same procedure except for addition of Cinnamomin, but no channel activity was found (n = 8). All the results below are obtained from excised patches of giant liposomes. If the concentration of Cinnamomin was above 50 ng/ml in the process of reconstitution, sometimes, multi-channel activity with the same conductance was recorded from one patch (as shown in Fig. 1A). When the concentration of Cinnamomin was decreased to 10 ng/ml, usually only a single channel in a patch was obtained (Fig. 1B). The current±voltage relationship obtained by plotting the unit current against the holding potential is a straight line (Fig. 2). In symmetrical 100 mM KCl solution, the unit conductance was 90 25 pS (n = 12). In an asymmetrical solution, bath 150/pipette 100 mM KCl or bath 300/pipette 100 mM KCl, the unit conductance is 1402 7 pS (n = 18) and 155 pS (n = 3), respectively. 3.3. Cation selectivity In order to estimate the ion selectivity of this conductance, a single channel current is recorded in asymmetrical solutions at dierent holding potentials. The reversal potential is obtained from the current±voltage curve (Fig. 2). In bath 150/ pipette 100 mM KCl and bath 300/pipette 100 mM KCl, the reversal potentials are 10.4 20.6 mV (n = 18) and 26.5 2 0.5 mV (n = 3), respectively (Fig. 2), very close to the theoretical values of K + electrodes (10.6 and 27 mV, respectively). These are consistent with the results obtained from the planar lipid bilayer experiment. Whether Na+ can pass through the Cinnamomin channel is tested by replacement of K+ in the pipette solution with Na+ (bath 100 KCl/pipette 100 mM NaCl). In the Na+/K+ bi-ionic condition, the unit conductance does not change much (90 2 6.1 pS, n = 27), the reversal potential only has a 1±3 mV positive shift (n = 10) relative to the K+/K+ condition (Fig. 2). However, an obvious decrease in the channel open probability (Po) is encountered in all the patches. All of the Po are near 0.8 in the symmetric 100 mM KCl solution, but lower than 0.2 in the bath 100 mM KCl/pipette 100 mM NaCl solution system, at a voltage of 2100 mV, as shown in Fig. 4(C) as an example. No obvious voltagedependence of the Cinnamomin channel is observed in all patches of this work at a potential ranging from ÿ150 to +150 mV in the KCl solution system. In sixteen other experiments, when cations are completely replaced with NMDG at both sides of the bilayer, no channel activity can be found in a voltage range of 2100 mV (data not shown). In an asymmetric solution system, bath 100 KCl/ pipette 100 mM NMDG±Cl, in which all of the cations in the pipette are replaced by impermeable NMDG, the channel events can be observed only at a negative potential (n = 10, Fig. 3 as an example). These results demonstrate further the cation selectivity of the Cinnamomin channel.
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Fig. 1. Recordings of Cinnamomin-formed channels in bilayer membranes of giant liposomes. (A) There are three Cinnamomin-channels in one patch at least; (B) single channel recordings at various holding potentials from an excised patch of a giant liposome reconstituted with Cinnamomin. Solution system (mM): bath 150/pipette 100 KCl, solutions buered with 10 mM Hepes, pH 7.4, the number in the right of each trace: holding potential (mV); ®lter frequency, 3 kHz.
3.4. Two gating modes It is found that the transition rates between open and close are obviously dierent at dierent patches. In most patches (72/85), as shown in Fig. 1 and Fig. 4(A), the channel activity displayed a slow transition between open and closed states, however, in some patches (13/85, Fig. 4B), the transition of two
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Fig. 2. Current±voltage relationship obtained by plotting the unitary current against the holding potential (HP) in dierent solution systems (mM) buered with 10 mM Hepes, pH 7.4. The reversal potentials are very close to the theoretical values of K + electrode. The data in each curve is from one patch.
states is fast. Moreover, the transfer between the slow and the fast mode has never been observed in the same channel, even in a 1 h period of recording time.
4. Discussion In this work, Cinnamomin-formed channels in bilayer membranes are characterized by patch clamp recording. This is the ®rst report that a ribosome-
Fig. 3. Recording trace in an asymmetrical solution system (mM) with bath 100 KCl/pipette 100 NMDG-Cl, buered with 10 mM Hepes, pH 7.4, ®lter frequency 3 kHz.
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Fig. 4. (A and B) Slow and fast kinetics of Cinnamomin channels. There are dierent transition rates between open and closed states in the same solution system (mM): bath 100 KCl/pipette 100 KCl, buered with 10 Hepes, pH 7.4, ®lter frequency 3 kHz. (C) The eect of Na + on open probability of the channel. Po under each voltage is estimated from a recording period of 30±50 s.
inactivating protein can incorporate into a bilayer and form transmembrane channels. The results oer a reasonable explanation for the transmembrane mechanism of type II RIP and the toxicity dierence between type I and type II RIP. For two types of ribosome-inactivating protein, type II RIP is called cytotoxin because of its toxicity on the integral cell, type I RIP is hemitoxin because of its lower toxicity on the integral cell. The B-chain of type II RIP, a monovalence lectin, functions in binding the membrane receptor on the cell surface and allowing the entry of the A-chain (Barbieri et al., 1993). The results reported here indicate that Cinnamomin can form a cation channel in the bilayer. The viewpoint that the B-chain of type II may be the channel-forming chain is supported by the following data: (1) channel-forming properties of lectin in the lipid bilayer have been reported (Shi et al., 1992; Zhang and Shi, 1994); (2) some toxins containing two chains similar to type II RIP such as diphtheria, tetanus toxin, botulinum toxin or leukotoxin can form channels in the bilayer via their B-chain (Donovan et al., 1981; Boquet and Du¯ot, 1982; Sandvig and Olsnes, 1988; Schmid et al., 1994; Lear et al., 1995; Gouaux, 1997; Lanzrein et al., 1997); (3) trichosanthin, a type I RIP, has no channel-forming ability. However, ricin, a type II RIP, can form a channel in the lipid bilayer (Shi et al., unpublished data). The results that there are no signi®cant dierences at the reversal potential and conductance of the channel in both K+/K+ and K+/Na+ conditions indicate that the Cinnamonin channel passes Na+ and K+ with a similar permeability. It is unclear why Na+ aects the Po of the Cinnamomin channel. However, it is interesting that the Po of the Na+-dependent K+ channel from guinea-pig ventricular myocytes is [Na+]-dependent, reducing Po when decreasing [Na+]i (Mistry et al., 1997).
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In summary, the present results are the ®rst to demonstrate the channelformation of a type II ribosome-inactivating protein in a bilayer membrane and suggest that the B-chain of the protein may be responsible for the action.
Acknowledgements We thank Mr. R.G. Hu for his assistance in preparation of the Cinnamomin. This work was supported by the Natural Science Foundation of China.
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