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The Peptide Mastoparan Is a Potent Facilitator of the Mitochondrial Permeability Transition

The Peptide Mastoparan Is a Potent Facilitator of the Mitochondrial Permeability Transition Vol. 270, No.9, Issue of March 3, pp. 4923-4932, 1995 THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. The Peptide Mastoparan Is a Potent Facilitator of the Mitochondrial Permeability Transition* (Received for publication, August 22, 1994) Douglas R. Pfeiffer:j:, Tatyana I. Gudz, Sergei A. Novgorodov, and Warren L. Erdahl From the Department of Medical Biochemistry, College of Medicine, The Ohio State University, Columbus, Ohio 43210 and the Hormel Institute, University of Minnesota, Austin, Minnesota 55912 ation of the transition with cell injury mechanisms and the Mastoparan facilitates opening of the mitochondrial identification of ~yclosporin A (CSA)! as a potent inhibitor of permeability transition pore through an apparent bimo­ dal mechanism of action. In the submicromolar concen­ the phenomenon (11-14) has created a high level of interest in tration range, the action of mastoparan is dependent this aspect of mitochondrial research. upon the medium Ca + and phosphate concentration Many of the agents which induce the transition are toxins and is subject to inhibition by cyclosporin A. At concen­ and pharmacological agents not normally encountered in vivo trations above 1 /.LM, pore induction by mastoparan oc­ (1). The same is true for the known inhibitors (1). Both induc­ curs without an apparent Ca + requirement and in a ing agents and inhibitors are chemically diverse but display a cyclosporin A insensitive manner. Studies utilizing common activity in favoring an open or closed state of the phospholipid vesicles show that mastoparan perturbs permeability transition pore (PTP), respectively. It has proven bilayer membranes across both concentration ranges, difficult to explain the analogous actions of diverse compounds through a mechanism which is strongly dependent upon on the PTP. Bernardi and co-workers (15,16) have shown that transmembrane potential. However, solute size exclu­ membrane potential and matrix pH are central PTP regulators. sion studies suggest that the pores formed in mitochon­ Membrane surface potential may also be a central regulator (3, dria in response to both low and high concentrations of 17). It is thus possible that the chemical diversity/common mastoparan are the permeability transition pore. It is activity problem associated with the transition may arise, in proposed that low concentrations of mastoparan influ­ part, from the effects of transition regulators on bioenergetic ence the pore per se, with higher concentrations having parameters and membrane physical properties. the additional effect of depolarizing the mitochondrial Although many known transition effectors would not nor­ inner membrane through an action exerted upon the mally be encountered in cells, a number of activators (e.g. Ca +, lipid phase. It may be the combination of these effects Pi' acyl-CoA) and inhibitors (e.g. ADP, M~+, polyamines) are which allow pore opening in the absence of Ca + and in the presence of cyclosporin A, although other interpre­ physiological cell constituents. In seeking to understand regu­ tations remain viable. A comparison of the activities of lation ofthe transition in an intracellular setting, it then seems mastoparan and its analog, MP14, on mitochondria and reasonable to consider these agents as primary regulators and phospholipid vesicles provides an initial indication that to investigate how other cellular components and conditions a G-protein may participate in regulation of the perme­ affect their actions on the PTP. The present report demon­ ability transition pore. These studies draw attention to strates that the 14-amino acid peptide mastoparan has a potent peptides, in a broad sense, as potential pore regulators stimulatory effect on PTP opening when mitochondria are in­ in cells, under both physiological and pathological 2 cubated in the presence of Ca + and Pi' At somewhat higher conditions. concentrations, mastoparan appears to induce pore opening in a Ca + independent and CSA insensitive manner. This is the first report of a peptide exerting a regulatory influence on the The mitochondrial permeability transition remains an in­ transition and, as such, draws attention to a new class of completely understood phenomenon, in spite of the fact that it potential regulators of this phenomenon within cells. In this was first observed in the 1950s and has been studied exten­ report, the actions of mastoparan on the transition are also sively, particularly during the last 15 years (see Refs. 1-3 for compared to those of an analog, MPI4. Differences observed review). Recent work utilizing the patch clamp technique ap­ between the potency of these two peptides are consistent with plied to isolated mitochondria indicates that the transition is the hypothesis that components of cell signaling mechanisms, caused by the opening of large pores in the inner membrane 2 apart from Ca +, are involved in regulation ofthe PTP. Aspects (4-6). At present there is no known physiological function of of these data have been presented in abstract form (18). this phenomenon; however, it is clear that it occurs in situ, EXPERIMENTAL PROCEDURES under conditions of oxidative stress, and is an event that can be pivotal in the mechanisms leading to cell death (7-10). Associ- Reagents-Polyethylene glycols (PEGs) were obtained from Aldrich (average M = 400,600, 1,000, 1,500,2,000,3,400,8,000, and 10,000, or from Fluka (average M = 4,000 and 6,000). Mastoparan and alamethi­ cin were from Sigma. The mastoparan analog MP14 (see Table I for * This research was supported by United States Public Health Ser­ structure) was obtained from Quality Controlled Biochemicals, Inc. vice Grants HL 49182, HL 49181, and HL 36124 from the National (Hopkinton, MA). The purity (>95%) and sequence of this peptide were Institutes of Health, NHLBI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The abbreviations used are: CSA, cyclosporin A; PEG, polyethylene :j: To whom correspondence and reprint requests should be addressed: glycol; POPC, 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine; PTP, The Ohio State University, Dept. of Medical Biochemistry, 310A Ham­ permeability transition pore; TPP+, tetraphenylphosphonium cation; ilton Hall, 1645 Neil Ave., Columbus, OH 43210-1218. Tel.: 614-292­ VDAC, voltage-dependent anion channel; HPLC, high performance liq­ 8774; Fax: 614-292-4118. uid chromatography. This is an Open Access article under the CC BY license. 4924 Mitochondrial Permeability Transition Ca + (Mas) Mas 0.7 0.7 1.0 1.0 ::l. ::l. 1.3 0. 0. 1.3 ~ 0. 1.7 lil 1.7 lil 2.0 2.0 2.3 ~ w 2.3 2.7 2.7 3.0 3.0 3.3 3.3 a RLM RLM Ca + (Mas) 1 Mas 01 1 ._------------_._------~--------------_ ..-----._----- a I 4min 4mln t---------i FIG. 1. Stimulation of the permeability transition by mastopa­ FIG. 2. Stimulation of the permeability transition by mastopa­ ran; inhibition by CSA. Mitochondria were incubated at 0.5 mg of ran; inhibition by EGTA and oligomycin. Mitochondria were incu­ protein/ml and at 25°C. The medium contained 250 mM sucrose, 10 mM bated as described in the legend to Fig. 1, and TPP+ accumulation Hepes (Tris), 5 mM succinate (Tris), 5 mM Pi (Tris), pH 7.4, plus 3.3 J1-M (upper panel) and swelling (lower panel) were determined simulta­ TPP·C!. TPP+ accumulation and release (upper panel) and swelling neously as described under "Experimental Procedures." For all traces (lower panel) were determined simultaneously as described under "Ex­ (a-c), 1.0 J1-M mastoparan was added where indicated. For traces labeled perimental Procedures." CaC~ (dashed lines) or mastoparan (solid band c, the medium contained EGTA (0.5 mM) or oligomycin (1 J1-g/mg lines) were added where indicated at 25 nmol/mg of protein and 1.0 J1-M, protein), respectively, from the beginning of the incubation. respectively. For traces labeled a, the medium contained CSA at 0.5 J1-M from the beginning of the experiment. For traces labeled b, CSA was absent. to the spectrophotometer. Preparation of Phospholipid Vesicles and Determination of Vesicle confirmed by the supplier, following its synthesis by solid state Permeability-Freeze-thaw extruded POPC vesicles loaded with Quin-2 methods. Synthetic I-palmitoyl-2-01eoyl-sn-glycerophosphatidylcholine (K+) and Hepes (K+), pH 7.0, were prepared as described previously (24, (POPC) was obtained from Avanti Polar Lipids. Quin-2 was obtained 25). Briefly, 250 mg of POPC in chloroform was dried by rotation under from Molecular Probes and was deionized and converted to the K+ salt a nitrogen stream, to produce a film on the wall of a 25 X 150 mm as described previously (19). Other chemicals were obtained from com­ culture tube. Residual solvent was removed under high vacuum (4 h), mercial sources and were reagent grade or better. and the film was subsequently hydrated in 5 ml of medium containing Mitochondrial Preparations and Incubation Conditions-Liver mito­ 10 mM Hepes buffer and 5 mM Quin-2. The mixture was vortexed until chondria were prepared by a standard procedure (20) from male the entire film was suspended and the resulting multilamellar vesicles Sprague-Dawley rats weighing -250 g. Bovine serum albumin (2 mg! were frozen in a dry ice-acetone bath, thawed in lukewarm water, and ml) and EGTA (0.5 mM) were present during homogenization but were vortexed again. The freeze-thaw and vortexing procedure was repeated twice. Following this, the preparations were extruded three times excluded from the washing medium. For some experiments these prep­ arations were depleted of endogenous Ca + by the method of Wingrove through two stacked O.I-J1-m polycarbonate membrane filters. This step and Gunter (21). Unless otherwise specified, incubations were con­ was followed by six additional freeze-thaw cycles and eight additional ducted at 25°C, and 0.5 mg of protein/ml, in a medium containing 250 extrusions. The resulting unilamellar preparations were then applied mM sucrose, 10 mM Hepes (Tris), pH 7.4, 5 mM succinate (Tris), and to Sephadex G-50 minicolumns to remove extravesicular Quin-2 (26). rotenone (2 J1-M). Deviations from this medium and other reagents The columns, which were eluted by low speed centrifugation (26), had previously been equilibrated with 10 mM Hepes (Na+), pH 7.0, in 20 mM employed are described in the figure legends. The permeability transi­ tion was monitored by swelling measurements and by tetraphenylphos­ NaC!. The nominal concentrations of POPC in the final preparations phonium (TPP+) release (loss of membrane potential) which were de­ was 70-90 mM as determined by measurements of lipid phosphorus termined simultaneously with a Brinkmann probe colorimeter (22) and (27). Entrapped volume, K+ concentration, and the content of Quin-2 a TPP+ selective electrode (23), respectively. For some experiments, an were determined as described before (19, 24, 25). The permeability of POPC vesicles to Ca +/Quin-2 was monitored by Aminco DW2a spectrophotometer was used instead of the probe color­ incubating them in 10 mM Hepes (Na+), pH 7.0, containing 20 mM NaCI imeter to determine swelling. In these cases, the data were collected and processed with the aid of a computer system which was interfaced and 50 J1-M CaCI . The development of a permeable membrane was 2 4925 Mitochondrial Permeability Transition :::J 0 • • • • • • • .~ 40 50 0 10 20 30 0 10 20 30 40 50 [Ci+], nmoVmg protein '#. 0 2 4 6 8 10 0 2 4 6 8 10 [Pi]' mM FIG. 3. Effects of Ca + content and Pi concentration on the mastoparan-dependent opening of the permeability transition pore. Ca +-depleted mitochondria were incubated at 0.5 mg of protein/ml and at 25°C. The medium contained 250 mM sucrose, 10 mM Hepes (Tris), and 5 mM succinate (Tris), pH 7.4. Pore opening was monitored by swelling as described under "Experimental Procedures." A, the medium also contained 5 mM Pi (Tris) and CaCl as shown. Pi was present from the beginning, whereas CaCl was added at 1 min following the addition of 2 2 mitochondria. • , 1.0 f.LM mastoparan was added 1 min after the addition of CaCI . 0, mastoparan was not added. Values plotted are the percent of maximal swelling which had occurred at 10 min following the addition of mitochondria. B, same as A, except that Pi was deleted from the medium. C, same as A, except that the mitochondrial Ca + load was 25 nmol/mg protein, and the medium Pi concentration was varied as shown. D, same as C, except that Ca + was not added. indicated by formation of the Ca +.Quin-2 complex, which was deter­ electron microscopy as described before (28). Fractions of total enzyme mined by difference absorbance measurements, using the Aminco activities and Mg2+ released during incubation were determined by DW2a spectrophotometer operated in the dual wavelength mode (wave­ assaying the supernatants, also using established methods (29, 30). The length pair 264 versus 338 nm). The temperature was 25°C, and the release of nucleotides and other coenzymes from the matrix space were nominal POPC concentration was 300 f.LM. At this concentration, the determined by HPLC analysis of the sedimented mitochondria. Pellets entrapped volume is 0.6 f.Ll/ml (24). For some experiments, a membrane were extracted as described by Stocchi et ai. (31), whereas HPLC anal­ potential (inside negative) was imposed across the vesicle membrane by ysis was carried out as described by Novgorodov et ai. (17). adding valinomycin at 0.1 f.LM. Parallel experiments employing the RESULTS TPP+ electrode and application of the Nernst equation showed that the magnitude of the membrane potential produced in this way was ap­ CSA-sensitive and Insensitive Actions of Mastoparan on the proximately 150 mV. Permeability Transition-Fig. 1 demonstrates that 1.0 J.LM mas­ Other Methods-The osmotic pressure of solutions containing PEGs toparan can be used in place of exogenous Ca + to induce a was measured with a Wescor model 5500 vapor pressure osmometer. permeability transition in mitochondria incubated in the pres­ The instrument was calibrated in units of milliosmolal using standard ence of succinate and 5 mM Pi' Under these conditions, the solutions provided by Wescor. Because measurements made with this instrument are based upon the vapor pressure of solutions, the osmotic depolarization and swelling provoked by mastoparan arise pressure values are relatively free of artifacts related to the viscosity of from opening the PTP as indicated by the inhibitory action of PEG-containing solutions. CSA (Fig. 1). Oligomycin and EGTA also inhibit depolarization When examining mitochondrial ultrastructure or determining the and swelling induced by 1.0 J.LM mastoparan (Fig. 2). The action release of enzymes and matrix space solutes, aliquots taken from incu­ of oligomycin is further evidence that mastoparan facilitates bations were initially centrifuged to sediment the mitochondria (Eppen­ the transition, because oligomycin is a well-known inhibitor of dorfmicrocentrifuge, -13,000 x g, 3 min). For ultrastructural studies, pellets were then fixed, stained, and processed for examination by the phenomenon when it is induced by Pi (1, 17). The action of 4926 Mitochondrial Permeability Transition EGTA shows that induction of the transition by 1.0 p..M masto­ Mas paran requires the participation of endogenous Ca +. 0.3 Mitochondria depleted ofendogenous Ca + (21) were utilized to investigate the effect ofCa + content and Pi concentration on mastoparan-dependent induction of the transition. When Pi is present at a concentration of 5 mM, 1.0 p..M mastoparan mark­ 0.7 edly stimulates the transition across a range ofCa + loads (Fig. ::;: 3A), although little or no activity is seen in the absence of ::t. 1.0 exogenous Pi (Fig. 3B). In a similar way, when an intermediate Q. Q. Ca + load is employed, the action of mastoparan is dependent I- 1.3 "ii on Pi concentration (Fig. 3C) with little or no activity seen in the absence of exogenous Ca + (Fig. 3D). These findings dis­ 1.7 tinguish mastoparan from better known inducers of the tran­ 2.0 sition such as hydroperoxides, sulfhydryl reagents, and Pi' 2.3 Rather than inducing a rapid transition in Ca +-loaded mito­ 2.7 chondria, as do these other agents (1), or acting as a substitute 3.0 d 3.3 for Ca + per se, mastoparan enhances the action ofCa + and Pi which are both still required to obtain the transition. Mastoparan also differs from other inducers ofthe transition with respect to the inhibitory activity of CSA. While CSA pre­ RLM vents the action of Pi' hydroperoxides, and other agents for extended periods, and apparently without a sharp dependence Mas on inducing agent concentration (32, 33), increasing the con­ centration of mastoparan from 1 to 3 p..M eliminates the inhib­ itory action of CSA (Fig. 4). A comparison of Figs. 1 and 2 with Fig. 4 illustrates another point of interest. When a low concen­ tration of mastoparan is employed and the resulting transition is sensitive to CSA (Figs. 1 and 2), TPP+ release and swelling proceed after a lag period which is characteristic of the transi­ tion induced by most agents. At higher mastoparan concentra­ (\J '" 0 tions, where sensitivity to CSA is lacking or incomplete (Fig. 4), .!: 'ii • TPP+ release and, to some extent, swelling proceed without a ~~ significant lag period following mastoparan addition. The mastoparan analog MP14 (Table I) facilitates opening of the PTP in a manner analogous to the parent peptide. At relatively low concentrations, the actions of MP14 are depend­ ent upon the Ca + and Pi concentrations and are sensitive to 4min CSA, whereas at higher concentrations, MP14 actions are seen in the absence of free Ca + and are CSA insensitive (data not shown). Fig. 5 shows the CSA-sensitive and insensitive effects FIG. 4. CSA insensitive actions of mastoparan on mitochon­ of the two peptides on PTP opening (swelling response) as a dria. Incubations were conducted as described in the legend to Fig. 1 function of their concentrations. With mastoparan, it is seen with 0.5 fJ-M CSA present from the beginning. TTp· accumulation (upper panel) and swelling (lower panel) were determined simulta­ that the CSA insensitive activity occurs over a concentration neously as described under "Experimental Procedures." For traces la­ range which is approximately twice that required for the CSA­ beled a-d, mastoparan was added where indicated at the following sensitive activity. MP14 is less effective than mastoparan on a concentrations: a, 0 fJ-M; b, 1.0 J.LM; c, 2.0 J.LM; d, 3.0 J.LM. concentration basis, regardless ofwhether the CSA-sensitive or insensitive activities are considered. However, with the latter release and on mitochondrial ultrastructure. Prior to conduct­ peptide, the CSA-sensitive and insensitive concentration ing these studies, the effects of PEGs on the osmotic pressure of curves are offset by 4-5-fold (Fig. 5). mitochondrial incubation media were examined. This work was The Solute Size Exclusion Properties of Mastoparan and conducted because PEGs increase the osmotic pressure of so­ MP14-dependent Pores-A Ca + requirement and CSA sensi­ lutions in disproportion to their concentration (40), a property tivity are the primary biochemical identifiers ofthe permeabil­ which would complicate the use of PEGs to characterize the ity transition. In addition, it is known that mastoparan can pores in question. Fig. 6A shows that small, intermediate, and perturb phospholipid bilayers under certain conditions (e.g. relatively large PEGs increase the osmotic pressure of a dilute Refs. 34-37). These considerations, taken together with the buffer solution in marked excess of their molal concentration. data in Figs. 4 and 5, raise the question of whether the Ca + The extent of this nonideal behavior is a nonlinear function of independent and CSA insensitive depolarization and swelling the weight of PEG per volume of solution and is more extreme produced by these peptides arise from opening the PTP or from for the larger PEGs. Indeed, as the molecular weight increases, direct actions upon the membrane lipid phase. To investigate the weight per volume required to establish a given osmotic this point, the solute size exclusion properties of the pore in­ pressure becomes almost independent of the PEG molecular duced by Ca + plus Pi alone were compared to those ofthe pore weight (Fig. 6B). To maintain a constant osmotic pressure in induced by mastoparan, MP14, and by alamethicin. The latter agent is an established pore-forming peptide (38, 39). 2 The binding of a large number of water molecules per molecule of In the methods used, the effects ofPEGs ofvarious molecular PEG and/or other structural ordering of water, with a resulting reduc­ weights on the large amplitude swelling associated with the tion in the activity of water, are thought to be responsible for the nonideal osmotic properties of PEGs (40). transition were compared to their effects on matrix solute 4927 Mitochondrial Permeability Transition TABLE I The amino acid sequence ofmastoparan and its analog MP14 MP14 differs from mastoparan by a single amino acid substitution at position 12. Position Peptide 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu-NH Mastoparan 2 Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-LyS-Gly-Ile-Leu-NH MP14 FIG. 5. The concentration depend­ E" ence of mastoparan and MP14 effects .~ on mitochondria in the presence and (5 absence of CSA. Data were obtained from experiments like those shown in Fig. #. 4. e, mastoparan was employed in the .~ absence of eSA. _, same as e except that 0.5 J-tM eSA was present from the begin­ ning ofthe incubations. 0, MP14 was em­ ployed in the absence of eSA. D, same as ° except that 0.5 J-tM eSA was present from the beginning of the incubations. o 14 2 4 6 8 10 12 Peptide, f1M native interpretation, decreased swelling might represent PTP mitochondrial incubations containing PEGs (Figs. 7-9), the media were prepared by mixing the normal medium with 300 opening in a decreasing fraction ofthe mitochondria. The latter milliosmolal solutions of PEG dissolved in a dilute buffer, to interpretation must be considered because it is well known that yield the desired concentration of the polymer. The table con­ the transition occurs heterogeneously in mitochondrial popula­ tained within Fig. 6B shows the solution concentrations which tions (1). Furthermore, it is not clear if the PTP is a single yield a 300 milliosmolal osmotic pressure for all PEGs which molecular entity or the same entity in all mitochondria (17). were used. Fig. 6C shows that when the mitochondrial medium Fig. 8 distinguishes between the alternative interpretations. In is mixed with isoosmotic solutions of PEGs, the resulting os­ this figure, panels A-D, it is seen that mitochondrial ultra­ motic pressures remain relatively constant when the fraction of structure is relatively uniform when observed in samples osmotic pressure arising from PEG is varied from 0-100%. which were fixed after the swelling response reached comple­ Under these near isoosmotic conditions, low concentrations tion in the presence of 0, 3, 8, or 20 mM PEG. This structural of 3.4-kDa PEG markedly reduce the magnitude of swelling characteristic is not consistent with PTP opening in a variable response which occurs when the PTP is opened by the action of subfraction of the mitochondria (see Ref. 1). In addition, de­ Ca + plus Pi (Fig. 7). Based upon existing data, a 3.4-kDa PEG creased swelling produced by increasing concentrations of 3.4­ would be expected to pass through the outer membrane volt­ kDa PEG is not accompanied by a decreased release of matrix age-dependent anion channel (VDAC) (41) but not through the Mg2+ (Fig. 8E) nor that of matrix space nucleotides and other PTP (42-45). Several features of these swelling curves are cofactors, as determined by HPLC analysis (data not shown). noteworthy. For all but the highest PEG concentrations em­ Finally, malate dehydrogenase release was minimal or absent ployed, the curves are monophasic and produce a final absorb­ across the full range of PEG concentration investigated, al­ ance change which is relatively stable. The latter characteristic though a marked release of adenylate kinase activity was ob­ is taken to indicate that 3.4-kDa PEG does not slowly permeate served which was diminished by PEG. Taken together, these through the PTP. Near the high end of the concentration range, data strongly indicate that the concentration-dependent inhi­ some multiphasic behavior is seen. This might indicate that bition of swelling produced by 3.4-kDa PEG results from a movement of 3.4-kDa PEG through VDAC is somewhat re­ reduced matrix space expansion in all, or nearly all, of the stricted, as is discussed further below. Finally, the initial rate mitochondria and not from PTP opening in a decreasing frac­ of swelling is seen to be essentially constant as the PEG con­ tion of the mitochondria. In addition, the enzyme release data centration increases. This suggests that PEG does not impede show that the limited swelling which is observed in the pres­ the access of low molecular weight solutes to the PTP or oth­ ence of PEG does not reflect rupture of the inner membrane in erwise slow their rate of permeation. some of the mitochondria (absence of malate dehydrogenase The above interpretations are based upon the assumption release), although rupture ofthe outer membrane occurs and is that the decreased swelling produced by increasing PEG con­ subject to inhibition by 3.4-kDa PEG (adenylate kinase centrations results primarily from a decrease in the maximal release). matrix space volume which is attained following opening of With the above information as a background, the size exclu­ PTP and that the PTP opens in all mitochondria. By an alter- sion properties of "permeability transition pores" induced in 4928 Mitochondrial Permeability Transition B C 10 A 400 M.W. mM iii 1000 400 219 I (5 '? 8 600 189 0 E (/) ... 1000 150 (; X 1500 E E a. 2000 102 1: o i~:-.-.-.-.-._._._.~O 3400 64 e- 6 e- 'Q) ::l ::l ~o ,0-0 I (/) 4000 55 (/) 300 (/) ~ (/) ._~-o,%-o; ",,/ 600 6000 38 l!! l!! '... ..... ...- ....... ta 8000 28 a. a. -...-"" 1ססoo 1i 23 4 ,g ~ I ~ 0 :iE (/) 200 A/ A/ A_A/' 0 25 50 75 100 100 200 300 400 0 75 150 225 300 PEG Solution, % PEG, mg/ml PEG, mg/ml FIG. 6. The osmotic pressure properties ofPEG·containing solutions. A, measured osmotic pressure values of3 mM Hepes buffer, pH 7.4, containing increasing amounts of 0.6 (e)-, 3.4 (0)-, or 8.0 (.)-kDa PEG. The dashed lines in this panel show how osmotic pressure would vary if solutions of 0.6 (a)-, 3.4 (b)-, or 8.0 (c)-kDa PEG behaved ideally. These lines were located by preparing single solutions of each PEG utilizing weighed amounts of PEG and solvent. The molal concentration of the solution could then be calculated, providing a y axis coordinate for the solution. The x axis coordinate was located by noting the volume of each solution and dividing the weight of PEG used by the volume to yield milligrams of PEG/ml of solution. Straight lines were then drawn as defined by these points and the origin. B, the concentration of PEG in milligrams per ml of solution required to give an osmotic pressure of 300 mosM, as a function of PEG molecular weight. These values were determined from experiments like those shown in A. The table embedded in B shows the same data expressed numerically in concentration units of mM. C, measured osmotic pressure values of 0.6 (e)-, 3.4 (0)-, and 8.0 (.)-kDa PEG solutions progressively diluted with a mitochondrial incubation medium. As seen in the panel, both the initial PEG solutions and the mitochondrial incubation medium had osmotic pressures near 300 milliosmolal when measured individually. [PEGj,mM FIG. 7. Inhibition ofPTP-dependent swelling by selected concentrations of 3.4·kDa PEG. Mitochondria were in­ l------------------- 10 cubated at 1.0 mg of protein/ml and at \\'--------------------7.0 25 cC, in isoosmotic media which did not contain an exogenous respiratory sub- "------------------5.0 strate. The media were prepared by mix- ing 300 milliosmolal mannitol/sucrose/ -----------------4.0 Hepes (mitochondrial isolation medium) with a medium comprised of 64 mM 3.4- ----------------- 3.0 kDa PEG in 3 mM Hepes, pH 7.4 (also 300 milliosmolal) to yield the indicated con­ centrations of PEG, while maintaining a ------------- 2.0 constant osmotic pressure (see Fig. 6). P; was present at a final concentration of 2 mM. CaCl was added where shown, at 50 2 -----------1.0 nmol/mg protein, and swelling was subse- quently monitored as described under "Experimental Procedures." 3 min alamethicin pore is clearly larger, with PEG of molecular several ways were examined by observing the maximal extents of swelling which occur when the medium contains PEG of weight near 1700 being required to produce a comparable re­ duction in the extent of swelling (Fig. 9B). The remaining various molecular weights. The concentrations of PEGs were panels in Fig. 9 show the size exclusion properties of the pores selected and the solutions were prepared such that 40% of the induced by mastoparan and MP14 in the presence or absence of osmotic pressure was obtained from PEG, while the total os­ CSA. No marked differences are seen, although all of these motic pressure remained near 300 milliosmolal (Fig. 6). The pores may be slightly larger than the PTP as induced by Ca + PTP induced by Ca + plus Pi can be easily distinguished from the pore formed by alamethicin using this method. A compar­ plus Pi alone. ison of A and B of Fig. 9 illustrates this, together with the Mastoparan and MP14 Permeabilize Phospholipid Vesicles: the Effect of Membrane Potential-To aid in interpreting the approach taken when analyzing these data. The Ca + plus actions of mastoparan and MP14 on mitochondria, their effects Pcinduced PTP produces a swelling response which is one-half inhibited by PEG of a molecular weight near 650 (Fig. 9A). The on a model phospholipid membrane were investigated. POPC Mitochondrial Permeability Transition 4929 linear peptides were added in the case of the membrane poten­ tial present condition (data not shown). To analyze data like those shown in Fig. 10, trace B, the relative initial rate of Ca +·Quin-2 complex formation was estimated by fitting early portions of the progress curve (non­ linear least square techniques, see Ref. 19) to the expression: AT = A + Bt + Ct . In this expression, AT and A are the total o o and initial difference absorbance values, respectively, B is the initial rate, C is a correction factor for nonlinearity, and t is time. The right side panel of Fig. 10 shows the effect of masto­ paran and MP14 concentration on vesicle permeability as de­ termined by this method. No significant differences between the two peptides are apparent in the absence of a membrane potential. When a membrane potential is present, however, :0 mastoparan is markedly more effective than its analog. The .~ increment produced by membrane potential, compared to no membrane potential was 5.8- and 2.3-fold for mastoparan and "5 MP14, respectively. This panel also shows that for all cases oi examined, the relationship between peptide concentration and c: '" 40 vesicle permeability are linear. ., '" a: DISCUSSION Mastoparan is a 14-amino acid amphipathic peptide ob­ tained from wasp venom (47-53). It possesses a wide spectrum of pharmacological activities including mast cell degranulation (50), activation of G-protein-mediated mechanisms (54-58), 10 15 20 25 0 5 inhibition of calmodulin-mediated mechanisms (59-61), stim­ [PEGI. mM ulation of phospholipases A and C (55, 62, 63), stimulation or inhibition of cation-specific channels (64), and others (see Refs. FIG. 8. The fraction of mitochondria which undergo PTP open­ 51 and 65). Facilitation of the mitochondrial permeability tran­ ing as a function of 3.4-kDa PEG concentration. Incubations were conducted as described in the legend to Fig. 7. For all parameters, sition can now be added to this broad spectrum of activities. It samples were taken 8 min after the addition of Ca +, and mitochondria should be noted that this newly recognized action of mastopa­ were sedimented u ing a microcentrifuge (-13,000 x g for 3 min). For ran is marked at concentrations <1 J.LM (Fig. 5), whereas the A-D, the supernatants were removed and the pellets were fixed, em­ other activities are typically seen over a 5-100 J.LM range. Thus, bedded, and examined by electron microscopy as described previously (28) (total magnification = 3,000). The medium concentrations of 3.4­ the actions of mastoparan on mitochondria are the most potent kDa PEG were as follows: A, a mM; B, 3 mM; C, 8 mM; D, 20 mM. For E, described to date and may well be involved in the toxicological the PEG concentration was as shown. The extents of swelling (.), mechanism of this peptide, given the relationship between the malate dehydrogenase release (0), adenylate kinase release (ll.), and transition and the death of injured cells (see introduction to the Mg2+ release (A) were determined using the supernatants obtained after sedimenting mitochondria, as described under "Experimental text). It should also be noted that as a modulator of the tran­ Procedures." sition, mastoparan is more potent than most of the known agents (1). Whether or not the inner membrane pore(s) induced by both vesicles containing Quin-2 were prepared and incubated in a medium containing Ca +. Under the conditions employed, the low (:51 J.LM) and high (~2 J.LM) levels of mastoparan are in fact nominal phospholipid concentration equates approximately to the PTP is an important question because all other inducers a mitochondrial suspension at 1.5 mg of protein/ml, whereas require Ca + for activity, with the apparent exception of phen­ ylarsine oxide (66), and are poorly active in the presence of the entrapped volume (-0.6 J.LlIml) is similar to that of mito­ chondria in an isoosmotic medium at 0.8 mg of protein/ml (see CSA, even when they are used over a broad concentration range (1, 3). In the lower concentration range, pore induction Refs. 24 and 46). These vesicles contained an internal K+ concentration of -60 mM and were suspended in a Na+­ by mastoparan also requires Ca +, is facilitated by Pi' and is inhibited by CSA and by oligomycin (Figs. 1-3). Given these containing medium. Measurements carried out with a TPP+ electrode showed that the addition of valinomycin produced a characteristics, it is very likely that this pore is in fact the PTP. To test the identity of the pore formed in the higher mastopa­ membrane potential of -150 mV, inside negative, which is also similar to the situation existing with intact mitochondria (data ran concentration range, the solute size exclusion properties of several pores were compared (Fig. 9). By this criterion, the pore not shown). In this system, the development of a permeable membrane is indicated by formation of the Ca +·Quin-2 com­ induced by mastoparan in the presence of CSA is also the PTP as are the CSA-sensitive and insensitive pores induced by plex which was monitored by dual wavelength spectroscopy (see 19). Fig. 10 shows that these vesicles are impermeant to MP14. In considering these interpretations it must also be Ca + and Quin-2 under these conditions, in the presence or noted that mastoparan and MP14 perturb phospholipid bilay­ absence of a membrane potential, when mastoparan is absent ers by a mechanism which is membrane potential-dependent (Fig. 10) but otherwise unknown. It is thus possible that the (trace A). The addition of 2.0 J.LM mastoparan produces a slow permeation of Ca + and/or Quin-2 and a much faster perme­ peptide molecules per se form pores in the inner mitochondrial ation when a membrane potential is present (trace B). Similar membrane and that these pores, rather than the PTP, are results (not shown) were obtained with MP14. The rates of responsible for the CSA independent activities. While this in­ permeation in the presence or absence of a membrane potential terpretation remains viable, it seems improbable because it were independent of Ca + concentration over a range of 10­ would be highly fortuitous if the pores formed by both peptides 1000 J.L 1, were not significantly affected by CSA (1.0 J.LM), and were so similar to each other, and to the PTP, by the criterion were independent of the order in which valinomycin and the of solute size exclusion. 4930 Mitochondrial Permeability Transition 100 100 •. A B e. 80 80 '! , :----\~. , , \ ______J ~:::::::=.-. FIG. 9. Solute size exclusion proper­ 20 1700 ties of pores in mitochondria. Mito­ 20 _J ~_=~~....__. chondria were incubated at 0.5 mg of pro­ • tein/ml and at 25°C in -300 milliosmolal o "-'-----'----'_-'--....L.---L_.L.---'---'---' 0'-'---'-_"--'-_1.--1...--'_--'---"--' media containing 5 mM concentration o 2 345 6 7 8 o 2 345 6 7 8 each of succinate (Tris) and P, (Tris), pH 7.4.40% ofthe total osmotic pressure was derived from PEG and the remainder from mannitol/sucrose (3:1 mole ratio) 100 100 plus the other solutes described above. C 0 PEGs ranging from 0.6 kDa to 8 kDa were :;, employed, as illustrated in the figure, and E 80 80 these were mixed with mannitol/sucrose .~ to give the desired fraction of osmotic 60 60 pressure derived from PEG, while keep­ ing the total value near 300 milliosmolal "6 (see Fig. 6). Swelling (pore formation) was 40 40 initiated after a 2-min preincubation and allowed to proceed until the apparent ab­ __j ____.._.-.-I-t sorbance of the suspensions became con­ 20 20 stant. These final values, presented as a percent of the value obtained in the ab­ 0 0 sence of PEG, are plotted as a function of 0 2 3 4 5 6 7 8 0 2 3 4 5 6 7 8 the PEG molecular weight. Each value is the mean ± S.E. of three determinations, with each replicate value obtained using a separate mitochondrial preparation. Pore formation was induced as follows: A, 100 .0 CaCl (50 nmol/mg protein); B, alamethi­ cin (3.5 /Lg/ml); C, mastoparan (1.0 /LM); D, mastoparan (3.0 /LM) and the medium contained l/LM CSA; E, MP14 (3.0 /LM); F, MP14 (ll/LM) and the medium contained l/LM CSA. :\ ,. l \ 20 "- !=--- ....__.-. OLL.....-'----'--'---'---'----'---''--..J.-j o 2 3 4 5 6 7 8 2 345 6 7 8 PEG M.W. X 10. There is a second aspect of the solute size exclusion data The present results are not subject to some of the uncertain­ presented here which is of interest and which becomes appar­ ties in the earlier data, and, as a consequence, they show that ent when these data are compared to earlier studies in which when the pore is induced by Ca + plus Pi' increasing the PEGs were utilized to characterize the size exclusion properties molecular weight of PEG in the medium from 0.4 to 4 kDa of the PTP (42, 44, 45). There are disagreements between the produces a progressive decrease in swelling. This behavior is early studies which may reflect pore induction by differing unexpected because each point was obtained at a (essentially) agents, and/or technical considerations arising from unrecog­ constant osmotic pressure, with PEG providing the same frac­ nized osmotic pressure properties of PEGs, the use of a limited tion of that pressure, and represents an apparent equilibrium number of PEG molecular weights, and other factors. Haworth condition (i.e. the extents of swelling were not increasing sig­ and Hunter (induction by arsenate) (42) reported that 1.5- and nificantly at the time the values were taken). If the PTP is a 4-kDa PEGs are excluded, 0.6-kDa or smaller PEGs are per­ rigid structure, one would expect a sharper cutoff in the mo­ lecular weight of PEG which is fully excluded. If it is a flexible meant, whereas an intermediate condition exists with l-kDa PEG. Vercesi (induction by oxalacetate) (45) reported that structure which can impede yet pass solutes of variable size, swelling is eliminated by l-kDa PEG. Le-Quoc and Le-Quoc one would expect the smaller PEGs to decrease the rate of swelling instead ofproducing an intermediate and stable value. (induction by N-butylmaleimide) (44) found no permeation of 6-kDa PEG but progressively faster permeation of 4-, 1.5-, and The presence of molecular weight heterogeneity in the various 0.6-kDa PEG. Large fractions ofmatrix space enzyme activities samples of PEG, together with a sieving action by the PTP, were released under the conditions of their study and the cannot easily explain the observed behavior. According to the authors concluded that an association of VDAC with the ade­ manufacturer, this heterogeneity does not exceed 2:10% of the nine nucleotide translocase forms the PTP (44). stated value, and filtration experiments conducted with some 4931 Mitochondrial Permeability Transition ±Val 0.8 A -.-. ...._"' ...-.... , ............__ ""J"....... t'"...... , .., ••",,*....""......"'""... """"_.. ±Val 0.7 0.6 0.5 0.4 ~i 0.3 0.2 0.1 0.0 3 min 0 2 3 Peptide, /1M FIG. 10. Membrane potential-dependent and independent actions of mastoparan and MP14 on the permeability of POPC vesicles. Quin 2/HepesJK+ load POPC vesicles were prepared and incubated as described under "Experimental Procedures." The nominal POPC concen­ tration was 300 J.l.M, and the external medium contained 10 mM Hepes (Na+), pH 7.0, 20 mM NaCl, and 50 /LM CaCl • Increased membrane permeability was indicated by formation of the Ca +'Quin 2 complex, which was monitored by dual wavelength absorbance measurements made at 294 versus 338 nm (19). A (left panel), valinomycin (0.1 /LM) was added or not added, where indicated. B (left panel), same as A except that mastoparan (2.0 J.l.M) was added where indicated. Right panel, a summary plot ofthe initial rates of absorbance change as a function of mastoparan or MP14 concentration, as determined by fitting the data to the equation given under "Results." • and ., mastoparan and MP14, respectively, membrane potential present. 0 and 0, mastoparan and MP14, respectively, membrane potential absent. of the samples were consistent with this specification (data not and CSA insensitive manner. The present data cannot iden­ shown). At the higher end of the molecular mass range (4 and tify the allosteric site at which the peptides may act, although it is noted that mastoparan carries three positive charges, possibly 3.4 kDa), PEG may act, in part, because it is partially excluded by VDAC (41). This could relax the oncotic pressure whereas MP14 carries two (Table I). Thus, the relative po­ tency of these peptides correlates with positive charge and gradient across the outer membrane, contract the intermem­ they might act at a site which normally binds a cation. The brane space volume, and raise the protein concentration, so that reduced swelling occurs when the PTP is opened. This Ca + binding site is one possibility and an action at this site possible explanation is not applicable in the case ofthe smaller could explain why pore induction can occur in the absence of PEGs, however, since they clearly permeate VDAC (41). The Ca + when the peptide concentration is high. Possible dis­ existence of different size pores in individual mitochondria placement of CSA from its site of action by high concentra­ could explain the observed behavior, and differences in the tions of mastoparan is another possibility to consider. mechanisms which gate solute entry from opposite sides of the Analogies between regulation of the PTP and the N-methyl­ membrane might also explain this behavior. No choice between n-aspartic acid receptor channel suggest that the PTP belongs such explanations seems possible without further studies, to the super family of ligand gated ion channels and thus may which are now in progress. be regulated by covalent modification (see Ref. 3). Such systems Induction of the transition by mastoparan and MP14 may often involve G-proteins, and a mitochondrial G-protein has occur through PTP regulatory mechanisms which are already recently been identified and isolated (67, 68). According to a recognized (1-3). However, since these peptides have a wide recent report, MP14 retains the G-protein-mediated activities spectrum of pharmacological activities (see above), new po­ of mastoparan but has a diminished capacity to perturb mem­ tential regulatory mechanisms can also be considered. Re­ branes (69). It is for these reasons that the actions of MP14 and garding the established mechanisms, it does not appear that mastoparan on the PTP were compared in this study. MP14 is reducing membrane potential is the primary mode by which only slightly less active than mastoparan when inducing pore the peptides act under CSA-sensitive conditions. This is in­ opening in a CSA-sensitive manner, whereas there is a larger dicated by the TPP+ accumulation data in Figs. 1 and 2 which activity differential when the pore is induced in the presence of show that mastoparan produces only minor changes in mem­ CSA (Fig. 5). If the CSA insensitive induction involves mem­ brane potential when CSA, EGTA, or oligomycin are present brane perturbation (depolarization) as suggested above, then to inhibit pore opening. The same is true for MP14 (data not these data suggest regulation of the PTP through a G-protein. shown). Under CSA insensitive conditions, an early and ex­ The relative effectiveness of mastoparan and MP14 have not tensive release of TPP+ is seen and this seems to occur yet been compared in a variety of systems, however, and so it is somewhat faster than swelling (Fig. 4). This depolarization not clear how completely the G-protein-mediated and mem­ could be brought about by peptide molecules acting upon the brane perturbation-mediated activities of MP14 are distin­ membrane lipid phase as illustrated in Fig. 10. It is thought guished. In particular, it appears that a membrane perturba­ that no PTP effector dominates in the interactive system of tion activity differential between these two peptides is only pore regulation through allosteric and membranelbioener­ seen in the presence of a membrane potential (Fig. 10) and the getic mechanisms. Instead, the open/closed probability ap­ reason for this deserves further investigation. pears to be established as a sum of positive and negative The actions of mastoparan and MP14 on the PTP suggest actions exerted at a number of sites (17). Thus, the peptides that low concentrations of other amphipathic peptides could may act at an allosteric site, synergistically with Ca + and Pi regulate the PTP in cells. Such peptides are a product of proc­ acting their sites, under CSA-sensitive conditions. It could essing imported proteins and may accumulate in the mitochon­ then be the additional influence of depolarization produced drial matrix under some conditions. Extra- and intramatrix by higher peptide levels acting upon the membrane lipid space amphipathic peptides may also accumulate in injured phase which allows the pore to open in a Ca + independent cells due to proteolysis. These peptides may promote pore open- 4932 Mitochondrial Permeability Transition 264,7826-7830 ing, even in presence of eSA, and become a factor in maintain­ 34. Katsu, T., Kuroko, M., Morikawa, T., Sanchika, K, Yamaraka, H., Shinoda, S., ing the protection of injured cells afforded by eSA. and Fujita, Y. (1990) Biochim. Biophys. Acta 1027, 185-190 35. Tanimura, A., Matsumoto, Y., and Tojyo, Y. (1991) Biochem. Biophys. Res. Acknowledgments-We thank Wayne Anderson of the Hormel Insti­ Commun. 177,802-808 tute for carrying out the electron microscopy and Ronald Louters ofThe 36. Raynor, R. 1., Kim, Y.-S., Zheng, B., Vogler, W. R, and Kuo, J. F. (1992) FEES Lett. 307,275-279 Ohio State University for technical assistance in other areas. 37. 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Broekemeier, K M., and Pfeiffer, D. R. (1989) Biochem. Biophys. Res. 69. Danilenko, M., Worland, P., Carlson, B., Sausville, E. A, and Sharoni, Y. Commun. 163, 561-566 (1993) Biochem. Biophys. Res. Cornrnun. 196, 1296-1302 33. Broekemeier, K M., Dempsey, M. E., and Pfeiffer, D. R (1989) J. Bioi. Chem. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

The Peptide Mastoparan Is a Potent Facilitator of the Mitochondrial Permeability Transition

Pfeiffer, Douglas R.; Gudz, Tatyana I.; Novgorodov, Sergei A.; Erdahl, Warren L.
Journal of Biological Chemistry – Mar 1, 1995
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10.1074/jbc.270.9.4923
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Vol. 270, No.9, Issue of March 3, pp. 4923-4932, 1995 THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. The Peptide Mastoparan Is a Potent Facilitator of the Mitochondrial Permeability Transition* (Received for publication, August 22, 1994) Douglas R. Pfeiffer:j:, Tatyana I. Gudz, Sergei A. Novgorodov, and Warren L. Erdahl From the Department of Medical Biochemistry, College of Medicine, The Ohio State University, Columbus, Ohio 43210 and the Hormel Institute, University of Minnesota, Austin, Minnesota 55912 ation of the transition with cell injury mechanisms and the Mastoparan facilitates opening of the mitochondrial identification of ~yclosporin A (CSA)! as a potent inhibitor of permeability transition pore through an apparent bimo­ dal mechanism of action. In the submicromolar concen­ the phenomenon (11-14) has created a high level of interest in tration range, the action of mastoparan is dependent this aspect of mitochondrial research. upon the medium Ca + and phosphate concentration Many of the agents which induce the transition are toxins and is subject to inhibition by cyclosporin A. At concen­ and pharmacological agents not normally encountered in vivo trations above 1 /.LM, pore induction by mastoparan oc­ (1). The same is true for the known inhibitors (1). Both induc­ curs without an apparent Ca + requirement and in a ing agents and inhibitors are chemically diverse but display a cyclosporin A insensitive manner. Studies utilizing common activity in favoring an open or closed state of the phospholipid vesicles show that mastoparan perturbs permeability transition pore (PTP), respectively. It has proven bilayer membranes across both concentration ranges, difficult to explain the analogous actions of diverse compounds through a mechanism which is strongly dependent upon on the PTP. Bernardi and co-workers (15,16) have shown that transmembrane potential. However, solute size exclu­ membrane potential and matrix pH are central PTP regulators. sion studies suggest that the pores formed in mitochon­ Membrane surface potential may also be a central regulator (3, dria in response to both low and high concentrations of 17). It is thus possible that the chemical diversity/common mastoparan are the permeability transition pore. It is activity problem associated with the transition may arise, in proposed that low concentrations of mastoparan influ­ part, from the effects of transition regulators on bioenergetic ence the pore per se, with higher concentrations having parameters and membrane physical properties. the additional effect of depolarizing the mitochondrial Although many known transition effectors would not nor­ inner membrane through an action exerted upon the mally be encountered in cells, a number of activators (e.g. Ca +, lipid phase. It may be the combination of these effects Pi' acyl-CoA) and inhibitors (e.g. ADP, M~+, polyamines) are which allow pore opening in the absence of Ca + and in the presence of cyclosporin A, although other interpre­ physiological cell constituents. In seeking to understand regu­ tations remain viable. A comparison of the activities of lation ofthe transition in an intracellular setting, it then seems mastoparan and its analog, MP14, on mitochondria and reasonable to consider these agents as primary regulators and phospholipid vesicles provides an initial indication that to investigate how other cellular components and conditions a G-protein may participate in regulation of the perme­ affect their actions on the PTP. The present report demon­ ability transition pore. These studies draw attention to strates that the 14-amino acid peptide mastoparan has a potent peptides, in a broad sense, as potential pore regulators stimulatory effect on PTP opening when mitochondria are in­ in cells, under both physiological and pathological 2 cubated in the presence of Ca + and Pi' At somewhat higher conditions. concentrations, mastoparan appears to induce pore opening in a Ca + independent and CSA insensitive manner. This is the first report of a peptide exerting a regulatory influence on the The mitochondrial permeability transition remains an in­ transition and, as such, draws attention to a new class of completely understood phenomenon, in spite of the fact that it potential regulators of this phenomenon within cells. In this was first observed in the 1950s and has been studied exten­ report, the actions of mastoparan on the transition are also sively, particularly during the last 15 years (see Refs. 1-3 for compared to those of an analog, MPI4. Differences observed review). Recent work utilizing the patch clamp technique ap­ between the potency of these two peptides are consistent with plied to isolated mitochondria indicates that the transition is the hypothesis that components of cell signaling mechanisms, caused by the opening of large pores in the inner membrane 2 apart from Ca +, are involved in regulation ofthe PTP. Aspects (4-6). At present there is no known physiological function of of these data have been presented in abstract form (18). this phenomenon; however, it is clear that it occurs in situ, EXPERIMENTAL PROCEDURES under conditions of oxidative stress, and is an event that can be pivotal in the mechanisms leading to cell death (7-10). Associ- Reagents-Polyethylene glycols (PEGs) were obtained from Aldrich (average M = 400,600, 1,000, 1,500,2,000,3,400,8,000, and 10,000, or from Fluka (average M = 4,000 and 6,000). Mastoparan and alamethi­ cin were from Sigma. The mastoparan analog MP14 (see Table I for * This research was supported by United States Public Health Ser­ structure) was obtained from Quality Controlled Biochemicals, Inc. vice Grants HL 49182, HL 49181, and HL 36124 from the National (Hopkinton, MA). The purity (>95%) and sequence of this peptide were Institutes of Health, NHLBI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The abbreviations used are: CSA, cyclosporin A; PEG, polyethylene :j: To whom correspondence and reprint requests should be addressed: glycol; POPC, 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylcholine; PTP, The Ohio State University, Dept. of Medical Biochemistry, 310A Ham­ permeability transition pore; TPP+, tetraphenylphosphonium cation; ilton Hall, 1645 Neil Ave., Columbus, OH 43210-1218. Tel.: 614-292­ VDAC, voltage-dependent anion channel; HPLC, high performance liq­ 8774; Fax: 614-292-4118. uid chromatography. This is an Open Access article under the CC BY license. 4924 Mitochondrial Permeability Transition Ca + (Mas) Mas 0.7 0.7 1.0 1.0 ::l. ::l. 1.3 0. 0. 1.3 ~ 0. 1.7 lil 1.7 lil 2.0 2.0 2.3 ~ w 2.3 2.7 2.7 3.0 3.0 3.3 3.3 a RLM RLM Ca + (Mas) 1 Mas 01 1 ._------------_._------~--------------_ ..-----._----- a I 4min 4mln t---------i FIG. 1. Stimulation of the permeability transition by mastopa­ FIG. 2. Stimulation of the permeability transition by mastopa­ ran; inhibition by CSA. Mitochondria were incubated at 0.5 mg of ran; inhibition by EGTA and oligomycin. Mitochondria were incu­ protein/ml and at 25°C. The medium contained 250 mM sucrose, 10 mM bated as described in the legend to Fig. 1, and TPP+ accumulation Hepes (Tris), 5 mM succinate (Tris), 5 mM Pi (Tris), pH 7.4, plus 3.3 J1-M (upper panel) and swelling (lower panel) were determined simulta­ TPP·C!. TPP+ accumulation and release (upper panel) and swelling neously as described under "Experimental Procedures." For all traces (lower panel) were determined simultaneously as described under "Ex­ (a-c), 1.0 J1-M mastoparan was added where indicated. For traces labeled perimental Procedures." CaC~ (dashed lines) or mastoparan (solid band c, the medium contained EGTA (0.5 mM) or oligomycin (1 J1-g/mg lines) were added where indicated at 25 nmol/mg of protein and 1.0 J1-M, protein), respectively, from the beginning of the incubation. respectively. For traces labeled a, the medium contained CSA at 0.5 J1-M from the beginning of the experiment. For traces labeled b, CSA was absent. to the spectrophotometer. Preparation of Phospholipid Vesicles and Determination of Vesicle confirmed by the supplier, following its synthesis by solid state Permeability-Freeze-thaw extruded POPC vesicles loaded with Quin-2 methods. Synthetic I-palmitoyl-2-01eoyl-sn-glycerophosphatidylcholine (K+) and Hepes (K+), pH 7.0, were prepared as described previously (24, (POPC) was obtained from Avanti Polar Lipids. Quin-2 was obtained 25). Briefly, 250 mg of POPC in chloroform was dried by rotation under from Molecular Probes and was deionized and converted to the K+ salt a nitrogen stream, to produce a film on the wall of a 25 X 150 mm as described previously (19). Other chemicals were obtained from com­ culture tube. Residual solvent was removed under high vacuum (4 h), mercial sources and were reagent grade or better. and the film was subsequently hydrated in 5 ml of medium containing Mitochondrial Preparations and Incubation Conditions-Liver mito­ 10 mM Hepes buffer and 5 mM Quin-2. The mixture was vortexed until chondria were prepared by a standard procedure (20) from male the entire film was suspended and the resulting multilamellar vesicles Sprague-Dawley rats weighing -250 g. Bovine serum albumin (2 mg! were frozen in a dry ice-acetone bath, thawed in lukewarm water, and ml) and EGTA (0.5 mM) were present during homogenization but were vortexed again. The freeze-thaw and vortexing procedure was repeated twice. Following this, the preparations were extruded three times excluded from the washing medium. For some experiments these prep­ arations were depleted of endogenous Ca + by the method of Wingrove through two stacked O.I-J1-m polycarbonate membrane filters. This step and Gunter (21). Unless otherwise specified, incubations were con­ was followed by six additional freeze-thaw cycles and eight additional ducted at 25°C, and 0.5 mg of protein/ml, in a medium containing 250 extrusions. The resulting unilamellar preparations were then applied mM sucrose, 10 mM Hepes (Tris), pH 7.4, 5 mM succinate (Tris), and to Sephadex G-50 minicolumns to remove extravesicular Quin-2 (26). rotenone (2 J1-M). Deviations from this medium and other reagents The columns, which were eluted by low speed centrifugation (26), had previously been equilibrated with 10 mM Hepes (Na+), pH 7.0, in 20 mM employed are described in the figure legends. The permeability transi­ tion was monitored by swelling measurements and by tetraphenylphos­ NaC!. The nominal concentrations of POPC in the final preparations phonium (TPP+) release (loss of membrane potential) which were de­ was 70-90 mM as determined by measurements of lipid phosphorus termined simultaneously with a Brinkmann probe colorimeter (22) and (27). Entrapped volume, K+ concentration, and the content of Quin-2 a TPP+ selective electrode (23), respectively. For some experiments, an were determined as described before (19, 24, 25). The permeability of POPC vesicles to Ca +/Quin-2 was monitored by Aminco DW2a spectrophotometer was used instead of the probe color­ incubating them in 10 mM Hepes (Na+), pH 7.0, containing 20 mM NaCI imeter to determine swelling. In these cases, the data were collected and processed with the aid of a computer system which was interfaced and 50 J1-M CaCI . The development of a permeable membrane was 2 4925 Mitochondrial Permeability Transition :::J 0 • • • • • • • .~ 40 50 0 10 20 30 0 10 20 30 40 50 [Ci+], nmoVmg protein '#. 0 2 4 6 8 10 0 2 4 6 8 10 [Pi]' mM FIG. 3. Effects of Ca + content and Pi concentration on the mastoparan-dependent opening of the permeability transition pore. Ca +-depleted mitochondria were incubated at 0.5 mg of protein/ml and at 25°C. The medium contained 250 mM sucrose, 10 mM Hepes (Tris), and 5 mM succinate (Tris), pH 7.4. Pore opening was monitored by swelling as described under "Experimental Procedures." A, the medium also contained 5 mM Pi (Tris) and CaCl as shown. Pi was present from the beginning, whereas CaCl was added at 1 min following the addition of 2 2 mitochondria. • , 1.0 f.LM mastoparan was added 1 min after the addition of CaCI . 0, mastoparan was not added. Values plotted are the percent of maximal swelling which had occurred at 10 min following the addition of mitochondria. B, same as A, except that Pi was deleted from the medium. C, same as A, except that the mitochondrial Ca + load was 25 nmol/mg protein, and the medium Pi concentration was varied as shown. D, same as C, except that Ca + was not added. indicated by formation of the Ca +.Quin-2 complex, which was deter­ electron microscopy as described before (28). Fractions of total enzyme mined by difference absorbance measurements, using the Aminco activities and Mg2+ released during incubation were determined by DW2a spectrophotometer operated in the dual wavelength mode (wave­ assaying the supernatants, also using established methods (29, 30). The length pair 264 versus 338 nm). The temperature was 25°C, and the release of nucleotides and other coenzymes from the matrix space were nominal POPC concentration was 300 f.LM. At this concentration, the determined by HPLC analysis of the sedimented mitochondria. Pellets entrapped volume is 0.6 f.Ll/ml (24). For some experiments, a membrane were extracted as described by Stocchi et ai. (31), whereas HPLC anal­ potential (inside negative) was imposed across the vesicle membrane by ysis was carried out as described by Novgorodov et ai. (17). adding valinomycin at 0.1 f.LM. Parallel experiments employing the RESULTS TPP+ electrode and application of the Nernst equation showed that the magnitude of the membrane potential produced in this way was ap­ CSA-sensitive and Insensitive Actions of Mastoparan on the proximately 150 mV. Permeability Transition-Fig. 1 demonstrates that 1.0 J.LM mas­ Other Methods-The osmotic pressure of solutions containing PEGs toparan can be used in place of exogenous Ca + to induce a was measured with a Wescor model 5500 vapor pressure osmometer. permeability transition in mitochondria incubated in the pres­ The instrument was calibrated in units of milliosmolal using standard ence of succinate and 5 mM Pi' Under these conditions, the solutions provided by Wescor. Because measurements made with this instrument are based upon the vapor pressure of solutions, the osmotic depolarization and swelling provoked by mastoparan arise pressure values are relatively free of artifacts related to the viscosity of from opening the PTP as indicated by the inhibitory action of PEG-containing solutions. CSA (Fig. 1). Oligomycin and EGTA also inhibit depolarization When examining mitochondrial ultrastructure or determining the and swelling induced by 1.0 J.LM mastoparan (Fig. 2). The action release of enzymes and matrix space solutes, aliquots taken from incu­ of oligomycin is further evidence that mastoparan facilitates bations were initially centrifuged to sediment the mitochondria (Eppen­ the transition, because oligomycin is a well-known inhibitor of dorfmicrocentrifuge, -13,000 x g, 3 min). For ultrastructural studies, pellets were then fixed, stained, and processed for examination by the phenomenon when it is induced by Pi (1, 17). The action of 4926 Mitochondrial Permeability Transition EGTA shows that induction of the transition by 1.0 p..M masto­ Mas paran requires the participation of endogenous Ca +. 0.3 Mitochondria depleted ofendogenous Ca + (21) were utilized to investigate the effect ofCa + content and Pi concentration on mastoparan-dependent induction of the transition. When Pi is present at a concentration of 5 mM, 1.0 p..M mastoparan mark­ 0.7 edly stimulates the transition across a range ofCa + loads (Fig. ::;: 3A), although little or no activity is seen in the absence of ::t. 1.0 exogenous Pi (Fig. 3B). In a similar way, when an intermediate Q. Q. Ca + load is employed, the action of mastoparan is dependent I- 1.3 "ii on Pi concentration (Fig. 3C) with little or no activity seen in the absence of exogenous Ca + (Fig. 3D). These findings dis­ 1.7 tinguish mastoparan from better known inducers of the tran­ 2.0 sition such as hydroperoxides, sulfhydryl reagents, and Pi' 2.3 Rather than inducing a rapid transition in Ca +-loaded mito­ 2.7 chondria, as do these other agents (1), or acting as a substitute 3.0 d 3.3 for Ca + per se, mastoparan enhances the action ofCa + and Pi which are both still required to obtain the transition. Mastoparan also differs from other inducers ofthe transition with respect to the inhibitory activity of CSA. While CSA pre­ RLM vents the action of Pi' hydroperoxides, and other agents for extended periods, and apparently without a sharp dependence Mas on inducing agent concentration (32, 33), increasing the con­ centration of mastoparan from 1 to 3 p..M eliminates the inhib­ itory action of CSA (Fig. 4). A comparison of Figs. 1 and 2 with Fig. 4 illustrates another point of interest. When a low concen­ tration of mastoparan is employed and the resulting transition is sensitive to CSA (Figs. 1 and 2), TPP+ release and swelling proceed after a lag period which is characteristic of the transi­ tion induced by most agents. At higher mastoparan concentra­ (\J '" 0 tions, where sensitivity to CSA is lacking or incomplete (Fig. 4), .!: 'ii • TPP+ release and, to some extent, swelling proceed without a ~~ significant lag period following mastoparan addition. The mastoparan analog MP14 (Table I) facilitates opening of the PTP in a manner analogous to the parent peptide. At relatively low concentrations, the actions of MP14 are depend­ ent upon the Ca + and Pi concentrations and are sensitive to 4min CSA, whereas at higher concentrations, MP14 actions are seen in the absence of free Ca + and are CSA insensitive (data not shown). Fig. 5 shows the CSA-sensitive and insensitive effects FIG. 4. CSA insensitive actions of mastoparan on mitochon­ of the two peptides on PTP opening (swelling response) as a dria. Incubations were conducted as described in the legend to Fig. 1 function of their concentrations. With mastoparan, it is seen with 0.5 fJ-M CSA present from the beginning. TTp· accumulation (upper panel) and swelling (lower panel) were determined simulta­ that the CSA insensitive activity occurs over a concentration neously as described under "Experimental Procedures." For traces la­ range which is approximately twice that required for the CSA­ beled a-d, mastoparan was added where indicated at the following sensitive activity. MP14 is less effective than mastoparan on a concentrations: a, 0 fJ-M; b, 1.0 J.LM; c, 2.0 J.LM; d, 3.0 J.LM. concentration basis, regardless ofwhether the CSA-sensitive or insensitive activities are considered. However, with the latter release and on mitochondrial ultrastructure. Prior to conduct­ peptide, the CSA-sensitive and insensitive concentration ing these studies, the effects of PEGs on the osmotic pressure of curves are offset by 4-5-fold (Fig. 5). mitochondrial incubation media were examined. This work was The Solute Size Exclusion Properties of Mastoparan and conducted because PEGs increase the osmotic pressure of so­ MP14-dependent Pores-A Ca + requirement and CSA sensi­ lutions in disproportion to their concentration (40), a property tivity are the primary biochemical identifiers ofthe permeabil­ which would complicate the use of PEGs to characterize the ity transition. In addition, it is known that mastoparan can pores in question. Fig. 6A shows that small, intermediate, and perturb phospholipid bilayers under certain conditions (e.g. relatively large PEGs increase the osmotic pressure of a dilute Refs. 34-37). These considerations, taken together with the buffer solution in marked excess of their molal concentration. data in Figs. 4 and 5, raise the question of whether the Ca + The extent of this nonideal behavior is a nonlinear function of independent and CSA insensitive depolarization and swelling the weight of PEG per volume of solution and is more extreme produced by these peptides arise from opening the PTP or from for the larger PEGs. Indeed, as the molecular weight increases, direct actions upon the membrane lipid phase. To investigate the weight per volume required to establish a given osmotic this point, the solute size exclusion properties of the pore in­ pressure becomes almost independent of the PEG molecular duced by Ca + plus Pi alone were compared to those ofthe pore weight (Fig. 6B). To maintain a constant osmotic pressure in induced by mastoparan, MP14, and by alamethicin. The latter agent is an established pore-forming peptide (38, 39). 2 The binding of a large number of water molecules per molecule of In the methods used, the effects ofPEGs ofvarious molecular PEG and/or other structural ordering of water, with a resulting reduc­ weights on the large amplitude swelling associated with the tion in the activity of water, are thought to be responsible for the nonideal osmotic properties of PEGs (40). transition were compared to their effects on matrix solute 4927 Mitochondrial Permeability Transition TABLE I The amino acid sequence ofmastoparan and its analog MP14 MP14 differs from mastoparan by a single amino acid substitution at position 12. Position Peptide 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu-NH Mastoparan 2 Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-LyS-Gly-Ile-Leu-NH MP14 FIG. 5. The concentration depend­ E" ence of mastoparan and MP14 effects .~ on mitochondria in the presence and (5 absence of CSA. Data were obtained from experiments like those shown in Fig. #. 4. e, mastoparan was employed in the .~ absence of eSA. _, same as e except that 0.5 J-tM eSA was present from the begin­ ning ofthe incubations. 0, MP14 was em­ ployed in the absence of eSA. D, same as ° except that 0.5 J-tM eSA was present from the beginning of the incubations. o 14 2 4 6 8 10 12 Peptide, f1M native interpretation, decreased swelling might represent PTP mitochondrial incubations containing PEGs (Figs. 7-9), the media were prepared by mixing the normal medium with 300 opening in a decreasing fraction ofthe mitochondria. The latter milliosmolal solutions of PEG dissolved in a dilute buffer, to interpretation must be considered because it is well known that yield the desired concentration of the polymer. The table con­ the transition occurs heterogeneously in mitochondrial popula­ tained within Fig. 6B shows the solution concentrations which tions (1). Furthermore, it is not clear if the PTP is a single yield a 300 milliosmolal osmotic pressure for all PEGs which molecular entity or the same entity in all mitochondria (17). were used. Fig. 6C shows that when the mitochondrial medium Fig. 8 distinguishes between the alternative interpretations. In is mixed with isoosmotic solutions of PEGs, the resulting os­ this figure, panels A-D, it is seen that mitochondrial ultra­ motic pressures remain relatively constant when the fraction of structure is relatively uniform when observed in samples osmotic pressure arising from PEG is varied from 0-100%. which were fixed after the swelling response reached comple­ Under these near isoosmotic conditions, low concentrations tion in the presence of 0, 3, 8, or 20 mM PEG. This structural of 3.4-kDa PEG markedly reduce the magnitude of swelling characteristic is not consistent with PTP opening in a variable response which occurs when the PTP is opened by the action of subfraction of the mitochondria (see Ref. 1). In addition, de­ Ca + plus Pi (Fig. 7). Based upon existing data, a 3.4-kDa PEG creased swelling produced by increasing concentrations of 3.4­ would be expected to pass through the outer membrane volt­ kDa PEG is not accompanied by a decreased release of matrix age-dependent anion channel (VDAC) (41) but not through the Mg2+ (Fig. 8E) nor that of matrix space nucleotides and other PTP (42-45). Several features of these swelling curves are cofactors, as determined by HPLC analysis (data not shown). noteworthy. For all but the highest PEG concentrations em­ Finally, malate dehydrogenase release was minimal or absent ployed, the curves are monophasic and produce a final absorb­ across the full range of PEG concentration investigated, al­ ance change which is relatively stable. The latter characteristic though a marked release of adenylate kinase activity was ob­ is taken to indicate that 3.4-kDa PEG does not slowly permeate served which was diminished by PEG. Taken together, these through the PTP. Near the high end of the concentration range, data strongly indicate that the concentration-dependent inhi­ some multiphasic behavior is seen. This might indicate that bition of swelling produced by 3.4-kDa PEG results from a movement of 3.4-kDa PEG through VDAC is somewhat re­ reduced matrix space expansion in all, or nearly all, of the stricted, as is discussed further below. Finally, the initial rate mitochondria and not from PTP opening in a decreasing frac­ of swelling is seen to be essentially constant as the PEG con­ tion of the mitochondria. In addition, the enzyme release data centration increases. This suggests that PEG does not impede show that the limited swelling which is observed in the pres­ the access of low molecular weight solutes to the PTP or oth­ ence of PEG does not reflect rupture of the inner membrane in erwise slow their rate of permeation. some of the mitochondria (absence of malate dehydrogenase The above interpretations are based upon the assumption release), although rupture ofthe outer membrane occurs and is that the decreased swelling produced by increasing PEG con­ subject to inhibition by 3.4-kDa PEG (adenylate kinase centrations results primarily from a decrease in the maximal release). matrix space volume which is attained following opening of With the above information as a background, the size exclu­ PTP and that the PTP opens in all mitochondria. By an alter- sion properties of "permeability transition pores" induced in 4928 Mitochondrial Permeability Transition B C 10 A 400 M.W. mM iii 1000 400 219 I (5 '? 8 600 189 0 E (/) ... 1000 150 (; X 1500 E E a. 2000 102 1: o i~:-.-.-.-.-._._._.~O 3400 64 e- 6 e- 'Q) ::l ::l ~o ,0-0 I (/) 4000 55 (/) 300 (/) ~ (/) ._~-o,%-o; ",,/ 600 6000 38 l!! l!! '... ..... ...- ....... ta 8000 28 a. a. -...-"" 1ססoo 1i 23 4 ,g ~ I ~ 0 :iE (/) 200 A/ A/ A_A/' 0 25 50 75 100 100 200 300 400 0 75 150 225 300 PEG Solution, % PEG, mg/ml PEG, mg/ml FIG. 6. The osmotic pressure properties ofPEG·containing solutions. A, measured osmotic pressure values of3 mM Hepes buffer, pH 7.4, containing increasing amounts of 0.6 (e)-, 3.4 (0)-, or 8.0 (.)-kDa PEG. The dashed lines in this panel show how osmotic pressure would vary if solutions of 0.6 (a)-, 3.4 (b)-, or 8.0 (c)-kDa PEG behaved ideally. These lines were located by preparing single solutions of each PEG utilizing weighed amounts of PEG and solvent. The molal concentration of the solution could then be calculated, providing a y axis coordinate for the solution. The x axis coordinate was located by noting the volume of each solution and dividing the weight of PEG used by the volume to yield milligrams of PEG/ml of solution. Straight lines were then drawn as defined by these points and the origin. B, the concentration of PEG in milligrams per ml of solution required to give an osmotic pressure of 300 mosM, as a function of PEG molecular weight. These values were determined from experiments like those shown in A. The table embedded in B shows the same data expressed numerically in concentration units of mM. C, measured osmotic pressure values of 0.6 (e)-, 3.4 (0)-, and 8.0 (.)-kDa PEG solutions progressively diluted with a mitochondrial incubation medium. As seen in the panel, both the initial PEG solutions and the mitochondrial incubation medium had osmotic pressures near 300 milliosmolal when measured individually. [PEGj,mM FIG. 7. Inhibition ofPTP-dependent swelling by selected concentrations of 3.4·kDa PEG. Mitochondria were in­ l------------------- 10 cubated at 1.0 mg of protein/ml and at \\'--------------------7.0 25 cC, in isoosmotic media which did not contain an exogenous respiratory sub- "------------------5.0 strate. The media were prepared by mix- ing 300 milliosmolal mannitol/sucrose/ -----------------4.0 Hepes (mitochondrial isolation medium) with a medium comprised of 64 mM 3.4- ----------------- 3.0 kDa PEG in 3 mM Hepes, pH 7.4 (also 300 milliosmolal) to yield the indicated con­ centrations of PEG, while maintaining a ------------- 2.0 constant osmotic pressure (see Fig. 6). P; was present at a final concentration of 2 mM. CaCl was added where shown, at 50 2 -----------1.0 nmol/mg protein, and swelling was subse- quently monitored as described under "Experimental Procedures." 3 min alamethicin pore is clearly larger, with PEG of molecular several ways were examined by observing the maximal extents of swelling which occur when the medium contains PEG of weight near 1700 being required to produce a comparable re­ duction in the extent of swelling (Fig. 9B). The remaining various molecular weights. The concentrations of PEGs were panels in Fig. 9 show the size exclusion properties of the pores selected and the solutions were prepared such that 40% of the induced by mastoparan and MP14 in the presence or absence of osmotic pressure was obtained from PEG, while the total os­ CSA. No marked differences are seen, although all of these motic pressure remained near 300 milliosmolal (Fig. 6). The pores may be slightly larger than the PTP as induced by Ca + PTP induced by Ca + plus Pi can be easily distinguished from the pore formed by alamethicin using this method. A compar­ plus Pi alone. ison of A and B of Fig. 9 illustrates this, together with the Mastoparan and MP14 Permeabilize Phospholipid Vesicles: the Effect of Membrane Potential-To aid in interpreting the approach taken when analyzing these data. The Ca + plus actions of mastoparan and MP14 on mitochondria, their effects Pcinduced PTP produces a swelling response which is one-half inhibited by PEG of a molecular weight near 650 (Fig. 9A). The on a model phospholipid membrane were investigated. POPC Mitochondrial Permeability Transition 4929 linear peptides were added in the case of the membrane poten­ tial present condition (data not shown). To analyze data like those shown in Fig. 10, trace B, the relative initial rate of Ca +·Quin-2 complex formation was estimated by fitting early portions of the progress curve (non­ linear least square techniques, see Ref. 19) to the expression: AT = A + Bt + Ct . In this expression, AT and A are the total o o and initial difference absorbance values, respectively, B is the initial rate, C is a correction factor for nonlinearity, and t is time. The right side panel of Fig. 10 shows the effect of masto­ paran and MP14 concentration on vesicle permeability as de­ termined by this method. No significant differences between the two peptides are apparent in the absence of a membrane potential. When a membrane potential is present, however, :0 mastoparan is markedly more effective than its analog. The .~ increment produced by membrane potential, compared to no membrane potential was 5.8- and 2.3-fold for mastoparan and "5 MP14, respectively. This panel also shows that for all cases oi examined, the relationship between peptide concentration and c: '" 40 vesicle permeability are linear. ., '" a: DISCUSSION Mastoparan is a 14-amino acid amphipathic peptide ob­ tained from wasp venom (47-53). It possesses a wide spectrum of pharmacological activities including mast cell degranulation (50), activation of G-protein-mediated mechanisms (54-58), 10 15 20 25 0 5 inhibition of calmodulin-mediated mechanisms (59-61), stim­ [PEGI. mM ulation of phospholipases A and C (55, 62, 63), stimulation or inhibition of cation-specific channels (64), and others (see Refs. FIG. 8. The fraction of mitochondria which undergo PTP open­ 51 and 65). Facilitation of the mitochondrial permeability tran­ ing as a function of 3.4-kDa PEG concentration. Incubations were conducted as described in the legend to Fig. 7. For all parameters, sition can now be added to this broad spectrum of activities. It samples were taken 8 min after the addition of Ca +, and mitochondria should be noted that this newly recognized action of mastopa­ were sedimented u ing a microcentrifuge (-13,000 x g for 3 min). For ran is marked at concentrations <1 J.LM (Fig. 5), whereas the A-D, the supernatants were removed and the pellets were fixed, em­ other activities are typically seen over a 5-100 J.LM range. Thus, bedded, and examined by electron microscopy as described previously (28) (total magnification = 3,000). The medium concentrations of 3.4­ the actions of mastoparan on mitochondria are the most potent kDa PEG were as follows: A, a mM; B, 3 mM; C, 8 mM; D, 20 mM. For E, described to date and may well be involved in the toxicological the PEG concentration was as shown. The extents of swelling (.), mechanism of this peptide, given the relationship between the malate dehydrogenase release (0), adenylate kinase release (ll.), and transition and the death of injured cells (see introduction to the Mg2+ release (A) were determined using the supernatants obtained after sedimenting mitochondria, as described under "Experimental text). It should also be noted that as a modulator of the tran­ Procedures." sition, mastoparan is more potent than most of the known agents (1). Whether or not the inner membrane pore(s) induced by both vesicles containing Quin-2 were prepared and incubated in a medium containing Ca +. Under the conditions employed, the low (:51 J.LM) and high (~2 J.LM) levels of mastoparan are in fact nominal phospholipid concentration equates approximately to the PTP is an important question because all other inducers a mitochondrial suspension at 1.5 mg of protein/ml, whereas require Ca + for activity, with the apparent exception of phen­ ylarsine oxide (66), and are poorly active in the presence of the entrapped volume (-0.6 J.LlIml) is similar to that of mito­ chondria in an isoosmotic medium at 0.8 mg of protein/ml (see CSA, even when they are used over a broad concentration range (1, 3). In the lower concentration range, pore induction Refs. 24 and 46). These vesicles contained an internal K+ concentration of -60 mM and were suspended in a Na+­ by mastoparan also requires Ca +, is facilitated by Pi' and is inhibited by CSA and by oligomycin (Figs. 1-3). Given these containing medium. Measurements carried out with a TPP+ electrode showed that the addition of valinomycin produced a characteristics, it is very likely that this pore is in fact the PTP. To test the identity of the pore formed in the higher mastopa­ membrane potential of -150 mV, inside negative, which is also similar to the situation existing with intact mitochondria (data ran concentration range, the solute size exclusion properties of several pores were compared (Fig. 9). By this criterion, the pore not shown). In this system, the development of a permeable membrane is indicated by formation of the Ca +·Quin-2 com­ induced by mastoparan in the presence of CSA is also the PTP as are the CSA-sensitive and insensitive pores induced by plex which was monitored by dual wavelength spectroscopy (see 19). Fig. 10 shows that these vesicles are impermeant to MP14. In considering these interpretations it must also be Ca + and Quin-2 under these conditions, in the presence or noted that mastoparan and MP14 perturb phospholipid bilay­ absence of a membrane potential, when mastoparan is absent ers by a mechanism which is membrane potential-dependent (Fig. 10) but otherwise unknown. It is thus possible that the (trace A). The addition of 2.0 J.LM mastoparan produces a slow permeation of Ca + and/or Quin-2 and a much faster perme­ peptide molecules per se form pores in the inner mitochondrial ation when a membrane potential is present (trace B). Similar membrane and that these pores, rather than the PTP, are results (not shown) were obtained with MP14. The rates of responsible for the CSA independent activities. While this in­ permeation in the presence or absence of a membrane potential terpretation remains viable, it seems improbable because it were independent of Ca + concentration over a range of 10­ would be highly fortuitous if the pores formed by both peptides 1000 J.L 1, were not significantly affected by CSA (1.0 J.LM), and were so similar to each other, and to the PTP, by the criterion were independent of the order in which valinomycin and the of solute size exclusion. 4930 Mitochondrial Permeability Transition 100 100 •. A B e. 80 80 '! , :----\~. , , \ ______J ~:::::::=.-. FIG. 9. Solute size exclusion proper­ 20 1700 ties of pores in mitochondria. Mito­ 20 _J ~_=~~....__. chondria were incubated at 0.5 mg of pro­ • tein/ml and at 25°C in -300 milliosmolal o "-'-----'----'_-'--....L.---L_.L.---'---'---' 0'-'---'-_"--'-_1.--1...--'_--'---"--' media containing 5 mM concentration o 2 345 6 7 8 o 2 345 6 7 8 each of succinate (Tris) and P, (Tris), pH 7.4.40% ofthe total osmotic pressure was derived from PEG and the remainder from mannitol/sucrose (3:1 mole ratio) 100 100 plus the other solutes described above. C 0 PEGs ranging from 0.6 kDa to 8 kDa were :;, employed, as illustrated in the figure, and E 80 80 these were mixed with mannitol/sucrose .~ to give the desired fraction of osmotic 60 60 pressure derived from PEG, while keep­ ing the total value near 300 milliosmolal "6 (see Fig. 6). Swelling (pore formation) was 40 40 initiated after a 2-min preincubation and allowed to proceed until the apparent ab­ __j ____.._.-.-I-t sorbance of the suspensions became con­ 20 20 stant. These final values, presented as a percent of the value obtained in the ab­ 0 0 sence of PEG, are plotted as a function of 0 2 3 4 5 6 7 8 0 2 3 4 5 6 7 8 the PEG molecular weight. Each value is the mean ± S.E. of three determinations, with each replicate value obtained using a separate mitochondrial preparation. Pore formation was induced as follows: A, 100 .0 CaCl (50 nmol/mg protein); B, alamethi­ cin (3.5 /Lg/ml); C, mastoparan (1.0 /LM); D, mastoparan (3.0 /LM) and the medium contained l/LM CSA; E, MP14 (3.0 /LM); F, MP14 (ll/LM) and the medium contained l/LM CSA. :\ ,. l \ 20 "- !=--- ....__.-. OLL.....-'----'--'---'---'----'---''--..J.-j o 2 3 4 5 6 7 8 2 345 6 7 8 PEG M.W. X 10. There is a second aspect of the solute size exclusion data The present results are not subject to some of the uncertain­ presented here which is of interest and which becomes appar­ ties in the earlier data, and, as a consequence, they show that ent when these data are compared to earlier studies in which when the pore is induced by Ca + plus Pi' increasing the PEGs were utilized to characterize the size exclusion properties molecular weight of PEG in the medium from 0.4 to 4 kDa of the PTP (42, 44, 45). There are disagreements between the produces a progressive decrease in swelling. This behavior is early studies which may reflect pore induction by differing unexpected because each point was obtained at a (essentially) agents, and/or technical considerations arising from unrecog­ constant osmotic pressure, with PEG providing the same frac­ nized osmotic pressure properties of PEGs, the use of a limited tion of that pressure, and represents an apparent equilibrium number of PEG molecular weights, and other factors. Haworth condition (i.e. the extents of swelling were not increasing sig­ and Hunter (induction by arsenate) (42) reported that 1.5- and nificantly at the time the values were taken). If the PTP is a 4-kDa PEGs are excluded, 0.6-kDa or smaller PEGs are per­ rigid structure, one would expect a sharper cutoff in the mo­ lecular weight of PEG which is fully excluded. If it is a flexible meant, whereas an intermediate condition exists with l-kDa PEG. Vercesi (induction by oxalacetate) (45) reported that structure which can impede yet pass solutes of variable size, swelling is eliminated by l-kDa PEG. Le-Quoc and Le-Quoc one would expect the smaller PEGs to decrease the rate of swelling instead ofproducing an intermediate and stable value. (induction by N-butylmaleimide) (44) found no permeation of 6-kDa PEG but progressively faster permeation of 4-, 1.5-, and The presence of molecular weight heterogeneity in the various 0.6-kDa PEG. Large fractions ofmatrix space enzyme activities samples of PEG, together with a sieving action by the PTP, were released under the conditions of their study and the cannot easily explain the observed behavior. According to the authors concluded that an association of VDAC with the ade­ manufacturer, this heterogeneity does not exceed 2:10% of the nine nucleotide translocase forms the PTP (44). stated value, and filtration experiments conducted with some 4931 Mitochondrial Permeability Transition ±Val 0.8 A -.-. ...._"' ...-.... , ............__ ""J"....... t'"...... , .., ••",,*....""......"'""... """"_.. ±Val 0.7 0.6 0.5 0.4 ~i 0.3 0.2 0.1 0.0 3 min 0 2 3 Peptide, /1M FIG. 10. Membrane potential-dependent and independent actions of mastoparan and MP14 on the permeability of POPC vesicles. Quin 2/HepesJK+ load POPC vesicles were prepared and incubated as described under "Experimental Procedures." The nominal POPC concen­ tration was 300 J.l.M, and the external medium contained 10 mM Hepes (Na+), pH 7.0, 20 mM NaCl, and 50 /LM CaCl • Increased membrane permeability was indicated by formation of the Ca +'Quin 2 complex, which was monitored by dual wavelength absorbance measurements made at 294 versus 338 nm (19). A (left panel), valinomycin (0.1 /LM) was added or not added, where indicated. B (left panel), same as A except that mastoparan (2.0 J.l.M) was added where indicated. Right panel, a summary plot ofthe initial rates of absorbance change as a function of mastoparan or MP14 concentration, as determined by fitting the data to the equation given under "Results." • and ., mastoparan and MP14, respectively, membrane potential present. 0 and 0, mastoparan and MP14, respectively, membrane potential absent. of the samples were consistent with this specification (data not and CSA insensitive manner. The present data cannot iden­ shown). At the higher end of the molecular mass range (4 and tify the allosteric site at which the peptides may act, although it is noted that mastoparan carries three positive charges, possibly 3.4 kDa), PEG may act, in part, because it is partially excluded by VDAC (41). This could relax the oncotic pressure whereas MP14 carries two (Table I). Thus, the relative po­ tency of these peptides correlates with positive charge and gradient across the outer membrane, contract the intermem­ they might act at a site which normally binds a cation. The brane space volume, and raise the protein concentration, so that reduced swelling occurs when the PTP is opened. This Ca + binding site is one possibility and an action at this site possible explanation is not applicable in the case ofthe smaller could explain why pore induction can occur in the absence of PEGs, however, since they clearly permeate VDAC (41). The Ca + when the peptide concentration is high. Possible dis­ existence of different size pores in individual mitochondria placement of CSA from its site of action by high concentra­ could explain the observed behavior, and differences in the tions of mastoparan is another possibility to consider. mechanisms which gate solute entry from opposite sides of the Analogies between regulation of the PTP and the N-methyl­ membrane might also explain this behavior. No choice between n-aspartic acid receptor channel suggest that the PTP belongs such explanations seems possible without further studies, to the super family of ligand gated ion channels and thus may which are now in progress. be regulated by covalent modification (see Ref. 3). Such systems Induction of the transition by mastoparan and MP14 may often involve G-proteins, and a mitochondrial G-protein has occur through PTP regulatory mechanisms which are already recently been identified and isolated (67, 68). According to a recognized (1-3). However, since these peptides have a wide recent report, MP14 retains the G-protein-mediated activities spectrum of pharmacological activities (see above), new po­ of mastoparan but has a diminished capacity to perturb mem­ tential regulatory mechanisms can also be considered. Re­ branes (69). It is for these reasons that the actions of MP14 and garding the established mechanisms, it does not appear that mastoparan on the PTP were compared in this study. MP14 is reducing membrane potential is the primary mode by which only slightly less active than mastoparan when inducing pore the peptides act under CSA-sensitive conditions. This is in­ opening in a CSA-sensitive manner, whereas there is a larger dicated by the TPP+ accumulation data in Figs. 1 and 2 which activity differential when the pore is induced in the presence of show that mastoparan produces only minor changes in mem­ CSA (Fig. 5). If the CSA insensitive induction involves mem­ brane potential when CSA, EGTA, or oligomycin are present brane perturbation (depolarization) as suggested above, then to inhibit pore opening. The same is true for MP14 (data not these data suggest regulation of the PTP through a G-protein. shown). Under CSA insensitive conditions, an early and ex­ The relative effectiveness of mastoparan and MP14 have not tensive release of TPP+ is seen and this seems to occur yet been compared in a variety of systems, however, and so it is somewhat faster than swelling (Fig. 4). This depolarization not clear how completely the G-protein-mediated and mem­ could be brought about by peptide molecules acting upon the brane perturbation-mediated activities of MP14 are distin­ membrane lipid phase as illustrated in Fig. 10. It is thought guished. In particular, it appears that a membrane perturba­ that no PTP effector dominates in the interactive system of tion activity differential between these two peptides is only pore regulation through allosteric and membranelbioener­ seen in the presence of a membrane potential (Fig. 10) and the getic mechanisms. Instead, the open/closed probability ap­ reason for this deserves further investigation. pears to be established as a sum of positive and negative The actions of mastoparan and MP14 on the PTP suggest actions exerted at a number of sites (17). Thus, the peptides that low concentrations of other amphipathic peptides could may act at an allosteric site, synergistically with Ca + and Pi regulate the PTP in cells. Such peptides are a product of proc­ acting their sites, under CSA-sensitive conditions. It could essing imported proteins and may accumulate in the mitochon­ then be the additional influence of depolarization produced drial matrix under some conditions. Extra- and intramatrix by higher peptide levels acting upon the membrane lipid space amphipathic peptides may also accumulate in injured phase which allows the pore to open in a Ca + independent cells due to proteolysis. These peptides may promote pore open- 4932 Mitochondrial Permeability Transition 264,7826-7830 ing, even in presence of eSA, and become a factor in maintain­ 34. Katsu, T., Kuroko, M., Morikawa, T., Sanchika, K, Yamaraka, H., Shinoda, S., ing the protection of injured cells afforded by eSA. and Fujita, Y. (1990) Biochim. Biophys. Acta 1027, 185-190 35. Tanimura, A., Matsumoto, Y., and Tojyo, Y. (1991) Biochem. Biophys. Res. Acknowledgments-We thank Wayne Anderson of the Hormel Insti­ Commun. 177,802-808 tute for carrying out the electron microscopy and Ronald Louters ofThe 36. Raynor, R. 1., Kim, Y.-S., Zheng, B., Vogler, W. R, and Kuo, J. F. (1992) FEES Lett. 307,275-279 Ohio State University for technical assistance in other areas. 37. 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