(Received for publication, August 22, 1994)
From the
Mastoparan facilitates opening of the mitochondrial permeability
transition pore through an apparent bimodal mechanism of action. In the
submicromolar concentration range, the action of mastoparan is
dependent upon the medium Ca and phosphate
concentration and is subject to inhibition by cyclosporin A. At
concentrations above 1 µM, pore induction by mastoparan
occurs without an apparent Ca
requirement and in a
cyclosporin A insensitive manner. Studies utilizing phospholipid
vesicles show that mastoparan perturbs bilayer membranes across both
concentration ranges, through a mechanism which is strongly dependent
upon transmembrane potential. However, solute size exclusion studies
suggest that the pores formed in mitochondria in response to both low
and high concentrations of mastoparan are the permeability transition
pore. It is proposed that low concentrations of mastoparan influence
the pore per se, with higher concentrations having the
additional effect of depolarizing the mitochondrial inner membrane
through an action exerted upon the lipid phase. It may be the
combination of these effects which allow pore opening in the absence of
Ca
and in the presence of cyclosporin A, although
other interpretations remain viable. A comparison of the activities of
mastoparan and its analog, MP14, on mitochondria and phospholipid
vesicles provides an initial indication that a G-protein may
participate in regulation of the permeability transition pore. These
studies draw attention to peptides, in a broad sense, as potential pore
regulators in cells, under both physiological and pathological
conditions.
The mitochondrial permeability transition remains an
incompletely understood phenomenon, in spite of the fact that it was
first observed in the 1950s and has been studied extensively,
particularly during the last 15 years (see (1, 2, 3) for review). Recent work utilizing
the patch clamp technique applied to isolated mitochondria indicates
that the transition is caused by the opening of large pores in the
inner membrane(4, 5, 6) . At present there is
no known physiological function of this phenomenon; however, it is
clear that it occurs in situ, under conditions of oxidative
stress, and is an event that can be pivotal in the mechanisms leading
to cell death(7, 8, 9, 10) .
Association of the transition with cell injury mechanisms and the
identification of cyclosporin A (CSA) ()as a potent
inhibitor of the phenomenon (11, 12, 13, 14) has created a high
level of interest in this aspect of mitochondrial research.
Many of the agents which induce the transition are toxins and pharmacological agents not normally encountered in vivo(1) . The same is true for the known inhibitors(1) . Both inducing agents and inhibitors are chemically diverse but display a common activity in favoring an open or closed state of the permeability transition pore (PTP), respectively. It has proven difficult to explain the analogous actions of diverse compounds on the PTP. Bernardi and co-workers (15, 16) have shown that membrane potential and matrix pH are central PTP regulators. Membrane surface potential may also be a central regulator (3, 17) . It is thus possible that the chemical diversity/common activity problem associated with the transition may arise, in part, from the effects of transition regulators on bioenergetic parameters and membrane physical properties.
Although many known transition effectors would not normally be
encountered in cells, a number of activators (e.g. Ca, P
, acyl-CoA) and inhibitors (e.g. ADP, Mg
, polyamines) are physiological
cell constituents. In seeking to understand regulation of the
transition in an intracellular setting, it then seems reasonable to
consider these agents as primary regulators and to investigate how
other cellular components and conditions affect their actions on the
PTP. The present report demonstrates that the 14-amino acid peptide
mastoparan has a potent stimulatory effect on PTP opening when
mitochondria are incubated in the presence of Ca
and
P
. At somewhat higher 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 transition and, as such, draws attention to
a new class of potential regulators of this phenomenon within cells. In
this report, the actions of mastoparan on the transition are also
compared to those of an analog, MP14. Differences observed between the
potency of these two peptides are consistent with the hypothesis that
components of cell signaling mechanisms, apart from
Ca
, are involved in regulation of the PTP. Aspects of
these data have been presented in abstract form(18) .
The
permeability of POPC vesicles to Ca/Quin-2 was
monitored by incubating them in 10 mM Hepes
(Na
), pH 7.0, containing 20 mM NaCl and 50
µM CaCl
. The development of a permeable
membrane was indicated by formation of the
Ca
Quin-2 complex, which was determined by
difference absorbance measurements, using the Aminco DW2a
spectrophotometer operated in the dual wavelength mode (wavelength pair
264 versus 338 nm). The temperature was 25 °C, and the
nominal POPC concentration was 300 µM. At this
concentration, the entrapped volume is 0.6 µl/ml(24) . For
some experiments, a membrane potential (inside negative) was imposed
across the vesicle membrane by adding valinomycin at 0.1
µM. Parallel experiments employing the TPP
electrode and application of the Nernst equation showed that the
magnitude of the membrane potential produced in this way was
approximately 150 mV.
When examining mitochondrial
ultrastructure or determining the release of enzymes and matrix space
solutes, aliquots taken from incubations were initially centrifuged to
sediment the mitochondria (Eppendorf microcentrifuge, 13,000
g, 3 min). For ultrastructural studies, pellets were
then fixed, stained, and processed for examination by electron
microscopy as described before(28) . Fractions of total enzyme
activities and Mg
released during incubation were
determined by assaying the supernatants, also using established
methods(29, 30) . The release of nucleotides and other
coenzymes from the matrix space were determined by HPLC analysis of the
sedimented mitochondria. Pellets were extracted as described by Stocchi et al.(31) , whereas HPLC analysis was carried out as
described by Novgorodov et al.(17) .
Figure 1:
Stimulation of the permeability
transition by mastoparan; inhibition by CSA. Mitochondria were
incubated at 0.5 mg of protein/ml and at 25 °C. The medium
contained 250 mM sucrose, 10 mM Hepes (Tris), 5
mM succinate (Tris), 5 mM P (Tris), pH
7.4, plus 3.3 µM TPP
Cl. TPP
accumulation and release (upper panel) and swelling (lower panel) were determined simultaneously as described
under ``Experimental Procedures.'' CaCl
(dashed lines) or mastoparan (solid lines) were
added where indicated at 25 nmol/mg of protein and 1.0 µM,
respectively. For traces labeled a, the medium
contained CSA at 0.5 µM from the beginning of the
experiment. For traces labeled b, CSA was
absent.
Figure 2:
Stimulation of the permeability transition
by mastoparan; inhibition by EGTA and oligomycin. Mitochondria were
incubated as described in the legend to Fig. 1, and
TPP accumulation (upper panel) and swelling (lower panel) were determined simultaneously as described
under ``Experimental Procedures.'' For all traces (a-c), 1.0 µM mastoparan was added
where indicated. For traces labeled b and c,
the medium contained EGTA (0.5 mM) or oligomycin (1 µg/mg
protein), respectively, from the beginning of the
incubation.
Mitochondria
depleted of endogenous Ca(21) were utilized
to investigate the effect of Ca
content and P
concentration on mastoparan-dependent induction of the
transition. When P
is present at a concentration of 5
mM, 1.0 µM mastoparan markedly stimulates the
transition across a range of Ca
loads (Fig. 3A), although little or no activity is seen in
the absence of exogenous P
(Fig. 3B). In a
similar way, when an intermediate Ca
load is
employed, the action of mastoparan is dependent on P
concentration (Fig. 3C) with little or no
activity seen in the absence of exogenous Ca
(Fig. 3D). These findings distinguish mastoparan
from better known inducers of the transition such as hydroperoxides,
sulfhydryl reagents, and P
. Rather than inducing a rapid
transition in Ca
-loaded mitochondria, as do these
other agents(1) , or acting as a substitute for Ca
per se, mastoparan enhances the action of Ca
and P
which are both still required to obtain the
transition.
Figure 3:
Effects of Ca content
and P
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 P
(Tris) and CaCl
as shown. P
was present from the beginning, whereas CaCl
was
added at 1 min following the addition of mitochondria.
, 1.0
µM mastoparan was added 1 min after the addition of
CaCl
.
, 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 P
was deleted from the medium. C, same as A, except that the mitochondrial Ca
load was
25 nmol/mg protein, and the medium P
concentration was
varied as shown. D, same as C, except that
Ca
was not added.
Mastoparan also differs from other inducers of the
transition with respect to the inhibitory activity of CSA. While CSA
prevents the action of P, hydroperoxides, and other agents
for extended periods, and apparently without a sharp dependence on
inducing agent concentration(32, 33) , increasing the
concentration of mastoparan from 1 to 3 µM eliminates the
inhibitory action of CSA (Fig. 4). A comparison of Fig. 1and Fig. 2with Fig. 4illustrates another
point of interest. When a low concentration of mastoparan is employed
and the resulting transition is sensitive to CSA ( Fig. 1and Fig. 2), TPP
release and swelling proceed after
a lag period which is characteristic of the transition induced by most
agents. At higher mastoparan concentrations, where sensitivity to CSA
is lacking or incomplete (Fig. 4), TPP
release
and, to some extent, swelling proceed without a significant lag period
following mastoparan addition.
Figure 4:
CSA insensitive actions of mastoparan on
mitochondria. Incubations were conducted as described in the legend to Fig. 1with 0.5 µM CSA present from the beginning.
TTP accumulation (upper panel) and swelling (lower panel) were determined simultaneously as described
under ``Experimental Procedures.'' For traces labeled a-d, mastoparan was added where indicated
at the following concentrations: a, 0 µM; b, 1.0 µM; c, 2.0 µM; d, 3.0 µM.
The mastoparan analog MP14 (Table 1) facilitates opening of the PTP in a manner analogous to
the parent peptide. At relatively low concentrations, the actions of
MP14 are dependent upon the Ca and P
concentrations and are sensitive to CSA, whereas at higher
concentrations, MP14 actions are seen in the absence of free
Ca
and are CSA insensitive (data not shown). Fig. 5shows the CSA-sensitive and insensitive effects of the two
peptides on PTP opening (swelling response) as a function of their
concentrations. With mastoparan, it is seen that the CSA insensitive
activity occurs over a concentration range which is approximately twice
that required for the CSA-sensitive activity. MP14 is less effective
than mastoparan on a concentration basis, regardless of whether the
CSA-sensitive or insensitive activities are considered. However, with
the latter peptide, the CSA-sensitive and insensitive concentration
curves are offset by 4-5-fold (Fig. 5).
Figure 5:
The concentration dependence of mastoparan
and MP14 effects on mitochondria in the presence and absence of CSA.
Data were obtained from experiments like those shown in Fig. 4.
, mastoparan was employed in the absence of CSA.
, same as
except that 0.5 µM CSA was present from the
beginning of the incubations.
, MP14 was employed in the absence
of CSA.
, same as
except that 0.5 µM CSA was
present from the beginning of the
incubations.
In the
methods used, the effects of PEGs of various molecular weights on the
large amplitude swelling associated with the transition were compared
to their effects on matrix solute release and on mitochondrial
ultrastructure. Prior to conducting these studies, the effects of PEGs
on the osmotic pressure of mitochondrial incubation media were
examined. This work was conducted because PEGs increase the osmotic
pressure of solutions in disproportion to their
concentration(40) , a property which would complicate the use
of PEGs to characterize the pores in question. Fig. 6A shows
that small, intermediate, and relatively large PEGs increase the
osmotic pressure of a dilute buffer solution in marked excess of their
molal concentration. The extent of this nonideal behavior is a
nonlinear function of the weight of PEG per volume of solution and is
more extreme for the larger PEGs. Indeed, as the molecular weight
increases, the weight per volume required to establish a given osmotic
pressure becomes almost independent of the PEG molecular weight (Fig. 6B). ()To maintain a constant osmotic
pressure in mitochondrial incubations containing PEGs (Fig. 7Fig. 8Fig. 9), the media were prepared by
mixing the normal medium with 300 milliosmolal solutions of PEG
dissolved in a dilute buffer, to yield the desired concentration of the
polymer. The table contained within Fig. 6B shows the
solution concentrations which yield a 300 milliosmolal osmotic pressure
for all PEGs which were used. Fig. 6C shows that when
the mitochondrial medium is mixed with isoosmotic solutions of PEGs,
the resulting osmotic pressures remain relatively constant when the
fraction of osmotic pressure arising from PEG is varied from
0-100%.
Figure 6:
The osmotic pressure properties of
PEG-containing solutions. A, measured osmotic pressure values
of 3 mM Hepes buffer, pH 7.4, containing increasing amounts of
0.6 ()-, 3.4 (
)-, 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 (
)-, 3.4
(
)-, 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.
Figure 7:
Inhibition of PTP-dependent swelling by
selected concentrations of 3.4-kDa PEG. Mitochondria were incubated at
1.0 mg of protein/ml and at 25 °C, in isoosmotic media which did
not contain an exogenous respiratory substrate. The media were prepared
by mixing 300 milliosmolal mannitol/sucrose/Hepes (mitochondrial
isolation medium) with a medium comprised of 64 mM 3.4-kDa PEG
in 3 mM Hepes, pH 7.4 (also 300 milliosmolal) to yield the
indicated concentrations of PEG, while maintaining a constant osmotic
pressure (see Fig. 6). P was present at a final
concentration of 2 mM. CaCl
was added where shown,
at 50 nmol/mg protein, and swelling was subsequently monitored as
described under ``Experimental
Procedures.''
Figure 8:
The fraction of mitochondria which undergo
PTP opening as a function of 3.4-kDa PEG concentration. Incubations
were conducted as described in the legend to Fig. 7. For all
parameters, samples were taken 8 min after the addition of
Ca, and mitochondria were sedimented using a
microcentrifuge (
13,000
g for 3 min). For A-D, the supernatants were removed and the pellets were
fixed, embedded, and examined by electron microscopy as described
previously (28) (total magnification = 3,000). The
medium concentrations of 3.4-kDa PEG were as follows: A, 0
mM; B, 3 mM; C, 8 mM; D, 20 mM. For E, the PEG concentration was
as shown. The extents of swelling (
), malate dehydrogenase
release (
), adenylate kinase release (
), and Mg
release (
) were determined using the supernatants obtained
after sedimenting mitochondria, as described under ``Experimental
Procedures.''
Figure 9:
Solute size exclusion properties of pores
in mitochondria. Mitochondria were incubated at 0.5 mg of protein/ml
and at 25 °C in 300 milliosmolal media containing 5 mM concentration each of succinate (Tris) and P
(Tris),
pH 7.4. 40% of the total osmotic pressure was derived from PEG and the
remainder from mannitol/sucrose (3:1 mole ratio) plus the other solutes
described above. PEGs ranging from 0.6 kDa to 8 kDa were employed, as
illustrated in the figure, and these were mixed with mannitol/sucrose
to give the desired fraction of osmotic pressure derived from PEG,
while keeping the total value near 300 milliosmolal (see Fig. 6). Swelling (pore formation) was initiated after a 2-min
preincubation and allowed to proceed until the apparent absorbance of
the suspensions became constant. These final values, presented as a
percent of the value obtained in the absence of PEG, are plotted as a
function of 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, CaCl
(50 nmol/mg protein); B,
alamethicin (3.5 µg/ml); C, mastoparan (1.0
µM); D, mastoparan (3.0 µM) and the
medium contained 1 µM CSA; E, MP14 (3.0
µM); F, MP14 (11 µM) and the medium
contained 1 µM CSA.
Under these near isoosmotic conditions, low
concentrations of 3.4-kDa PEG markedly reduce the magnitude of swelling
response which occurs when the PTP is opened by the action of
Ca plus P
(Fig. 7). Based upon
existing data, a 3.4-kDa PEG would be expected to pass through the
outer membrane voltage-dependent anion channel (VDAC) (41) but
not through the PTP (42, 43, 44, 45) . Several features
of these swelling curves are noteworthy. For all but the highest PEG
concentrations employed, the curves are monophasic and produce a final
absorbance change which is relatively stable. The latter characteristic
is taken to indicate that 3.4-kDa PEG does not slowly permeate through
the PTP. Near the high end of the concentration range, some multiphasic
behavior is seen. This might indicate that movement of 3.4-kDa PEG
through VDAC is somewhat restricted, as is discussed further below.
Finally, the initial rate of swelling is seen to be essentially
constant as the PEG concentration increases. This suggests that PEG
does not impede the access of low molecular weight solutes to the PTP
or otherwise slow their rate of permeation.
The above
interpretations are based upon the assumption that the decreased
swelling produced by increasing PEG concentrations results primarily
from a decrease in the maximal matrix space volume which is attained
following opening of PTP and that the PTP opens in all mitochondria. By
an alternative interpretation, decreased swelling might represent PTP
opening in a decreasing fraction of the mitochondria. The latter
interpretation must be considered because it is well known that the
transition occurs heterogeneously in mitochondrial
populations(1) . Furthermore, it is not clear if the PTP is a
single molecular entity or the same entity in all mitochondria (17) . Fig. 8distinguishes between the alternative
interpretations. In this figure, panels A-D, it is seen
that mitochondrial ultrastructure is relatively uniform when observed
in samples which were fixed after the swelling response reached
completion in the presence of 0, 3, 8, or 20 mM PEG. This
structural characteristic is not consistent with PTP opening in a
variable subfraction of the mitochondria (see (1) ). In
addition, decreased swelling produced by increasing concentrations of
3.4-kDa PEG is not accompanied by a decreased release of matrix
Mg (Fig. 8E) nor that of matrix space
nucleotides and other cofactors, as determined by HPLC analysis (data
not shown). Finally, malate dehydrogenase release was minimal or absent
across the full range of PEG concentration investigated, although a
marked release of adenylate kinase activity was observed which was
diminished by PEG. Taken together, these data strongly indicate that
the concentration-dependent inhibition of swelling produced by 3.4-kDa
PEG results from a reduced matrix space expansion in all, or nearly
all, of the mitochondria and not from PTP opening in a decreasing
fraction of the mitochondria. In addition, the enzyme release data show
that the limited swelling which is observed in the presence of PEG does
not reflect rupture of the inner membrane in some of the mitochondria
(absence of malate dehydrogenase release), although rupture of the
outer membrane occurs and is subject to inhibition by 3.4-kDa PEG
(adenylate kinase release).
With the above information as a
background, the size exclusion properties of ``permeability
transition pores'' induced in several ways were examined by
observing the maximal extents of swelling which occur when the medium
contains PEG of various molecular weights. The concentrations of PEGs
were selected and the solutions were prepared such that 40% of the
osmotic pressure was obtained from PEG, while the total osmotic
pressure remained near 300 milliosmolal (Fig. 6). The PTP
induced by Ca plus P
can be easily
distinguished from the pore formed by alamethicin using this method. A
comparison of A and B of Fig. 9illustrates
this, together with the approach taken when analyzing these data. The
Ca
plus P
-induced PTP produces a swelling
response which is one-half inhibited by PEG of a molecular weight near
650 (Fig. 9A). The alamethicin pore is clearly larger,
with PEG of molecular weight near 1700 being required to produce a
comparable reduction in the extent of swelling (Fig. 9B). The remaining panels in Fig. 9show
the size exclusion properties of the pores induced by mastoparan and
MP14 in the presence or absence of CSA. No marked differences are seen,
although all of these pores may be slightly larger than the PTP as
induced by Ca
plus P
alone.
Figure 10:
Membrane potential-dependent and
independent actions of mastoparan and MP14 on the permeability of POPC
vesicles. Quin 2/Hepes/K load POPC vesicles were
prepared and incubated as described under ``Experimental
Procedures.'' The nominal POPC concentration was 300
µM, and the external medium contained 10 mM Hepes
(Na
), pH 7.0, 20 mM NaCl, and 50 µM 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 µM) was added or not added, where
indicated. B (left panel), same as A except
that mastoparan (2.0 µM) was added where indicated. Right panel, a summary plot of the 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.
and
, mastoparan and MP14,
respectively, membrane potential absent.
To analyze
data like those shown in Fig. 10, trace B, the relative
initial rate of CaQuin-2 complex formation was
estimated by fitting early portions of the progress curve (nonlinear
least square techniques, see (19) ) to the expression: A
= A
+ Bt + Ct
. In this expression, A
and A
are the total 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. 10shows the effect of mastoparan and MP14 concentration on
vesicle permeability as determined 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,
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 MP14, respectively. This panel
also shows that for all cases examined, the relationship between
peptide concentration and vesicle permeability are linear.
Mastoparan is a 14-amino acid amphipathic peptide obtained
from wasp
venom(47, 48, 49, 50, 51, 52, 53) .
It possesses a wide spectrum of pharmacological activities including
mast cell degranulation(50) , activation of G-protein-mediated
mechanisms(54, 55, 56, 57, 58) ,
inhibition of calmodulin-mediated
mechanisms(59, 60, 61) , stimulation of
phospholipases A and
C(55, 62, 63) , stimulation or inhibition of
cation-specific channels(64) , and others (see (51) and (65) ). Facilitation of the mitochondrial
permeability transition can now be added to this broad spectrum of
activities. It should be noted that this newly recognized action of
mastoparan is marked at concentrations <1 µM (Fig. 5), whereas the other activities are typically seen
over a 5-100 µM range. Thus, the actions of
mastoparan on mitochondria are the most potent described to date and
may well be involved in the toxicological mechanism of this peptide,
given the relationship between the transition and the death of injured
cells (see introduction to the text). It should also be noted that as a
modulator of the transition, mastoparan is more potent than most of the
known agents(1) .
Whether or not the inner membrane pore(s)
induced by both low (1 µM) and high (
2
µM) levels of mastoparan are in fact the PTP is an
important question because all other inducers require Ca
for activity, with the apparent exception of phenylarsine
oxide(66) , and are poorly active in the presence of CSA, even
when they are used over a broad concentration
range(1, 3) . In the lower concentration range, pore
induction by mastoparan also requires Ca
, is
facilitated by P
, and is inhibited by CSA and by oligomycin (Fig. 1Fig. 2Fig. 3). Given these characteristics,
it is very likely that this pore is in fact the PTP. To test the
identity of the pore formed in the higher mastoparan concentration
range, the solute size exclusion properties of several pores were
compared (Fig. 9). By this criterion, the pore induced by
mastoparan in the presence of CSA is also the PTP as are the
CSA-sensitive and insensitive pores induced by MP14. In considering
these interpretations it must also be noted that mastoparan and MP14
perturb phospholipid bilayers by a mechanism which is membrane
potential-dependent (Fig. 10) but otherwise unknown. It is thus
possible that the peptide molecules per se form pores in the
inner mitochondrial membrane and that these pores, rather than the PTP,
are responsible for the CSA independent activities. While this
interpretation remains viable, it seems improbable because it would be
highly fortuitous if the pores formed by both peptides were so similar
to each other, and to the PTP, by the criterion of solute size
exclusion.
There is a second aspect of the solute size exclusion data presented here which is of interest and which becomes apparent when these data are compared to earlier studies in which PEGs were utilized to characterize the size exclusion properties of the PTP(42, 44, 45) . There are disagreements between the early studies which may reflect pore induction by differing agents, and/or technical considerations arising from unrecognized osmotic pressure properties of PEGs, the use of a limited number of PEG molecular weights, and other factors. Haworth and Hunter (induction by arsenate) (42) reported that 1.5- and 4-kDa PEGs are excluded, 0.6-kDa or smaller PEGs are permeant, whereas an intermediate condition exists with 1-kDa PEG. Vercesi (induction by oxalacetate) (45) reported that swelling is eliminated by 1-kDa PEG. Lê-Quôc and Lê-Quôc (induction by N-butylmaleimide) (44) found no permeation of 6-kDa PEG but progressively faster permeation of 4-, 1.5-, and 0.6-kDa PEG. Large fractions of matrix space enzyme activities were released under the conditions of their study and the authors concluded that an association of VDAC with the adenine nucleotide translocase forms the PTP(44) .
The present results are not subject to some of the
uncertainties in the earlier data, and, as a consequence, they show
that when the pore is induced by Ca plus
P
, increasing the molecular weight of PEG in the medium
from 0.4 to 4 kDa produces a progressive decrease in swelling. This
behavior is unexpected because each point was obtained at a
(essentially) constant osmotic pressure, with PEG providing the same
fraction of that pressure, and represents an apparent equilibrium
condition (i.e. the extents of swelling were not increasing
significantly at the time the values were taken). If the PTP is a rigid
structure, one would expect a sharper cutoff in the molecular weight of
PEG which is fully excluded. If it is a flexible structure which can
impede yet pass solutes of variable size, one would expect the smaller
PEGs to decrease the rate of swelling instead of producing an
intermediate and stable value. The presence of molecular weight
heterogeneity in the various samples of PEG, together with a sieving
action by the PTP, cannot easily explain the observed behavior.
According to the manufacturer, this heterogeneity does not exceed
±10% of the stated value, and filtration experiments conducted
with some of the samples were consistent with this specification (data
not shown). At the higher end of the molecular mass range (4 and
possibly 3.4 kDa), PEG may act, in part, because it is partially
excluded by VDAC(41) . This could relax the oncotic pressure
gradient across the outer membrane, contract the intermembrane space
volume, and raise the protein concentration, so that reduced swelling
occurs when the PTP is opened. This possible explanation is not
applicable in the case of the smaller PEGs, however, since they clearly
permeate VDAC(41) . The existence of different size pores in
individual mitochondria could explain the observed behavior, and
differences in the mechanisms which gate solute entry from opposite
sides of the membrane might also explain this behavior. No choice
between such explanations seems possible without further studies, which
are now in progress.
Induction of the transition by mastoparan and
MP14 may occur through PTP regulatory mechanisms which are already
recognized(1, 2, 3) . However, since these
peptides have a wide spectrum of pharmacological activities (see
above), new potential regulatory mechanisms can also be considered.
Regarding the established mechanisms, it does not appear that reducing
membrane potential is the primary mode by which the peptides act under
CSA-sensitive conditions. This is indicated by the TPP accumulation data in Fig. 1and Fig. 2which show
that mastoparan produces only minor changes in membrane potential when
CSA, EGTA, or oligomycin are present to inhibit pore opening. The same
is true for MP14 (data not shown). Under CSA insensitive conditions, an
early and extensive release of TPP
is seen and this
seems to occur somewhat faster than swelling (Fig. 4). This
depolarization could be brought about by peptide molecules acting upon
the membrane lipid phase as illustrated in Fig. 10. It is
thought that no PTP effector dominates in the interactive system of
pore regulation through allosteric and membrane/bioenergetic
mechanisms. Instead, the open/closed probability appears to be
established as a sum of positive and negative actions exerted at a
number of sites(17) . Thus, the peptides may act at an
allosteric site, synergistically with Ca
and P
acting their sites, under CSA-sensitive conditions. It could then
be the additional influence of depolarization produced by higher
peptide levels acting upon the membrane lipid phase which allows the
pore to open in a Ca
independent and CSA insensitive
manner. The present data cannot identify the allosteric site at which
the peptides may act, although it is noted that mastoparan carries
three positive charges, whereas MP14 carries two (Table 1). Thus,
the relative potency of these peptides correlates with positive charge
and they might act at a site which normally binds a cation. The
Ca
binding site is one possibility and an action at
this site could explain why pore induction can occur in the absence of
Ca
when the peptide concentration is high. Possible
displacement of CSA from its site of action by high concentrations of
mastoparan is another possibility to consider.
Analogies between regulation of the PTP and the N-methyl-D-aspartic acid receptor channel suggest that the PTP belongs to the super family of ligand gated ion channels and thus may be regulated by covalent modification (see (3) ). Such systems often involve G-proteins, and a mitochondrial G-protein has recently been identified and isolated(67, 68) . According to a recent report, MP14 retains the G-protein-mediated activities of mastoparan but has a diminished capacity to perturb membranes(69) . It is for these reasons that the actions of MP14 and mastoparan on the PTP were compared in this study. MP14 is only slightly less active than mastoparan when inducing pore opening in a CSA-sensitive manner, whereas there is a larger activity differential when the pore is induced in the presence of CSA (Fig. 5). If the CSA insensitive induction involves membrane perturbation (depolarization) as suggested above, then these data suggest regulation of the PTP through a G-protein. The relative effectiveness of mastoparan and MP14 have not yet been compared in a variety of systems, however, and so it is not clear how completely the G-protein-mediated and membrane perturbation-mediated activities of MP14 are distinguished. In particular, it appears that a membrane perturbation activity differential between these two peptides is only seen in the presence of a membrane potential (Fig. 10) and the reason for this deserves further investigation.
The actions of mastoparan and MP14 on the PTP suggest that low concentrations of other amphipathic peptides could regulate the PTP in cells. Such peptides are a product of processing imported proteins and may accumulate in the mitochondrial matrix under some conditions. Extra- and intramatrix space amphipathic peptides may also accumulate in injured cells due to proteolysis. These peptides may promote pore opening, even in presence of CSA, and become a factor in maintaining the protection of injured cells afforded by CSA.