YMGME-05711; No.

of pages: 6; 4C: 3
Molecular Genetics and Metabolism xxx (2014) xxxxxx

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Molecular Genetics and Metabolism

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Opening up of plasmalemma type-1 VDAC to form apoptotic nd me

signal pathways is essential in early apoptosis Evidence from the
pathogenesis of cystic brosis resulting from failure of apoptotic cell
clearance followed by sterile inammation
Friedrich P. Thinnes
Baumschulenweg 5, D-37083 Gttingen, Germany

a r t i c l e

i n f o

Article history:
Received 10 January 2014
Received in revised form 3 February 2014
Accepted 4 February 2014
Available online xxxx
Keywords:
Cystic brosis
Sterile inammation
Apoptosis
Find me signal
Plasmalemma VDAC-1

a b s t r a c t
Cell membrane-standing type-1 VDAC is involved in cell volume regulation and thus apoptosis. The channel has
been shown to gure as a pathway for osmolytes of varying classes, ATP included. An early event in apoptotic cell
death is the release of nd me signals by cells that enter the apoptotic process. ATP is one of those signals. Apoptotic cells this way attract phagocytes for an immunologically silent cell clearance. Thus, whenever apoptosis
fails by a blockade of plasmalemma type-1 VDAC processes of sterile inammation must be assumed for cell elimination. This is evident from a close look on the pathogenetic process of cystic brosis (CF). However, in normal
airway epithelia two different anion channels cooperate to guarantee an appropriate volume of airway surface
liquid (ASL) necessary for surface clearing: the cystic brosis conductance regulator (CFTR) and the outwardly
rectifying chloride channel (ORCC) complex also called alternate chloride channel and under the control of
the CFTR. There are arguments, that type-1 VDAC forms the channel part of the ORCC complex, and it has been
shown that CFTR and type-1 VDAC co-localize in the apical membranes of human surface respiratory epithelium.
In cystic brosis, the central cAMP-dependent regulation of ion and water transport via functional CFTR is lost.
Here, CFTR molecules do not reach the apical membranes of airway epithelia anymore or work in an insufcient
way, respectively. In addition, type-1 VDAC is no longer available to work as a nd me signal pathway. In consequence, clearing away of apoptotic cells is blocked. There are experimental data on the channel characteristics
of type-1 VDAC under the anion channel blocker DIDS (4,4-diisothiocyanato-stilbenedisulphonic acid) that argue
in favor of this hypothesis. Together, type-1 VDAC should be kept as a nd me signal pathway, which may give
way to several classes of such signals.
2014 Elsevier Inc. All rights reserved.

Contents
1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.
Channel active VDAC-1 reacts with DIDS in differing approaches
. .
1.2.
Type-1 VDAC is involved in regulatory volume decrease, RVD . . . .
1.3.
Plasmalemma VDAC-1 channels ATP . . . . . . . . . . . . . . .
1.4.
Opening of plasmalemma type-1 VDAC precedes caspase activation .
2.
Type-1 VDAC is expressed in permanent B-lymphocyte cell lines of CF people
3.
Onset of cystic brosis by failure of apoptosis . . . . . . . . . . . . . . .
4.
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.
Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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E-mail address: futhin@t-online.de.

URL: http://www.futhin.de.

http://dx.doi.org/10.1016/j.ymgme.2014.02.001
1096-7192/ 2014 Elsevier Inc. All rights reserved.

Please cite this article as: F.P. Thinnes, Opening up of plasmalemma type-1 VDAC to form apoptotic nd me signal pathways is essential in early
apoptosis Evidence from ..., Mol. Genet. Metab. (2014), http://dx.doi.org/10.1016/j.ymgme.2014.02.001

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F.P. Thinnes / Molecular Genetics and Metabolism xxx (2014) xxxxxx

1. Introduction
Regarding the classical cell death mechanisms, i.e. intrinsic and
extrinsic apoptotic pathways, the relevance of voltage dependent
anion-selective channels, VDACs, as resting in the outer mitochondrial
membranes is well established: blocking VDAC in this compartment
results in a blockade of apoptotic cell death [1]. Concerning VDAC in the
plasmalemma of several cell types, it has been demonstrated that type1 VDAC in this compartment forms part of the cell volume regulatory system. This was evidenced by experiments where either the regulatory volume decrease (RVD) of hypotonically stimulated cells or the apoptotic
volume decrease (AVD) of toxically stimulated cells, respectively, was
blocked by cell outside applied anti-VDAC-1 antibodies or the anion
channel inhibitor DIDS; for review see References [2,3]. In addition,
data on an opening up VDAC-1 in cell membranes by toxic stimuli,
e.g. staurosporine and amyloid A peptides, point to another type
of extrinsically stimulated cell death pathway, putatively relevant
for the pathogenesis of Alzheimer disease [3]. Finally, according to
a study using epithelial cells of VDAC1 knockout mice, cell
membrane-standing VDAC-1 has been proven to work as an ATP efux pathway [4]. However, VDAC is an archaic channel and can thus
be assumed to support housekeeping cell functions. A synopsis of
recent results on cell membrane-standing type-1 VDAC insinuates
to rmly schedule the channel as a nd me signal pathway,
which may give way to different classes of such signals [5,6].
Apoptotic cells are rarely seen in multicellular healthy individuals because highly effective clearance mechanisms quickly eliminate unwanted cells of varying provenance, e.g. excess cells built
in ontogenetic development, cells carrying intracellular bacteria
or viruses, transformed or malignant cells, toxically damaged
cells. According to recent studies the clearance process starts with
1) the sensing of corpses via nd me signals, which is followed
by 2) their recognition via eat me signals and their receptors,
3) the activation of signaling pathways that regulate cytoskeletal
rearrangement necessary for cell engulfment, and nally 4) the activity of phagocytes that keep cell clearance events immunologically silent by quick removal of apoptotic cells. However, this key step
of apoptosis, which is necessary for the maintenance of organismal
homeostasis, is nally executed via the internalization of dying
cells into membrane-bound vesicles, the phagosomes, by several
types of phagocytes which can be specialized or just neighboring
cells depending of the cell or tissue type affected.
According to an extensive survey by Hochreiter-Hufford and
Ravichandran [5] four apoptotic nd me signals are in discussion
and thought to establish chemotactic gradients stimulating the migration of phagocytes to the apoptotic cell: 1) fractalkine is a
membrane-associated protein that is released from apoptotic B
cells and neurons which directs macrophages to the dying targets.
2) Lysophosphatidylcholine (LPC) is discussed as a lipid type nd
me signal, and assumed to stimulate macrophage chemotaxis toward apoptotic cells. 3) There is some evidence that sphingosine1-phosphate (S1P) may work as another lipid type nd me signal
secreted by apoptotic cells. 4) Recently, the nucleotides ATP and
UTP entered the eld as a new class of apoptotic nd me signals.
Accordingly, small amounts of intracellular ATP and UTP are released
in early apoptosis to establish a gradient for monocyte attraction, putatively sensed by corresponding P2Y2 receptors. Nucleotides are readily
degraded by extracellular nucleotidases, and are thus unlikely to serve
as long-range nd me signals to phagocytes in circulation. They rather
will attract tissue resident macrophages, e.g. in epithelia. Concerning relevant nucleotide release pathways there are data referring to pannexin
channels, which are opened during apoptosis by caspase-dependent
cleavage. Here, I present data indicating that plasmalemma-integrated
type-1 VDAC, the putative channel part of the ORCC channel complex,
gures as another/co-operating apoptotic nd me signal pathway.
Excitingly the synopsis, graphically summarized by Fig. 1, may help to

better understand the pathogenesis of cystic brosis as a process resting

on sterile inammation.
1.1. Channel active VDAC-1 reacts with DIDS in differing approaches
Type-1 VDAC/VDAC-1, the gene product of VDAC1, when
reconstituted into lipid bilayer membranes, forms dened voltagedependent channels. Five minutes pre-incubation with 100 M DIDS altered the channel characteristics of the protein, now showing small irregular channels instead of distinct conductance steps. In addition, the
voltage-dependence of the channel was abolished by action of the agonist. In other words: DIDS changed the channel characteristics of VDAC1 without blocking it totally. On this experimental basis, taken together
with data on structural and functional similarities between human
VDAC-1 and porin preparations from other eukaryotic species, I asked
already in 1990 if cell membrane-integrated VDAC-1 may form the
channel part of a chloride channel complex, the alternate chloride
channel which is misregulated in cystic brosis [7]. By afnity chromatography it has meanwhile also been shown that channel active human
or bovine porin, reversibly, bind to the stilbene-disulfonate group of
DIDS. The data further support a direct interaction of VDAC-1 and
DIDS [8]. There are data on additional low molecular weight agonists
working on VDAC, which may be helpful in studies on the channel: cholesterol, ATP, Knig's polyanion, dextran sulfate, Ga3+, Al3+, Zn2+,
polyamines, compound 48/80, ruthenium red, uoxetine, cisplatin
[2,3], and on peptides e.g. BH4-BClXL peptides [911] and amyloid A
peptides, too [3].
1.2. Type-1 VDAC is involved in regulatory volume decrease, RVD
VDAC-1 shows multi-compartment expression. In plasma membranes electro-physiologically dened chloride/anion channel phenotypes exceed the number of molecularly dened chloride/anion
channels. However, VDAC-1 comprising channel complexes can be assumed to show varying phenotypes. Depending on the status of regulation these complexes may thus built up pathways for osmolytes of
several classes [2,3,12]. On this conceptual basis my laboratory has nally demonstrated that type-1 VDAC is part of the cell volume regulatory
system, playing its role in regulatory volume decrease (RVD) [13]. A
central event of this process is the opening up of a ubiquitous chloride/
anion channel, and blocking this pathway would abolish RVD. However,
cell outside applied monoclonal mouse anti-human type-1 porin antibodies blocked the RVD of HeLa cells, proving that VDAC-1 is involved
in the process. HeLa cells pre-incubated with the antibodies dramatically increased their volume within about 1 min after a stimulus by hypotonic Ringer solution, but did not move backward toward their starting
volume, thus indicating abolished RVD. To notice, corresponding effects
were induced by the established anion channel inhibitor DIDS [13] or
BH4BClXL peptides. Video-camera monitoring of cell size over time
was used in this direct and noninvasive approach [2,3,13; http://
www.futhin.de Supplement 1 includes the recording building the
basis of [13]]. The molecular identity of the channel phenotypes under
discussion in CF is still in debate (see below).
1.3. Plasmalemma VDAC-1 channels ATP
These functional data were, meanwhile, conrmed by a study on epithelial cells of VDAC1 knockout mice in Dr. Boucher's laboratory. In addition, this study demonstrated that type-1 VDAC contributes to the ATP
release from the murine cells under study [4]. The observation that cells,
VDAC1 being knocked out, still release some ATP points to additional
molecules involved in this function, e.g. to the CFTR, which from the beginning has been discussed as a modulator of the alternate chloride
channel with respect to cystic brosis therapy. For a discussion of this
aspect see l.c. [2,3]. Overall, type-1 VDAC channels can be kept as candidates to gure as pathways for the nd me signal ATP in epithelial

Please cite this article as: F.P. Thinnes, Opening up of plasmalemma type-1 VDAC to form apoptotic nd me signal pathways is essential in early
apoptosis Evidence from ..., Mol. Genet. Metab. (2014), http://dx.doi.org/10.1016/j.ymgme.2014.02.001

F.P. Thinnes / Molecular Genetics and Metabolism xxx (2014) xxxxxx

Fig. 1. A model suggested for the outwardly rectifying chloride channel complex including plasmalemma-integrated type-1 as a putative pathway for apoptotic nd me signals. The
channel complex is under the control of the cystic brosis gene product, the cystic brosis transmembrane conductance regulator (CFTR), and is in this way involved in the disturbed chloride ow in the case of CF-diseased epithelial cells. According to the model, in healthy resting cells plasmalemma-integrated human porin is kept closed by the interaction with one or more
modulators on the cytosolic side of the cell membrane. In this state, cell regulation makes no ATP available on the cell surface. If the cell is stimulated, ATP appears via the CFTR channel on
the outside of the cell and brings about a change in conformation of the ATP-bound VDAC-1/modulator unit which, for its part, leads to an opening of the channel. In the case of CF-diseased
cells, the CFTR molecule no longer reaches the apical membrane of epithelial cells, or only in a changed form. The absence of ATP on the cell surface results in the otherwise functional ORCC
channel no longer opening spontaneously. The model explains why the ORCC channel in the case of CF-diseased cells can be opened experimentally as a result of the extracellular application of ATP. The modied gure is taken from [2].

apoptotic cell death. In line, there is indication on a corresponding

function of VDAC-1 in red blood cells. Sridharan et al. summarized
their data on prostacyclin receptor mediated ATP release from erythrocytes via a model postulating the interaction of cell membrane-standing
VDAC and CFTR [14]. Concerning alternative apoptotic nd me signal
pathways, on the one hand, observations on varying channel phenotypes, set up by soluble Alzheimer A peptides applied to liposomes
or black membranes, i.e., amyloid made channels, have accumulated
since the early nineties of last century. Channels of this type are often assumed to explain apoptogenic effects on living cells in cell physiologic
settings. On the other hand, A peptides, alternatively, can be assumed to work as regulators of endogenous cell membranestanding channel molecules, i.e., amyloid regulated channels. Cell
membrane-integrated type-1 VDAC is a good candidate in this context. For a discussion of this issue see [15]. In addition, data of a recent paper argue in favor of pannexin as an ATP release pathway in
erythrocytes. The authors observing a second ATP pathway in their
study refer expressis verbis to VDAC as an ATP release pathway [16].
1.4. Opening of plasmalemma type-1 VDAC precedes caspase activation
Apoptotic cell death is an essential process in ontogenetic development and plays its role in the pathogenesis of degenerative diseases.
Apoptotic cells show several morphologic features: cell contraction in
isotonic surroundings (apoptotic volume decrease), nuclear fragmentation, blebbing and apoptotic body formation. To notice, cellular membranes of apoptotic cells stay intact to prevent massive protein release
and consequent inammation. However, in a rst step, efux of K+
and Cl ions leads to the shrinkage of the apoptotic cell followed by
water movement, activation of caspases and mitochondrial cytochrome
C shedding as established downstream reactions. Excitingly, Elinder
et al. presented electrophysiological and immunocytochemical evidences on an early activation of type-1 VDAC in the plasma membrane
of neurons under staurosporine stimulation. Again, cell outside applied
anti-VDAC-1 antibodies blocked VDAC-1 opening and thus inhibited
downstream apoptotic processes [17], and this is in good agreement
with the blockade of the RVD of hypotonically stimulated HeLa cells
(see above). In other words: to keep plasmalemma VDAC closed, blocks
apoptotic progress. Noteworthy, the expression level of VDAC-1 in

differentiated hippocampal neurons surmounts that one of neural

stem cells [18,19].
A series of studies on apoptosis of neuronal cells, elaborated by
Marin and colleges presented further evidence for the involvement of
VDAC at cell surface in apoptosis. First, a study on the toxic effect of amyloid A peptides on septal (SN56) and hippocampal (HT22) neurons
proved another time that blocking VDAC in cell membranes by two different anti-porin antibodies means preventing an apoptotic development of the cells. It also showed that VDAC and the estrogen receptor
(mER), in association with caveolin-1, co-localize and interact in
cell membrane caveolae [20]. Second, this relationship has been corroborated by the demonstration that VDAC-1 and mER are integrated in
caveolar lipid rafts [21,22].
A recent study of Pamenter and colleges assessed the impact of DIDS
on cellular viability by examining the morphology and functions of neuronal cells through 24 hour treatment with the VDAC-1 blocker DIDS. It
has been shown that control cells were unchanged, while cells under
DIDS clearly moved towards an apoptotic phenotype. Finally, in an apparent paradox, an overall apoptotic phenotype was stated while certain hallmarks of apoptosis were missing in DIDS treated cells, e.g.
mitochondrial ssion and loss of plasma membrane integrity [23]. It is
tempting to speculate that in this setting, which way however, an apoptotic process was initiated but could not be completed because the
VDAC blocker DIDS abolished the channel as a nd me signal pathway.
Finally, Dinnen and colleges demonstrated that pancreatic cancer
cell lines underwent an alternative form of cell death when treated
concomitantly with of arsenic trioxide, ascorbic acid and disulram.
The major effects observed were mediated through generation of intracellular reactive oxygen species and signicant decline in intracellular ATP. However, while N-acetyl cysteine and a superoxide
dismutase mimetic, prevented aponecrosis and restored intracellular ATP levels the VDAC blocker and shRNA of VDAC-1 (working on
the expression level of mitochondrial and plasmalemma type-1
VDAC) blocked cell death and ROS accumulation. Applied in vivo
the agonist mixture resulted in a reduction of mean tumor size and
the elimination of tumors of nude mice with PANC-1 xenografts.
The authors concluded that early caspase-independent apoptosis
was shifted to VDAC-mediated targeted aponecrosis by the addition of disulram to arsenic trioxide and ascorbic acid [24].

Please cite this article as: F.P. Thinnes, Opening up of plasmalemma type-1 VDAC to form apoptotic nd me signal pathways is essential in early
apoptosis Evidence from ..., Mol. Genet. Metab. (2014), http://dx.doi.org/10.1016/j.ymgme.2014.02.001

F.P. Thinnes / Molecular Genetics and Metabolism xxx (2014) xxxxxx

2. Type-1 VDAC is expressed in permanent B-lymphocyte cell lines of

CF people
Human typ1-VDAC, in my laboratory, has been puried and sequenced on the protein level four times starting from 1) enriched cell
membranes of the permanent B-lymphocyte cell line H2LCL [25], 2)
crude membranes of human muscle [26], 3) puried mitochondria
from the cell line H2LCL [27], and 4) enriched cell membranes of a permanent B-lymphocyte cell line of a CF patient [28]. We found identical
primary structures in each of the native type-1 VDAC preparations in
those studies: the molecule comprising 282 building blocks, and the
N-terminus being acetylated. Our 1989 data [25] were the basis for
many studies on the DNA level in the animal kingdom [29]. Also, shortly
after our rst report on the extra-mitochondrial expression of VDAC-1
in the plasmalemma of permanent human B-lymphocytes had been
published a topographic study demonstrated the co-localization of
VDAC-1 and CFTR in the apical domain of the ciliated cells of human surface respiratory epithelia [30]. Meanwhile we also reported on the cell
swelling behavior of two permanent CF B-lymphocyte cell lines thus
documenting differences in the RVD of the two CF cell lines and a corresponding healthy cell line under study [31,32].
3. Onset of cystic brosis by failure of apoptosis
The CF syndrome is characterized by exaggerated inammation,
progressive tissue damage, and chronic bacterial colonization, mainly
in the respiratory tract. According to an informative recent review on
apoptosis in cystic brosis cells [33], here the clearance of apoptotic epithelial cells is awed basically by disturbed chloride efux at the apical
membranes of epithelial cells. In the CF syndrome CFTR molecules carry
mutations that abolish a proper trafcking of the molecule to the plasmalemma or change its channel characteristics, respectively. In either
case the CFTR molecule fails as the regulator of the alternate chloride
channel mostly called outwardly rectifying chloride channel
(ORCC) at the apical membrane of respiratory epithelial cells, while in
healthy epithelia co-localized type-1 VDAC and the CFTR interact to
open up the ORCC complex, and thus generate adequate conditions for
epithelial surface uid control [3464].
Cell membrane-standing type-1 VDAC is a good candidate to form
the channel part of the ORCC complex misregulated in cystic brosis.
It can be stimulated by different approaches then showing different
phenotypes, and opening up this alternate chloride channel has
been discussed as a therapeutic escape since the early 1990s. Aside several antibody preparations that affect the channel in different ways
there are additional low molecular weight agonist and also peptides
working on VDAC-1, which may help in studies on the channel. E.g. uoxetine has been shown to react with type-1 VDAC in articial lipid bilayer membranes [3]; applied at endothelial cells the agonist blocks
volume-regulated anion channels and also Ca2+-activated chloride currents [45]. To notify, VDAC-1 carries a GxxxG motif in its helical Nterminal part that is relevant for channel gating. GxxxG motifs have
been shown to bind ATP or cholesterol [2,3]. Finally, type-1 VDAC is
also expressed in the endoplasmic reticulum (ER) here interacting
with IP3Rs to mediate the transmission of Ca2+ signals between the
ER and mitochondria, a process relevant in apoptosis [65,66].
Concerning the competitors of type-1 VDAC regarding the molecular
basis of volume regulated anion channels (VRACs, VSOACs) that include
the alternate chloride channel in cystic brosis a series of channels or
channel modulators has been discussed without reaching a consensus
[6568]. For a recent review of the different anion channel phenotypes
see [69].
4. Conclusion
Taken for granted that 1) VDAC-1 forms the channel part of the
ORCC complex in cell membranes, 2) opening up of ORCC precedes all

apoptotic reactions, and 3) VDAC-1 gures as a nd me signal pathway ne-tuned by the CFTR, recent assumptions on an involvement of
apoptotic processes in the pathogenesis of the cystic brosis syndrome
[33] can be specied. On the one hand, the basic defect in trafcking or
function of mutated forms of the CFTR renders CF cells rather sensitive
for apoptosis. On the other hand, apoptotic CF cells cannot nalize the
cell death process because the ORCC complex cannot open up without
help of functional CFTR in cell membrane. Together, in CF cells apoptosis
gets blocked and nd me signals cannot be released, a situation nally
superimposed by inammation and necrosis. In line with this view on
the pathogenesis of the cystic brosis syndrome is the observation
that in CF infant inammation occurs even in the absence of bacterial infection [33].
5. Outlook
Two recent papers state, on the on hand, that neuroinammation
and neuronal loss precede A plaque deposition in the hAPP-J20
mouse model of Alzheimer's disease [70], on the other hand, that extracellular amyloid beta 42 causes necrosis, inhibition of nuclear division,
and mitotic disruption [71]. From here it is tempting to speculate that
in Alzheimer's disease, too, incomplete apoptotic processes may play a
role in the pathogenesis of the syndrome. Finally, there is an early report
demonstrating that double-stranded DNA can be translocated across a
planar membrane containing puried mitochondrial porin [72] what
might be relevant for the initiation of necrotic cell development whenever apoptosis gets stuck.
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