To consider data on an interaction of type-1 VDAC and DIDS may help to further interpret the study recently presented by Pamenter et al. (2013) PLOS ONE 8, e60804.

1) "Porin 31HL" = type-1 VDAC = VDAC-1, the gene product of VDAC1, was reconstituted into lipid bilayer membranes, where it formed defined voltage-dependent channels. Five minutes pre-incubation with 100 microM DIDS altered the channel-forming properties of the protein, now showing small irregular channels instead of distinct steps. In addition, the voltage-dependence of the channel was abolished by the action of the agonist. In other words: DIDS changed channel characteristics of VDAC-1 without blocking it totally. On this experimental basis, taken together with data on functional and structural similarities between "Porin 31HL" and porin preparations from other human tissues and from other eukaryotic species, cell membrane-integrated VDAC-1 was first proposed to form the channel part of a chloride channel complex, which is defective in cystic fibrosis (1). The molecular identity of the “alternate chloride channel” in CF is still under debate.

2) By affinity chromatography it has been shown that channel active human or bovine porin, reversibly, binds to the stilbene-disulfonate group of DIDS. The data support a direct interaction of VDAC-1 and DIDS (2).

3) VDAC-1 shows multi-compartment expression, and it is found in the plasma lemma, the endoplasmic reticulum and outer mitochondrial membranes, too. In cell membranes electrophysiologically defined chloride/anion channel phenotypes exceed the number of molecularly known chloride channel molecules. However, type-1 VDAC comprising channel complexes might show varying phenotypes, this depending on the cell type studied or the regulatory status, respectively (3-5).

4) On this conceptual basis it has been demonstrated that VDAC-1 is part of the cell volume regulatory system, playing its role in regulatory volume decrease (RVD). A central event in RVD is the opening of a volume-sensitive chloride/anion channel(s), and blocking this pathway would abolish RVD. This has been shown by monoclonal mouse anti-human type-1 porin antibodies, thus proving that VDAC-1 is involved in the process. HeLa cells pre-incubated with these antibodies dramatically increase their volume within about 1 min after a hypotonic stimulus by 70 mM NaCl Ringer solution, but do not move back toward their starting volume, thus indicating abolished RVD. Corresponding effects are induced by the established anion channel inhibitor DIDS. Video camera monitoring of cell size over time was used as a direct and noninvasive approach (6; http://www.futhin.de). The data were confirmed by VDAC1 knock out mice, testing the involvement of VDAC-1 in ATP release and RVD of mouse epithelial cells. Taken together, the studies suggested that VDAC-1, directly or indirectly, contributes to ATP release from murine cells. However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function (7).

5) Apoptotic cell death is an essential process in the development of the central nervous system and in the pathogenesis of its degenerative diseases. In a first step, efflux of K(+) and Cl(-) ions leads to the shrinkage of the apoptotic cell (AVD) followed by activation of caspases. Excitingly, Elinder et al. (2005) presented electrophysiological and immuno-cytochemical evidences for the activation of VDAC in the plasma membrane of neurons, undergoing apoptosis induced by staurosporine. Anti-VDAC antibodies blocked the channel and inhibited downstream apoptotic processes (8). In other words: to keep plasmalemmal VDAC closed, blocks apoptotic progress. Noteworthy, the channel plays a critical role in differentiated hippocampal neurons but not in neural stem cells (9).

6) Three papers from Raquel Marin's laboratory presented further evidence for the involvement of VDAC at cell surface in apoptosis [10–12]. A first study on the toxic effect of amyloid Aß peptides on septal (SN56) and hippocampal (HT22) neurons, on the one hand, proved another time that blocking VDAC in cell membranes by two different anti-porin antibodies means preventing an apoptotic development of the cells. On the other hand, it showed that VDAC and the estrogen receptor α (mERα) in association with caveolin-1 co-localize and interact in cell membrane caveolae, mERα working towards neuroprotection [10]. The topographic relationship of the molecules was further specified by demonstrating that both are integrated in caveolar lipid rafts [11]. The Marin group meanwhile presented additional data to demonstrate that the interaction of VDAC and mERα in caveolae from human cortex is altered in Alzheimer disease [12], results which appear to be in line with an early 2D-electrophoresis report on differences in the VDAC amount of biopsies taken from normal or Alzheimer brains, respectively [13].

7) The relevance of VDAC-1 in the endoplasmic reticulum and outer mitochondrial membranes concerning apoptosis is well established. The compartments, furthermore, interact in the context of cellular calcium turnover, this in correlation with the plasma lemma.


To summarize: data referred to demonstrate an interaction of DIDS and type-1 VDAC in differing approaches. In artificial lipid bilayers the agonist alters the channel characteristics of the molecule without totally blocking it. Applied at the cell membrane of HeLa cells DIDS works as anti-VDAC-1 antibodies do: it blocks the regulatory volume decrease (RVD) of the cells by keeping VDAC-1 closed. Anti-VDAC-1 antibodies have also been shown to block the apoptotic volume decrease (AVD) of neuronal cells.

Conclusion

The study under consideration assessed the impact of DIDS on cellular viability by examining the morphology and function of neuronal cells through 24 hours treatment under DIDS. 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 fission and loss of plasma membrane integrity.

Taking the requirement of a working cell volume regulation in cell cultures for granted, from my point of view a clue on the apparent paradox discussed by Pamenter and colleges appears. Blocking the volume regulation of cells in culture should be hard to deal with and thus result in the induction of apoptotic processes. However, considering the data given above the apoptotic process should come to a standstill.

Finally, while working with DIDS in biological settings the time factor might be of high relevance.

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1) Thinnes FP, Schmid A, Benz R, Hilschmann N. Studies on human porin. III. Does the voltage-dependent anion channel "Porin 31HL" form part of the chloride channel complex, which is observed in different cells and thought to be affected in cystic fibrosis? Biol Chem Hoppe Seyler. 1990 Nov;371(11):1047-50.

2) Thinnes FP, Flörke H, Winkelbach H, Stadtmüller U, Heiden M, Karabinos A, Hesse D, Kratzin HD, Fleer E, Hilschmann N. Channel active mammalian porin, purified from crude membrane fractions of human B lymphocytes or bovine skeletal muscle, reversibly binds the stilbene-disulfonate group of the chloride channel blocker DIDS. Biol Chem Hoppe Seyler. 1994 May;375(5):315-22.

3) Thinnes FP. New findings concerning vertebrate porin II--on the relevance of glycine motifs of type-1 VDAC. Mol Genet Metab. 2013 Apr;108(4):212-24.

4) Thinnes FP, Reymann S. New findings concerning vertebrate porin. Naturwissenschaften. 1997 Nov;84(11):480-98. Review.

5) Morris AP, Frizzell RA. Ca(2+)-dependent Cl- channels in undifferentiated human colonic cells (HT-29). II. Regulation and rundown. Am J Physiol. 1993 Apr;264(4 Pt 1):C977-85.

6) Thinnes FP, Hellmann KP, Hellmann T, Merker R, Brockhaus-Pruchniewicz U, Schwarzer C, Walter G, Götz H, Hilschmann N. Studies on human porin XXII: cell membrane integrated human porin channels are involved in regulatory volume decrease (RVD) of HeLa cells. Mol Genet Metab. 2000 Apr;69(4):331-7.

7) Okada SF, O'Neal WK, Huang P, Nicholas RA, Ostrowski LE, Craigen WJ, Lazarowski ER, Boucher RC. Voltage-dependent anion channel-1 (VDAC-1) contributes to ATP release and cell volume regulation in murine cells. J Gen Physiol. 2004 Nov;124(5):513-26.

8) Elinder F, Akanda N, Tofighi R, Shimizu S, Tsujimoto Y, Orrenius S, Ceccatelli S. Opening of plasma membrane voltage-dependent anion channels (VDAC) precedes caspase cctivation in neuronal apoptosis induced by toxic stimuli. Cell Death Differ. 2005 Aug;12(8):1134-40.

9) Akanda N, Tofighi R, Brask J, Tamm C, Elinder F, Ceccatelli S. Voltage-dependent anion channels (VDAC) in the plasma membrane play a critical role in apoptosis in differentiated hippocampal neurons but not in neural stem cells. Cell Cycle. 2008 Oct;7(20):3225-34.

10) Marin R, Ramírez CM, González M, González-Muñoz E, Zorzano A, Camps M, Alonso R, Díaz M. Voltage-dependent anion channel (VDAC) participates in amyloid beta-induced toxicity and interacts with plasma membrane estrogen receptor alpha in septal and hippocampal neurons. Mol Membr Biol. 2007 Mar-Apr;24(2):148-60.

11) Marin R, Ramírez C, Morales A, González M, Alonso R, Díaz M. Modulation of Abeta-induced neurotoxicity by estrogen receptor alpha and other associated proteins in lipid rafts. Steroids. 2008 Oct;73(9-10):992-6.

12) Ramírez CM, González M, Díaz M, Alonso R, Ferrer I, Santpere G, Puig B, Meyer G, Marin R. VDAC and ERalpha interaction in caveolae from human cortex is altered in Alzheimer's disease. Mol Cell Neurosci. 2009 Nov;42(3):172-83

13) Yoo BC, Fountoulakis M, Cairns N, Lubec G. Changes of voltage-dependent anion-selective channel proteins VDAC1 and VDAC2 brain levels in patients with Alzheimer's disease and Down syndrome. 2001 Jan;22(1):172-9.