Studies on Human Porin XXII: Cell Membrane Integrated Human Porin Channels Are Involved in Regulatory Volume Decrease (RVD) of HeLa Cells

Molecular Genetics and Metabolism 69, 331–337 (2000) doi:10.1006/mgme.2000.2976, available online at http://www.idealibrary.com on

Studies on Human Porin XXII: Cell Membrane Integrated Human Porin Channels Are Involved in Regulatory Volume Decrease (RVD) of HeLa Cells Friedrich P. Thinnes,* Klaus P. Hellmann,* Thea Hellmann,* Rolf Merker,* Ulrike Brockhaus-Pruchniewicz,† Christian Schwarzer,* Go¨tz Walter,‡ Hilde Go¨tz,* and Norbert Hilschmann* *Abteilung Immunchemie and †Graphikabteilung, Max-Planck-Institut fu¨r experimentelle Medizin, Hermann-Rein-Strasse 3, D-37075 Go¨ttingen, Germany; and ‡DADE Behring Marburg, D-35001 Marburg, Germany Received December 29, 1999, and in revised form February 15, 2000

The expression of eukaryotic porin, voltage-dependent anion channels (VDAC) channels (1) in the plasmalemma of vertebrate cells is well established (2– 6). However, the channel’s function at this site is still under debate, mostly because its electrophysiological phenotype has not yet been defined. An initial clue came from a study on the interaction of the anion channel inhibitor DIDS and a putative chloride channel molecule in pig kidney with the molecular weight of mammalian porin (7). Next, we showed that the main channel characteristics of highly enriched human porin were significantly changed by 4,4⬘-diisothiocyanato-stilbene-2,2⬘-disulfonic acid (DIDS) and proposed that plasmalemma integrated porin forms part of a chloride channel complex affected in cystic fibrosis (8). Meanwhile, there is evidence that cell membrane integrated porin channels may figure as maxi-, midi-, or even mini-chloride channels, according to the electrophysiological approach used. On the one hand, maxi-chloride channels in excised patches of endothelial cells and astrocytes were closed down by antiporin antibodies applied to the cell exterior, the channels passing intermediate conductance states during closure (9,10). On the other hand, attached mode patch clamping studies of HT29 epithelial cells have shown that mini-chloride channels opened up to form maxi-chloride channels by simultaneous membrane permeabilization, the channels again crossing intermediate states (11). Maxi- and midi-,

Cell volume regulation receives increasing attention not only as the basis of regulatory volume increase or regulatory volume decrease (RVD) of cells in surroundings of changing osmolarity, but also appears to be relevant in cell proliferation, differentiation, and apoptosis. A central event in RVD is the opening of a volume-sensitive chloride/ anion channel(s), and blocking this pathway would abolish RVD. This is shown here with monoclonal mouse anti-human type-1 porin antibodies, proving that porin is involved in this process. HeLa cells preincubated 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. We had already accumulated evidence that plasmalemma integrated eukaryotic porin channels form chloride/anion channels in this cell compartment and that they are involved in cell volume regulation. Finally, the present data again demonstrate the suitability of our anti-porin antibodies in physiological studies. © 2000 Academic Press Key Words: osmoregulation; volume regulation; chloride channels; anion channels; channel regulation; monoclonal antibodies; human porin; voltagedependent anion channels (VDAC). 331

1096-7192/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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as well as mini-chloride channels, are discussed in the context of cell volume regulation (12). In the meantime, using the antibodies mentioned above (9,10,13–15), we provided initial data on the involvement of porin channels in chloride or taurine fluxes of hypotonically stimulated Xenopus laevis oocytes or HeLa cells (16,17). They also blocked cell swelling, induced by ionic gadolinium applied to human B lymphocytes and HeLa cells in isotonic and hypotonic NaCl or taurine Ringer solutions (18 –20). Here we demonstrate corresponding blocking effects of the anti-porin antibodies or DIDS on the regulatory volume decrease (RVD) reactions of hypotonically stimulated adherent HeLa cells. MATERIALS AND METHODS Cells. Adherent HeLa cells were cultured under standard conditions and finally seeded into tissue culture plates, with wells showing diameters of 15 mm (Greiner Labortechnik GmbH, Solingen, Germany, Cat. No. 662160). Solutions. Isotonic Ringer solutions (mM: 145 NaCl, 5 KCl, 2 CaCl 2, 1 MgCl 2, 10 glucose, 10 Hepes, adjusted to pH 7.4 by NaOH) showed osmolarities of about 300 mmol/kg. Hypotonicity was achieved by mixing equal volumes of isotonic and NaCl-free Ringer solutions, resulting in osmotic values around 200 mmol/kg. The buffer chemicals were of GR quality from Merck (Darmstadt, Germany). DIDS was from Sigma–Aldrich (Steinheim, Germany). Video camera monitoring. RVD measurements were made 1 or 2 days after cell seeding. Cells were washed twice by 1 ml of isotonic Ringer solution, covered with 1 ml of isotonic Ringer solution, microscopically focused, and finally stimulated by the addition of 1 ml of Ringer solution without any NaCl. Cell swelling behavior over time was monitored by a video camera (Kappa messtechnik GmbH, Gleichen, Germany, CF 15/4, RGB) on a ZEISS Axiovert 135 microscope. Pictures were taken every 5 or 10 s for 15 or 30 min. Monoclonal mouse anti-human type-1 porin antibodies (13–15; Calbiochem/Novabiochem Bad Soden/Ts., Germany) were applied before hypotonic cell stimulation by preincubation for 20 to 40 min. To measure the effect of DIDS, cells were preincubated with the agonist from stock solutions for 10 min. Experiments were carried out at room temperature.

RESULTS Cells suddenly exposed to hypotonic surroundings initially react with significant swelling by water entrance, which is driven by osmotic forces. However, within a minute they start to restore their original volume by a process called regulatory volume decrease, taking about 15 min. In order to initiate RVD, cells activate chloride channels, a reaction resulting in membrane depolarization and followed by opening of cation channels. Finally, the passive loss of inorganic ions, mostly K ⫹ and Cl ⫺ together with organic osmolytes, ends up in osmotically obliged water efflux from the cells and restoration of the volume at rest. Blocking of volume-sensitive anion channels would thus allow cell swelling but abolish RVD. Figure 1 represents essential steps of the swelling behavior of HeLa cells, hypotonically stimulated by a 70 mM NaCl Ringer solution in the presence of anti-porin antibodies, as they were documented by video camera monitoring of microscopic cell inspection over time. The optical approach used directly demonstrates changes in cell shape of the adherent cells in only two dimensions which, furthermore, should be minimized by cell fixation to the substrate. Movements into the third dimension are indicated at best indirectly by changes in optical focusing of cells at different moments during the experiments. However, it is evident from the series of graphs that HeLa cells under the conditions used, on the one hand, distinctly swell in about 1 min, a time course strongly indicating hypotonically induced cell swelling (Figs. 1a and 1b). On the other hand, there is no indication that the cells, monitored for 15 min after hypotonic stimulation, move backward toward their configuration at the start of the experiment (Figs. 1a and 1c), a strong indication for antibody blocked RVD. Measurements of one-dimensional dilatations of several cells at an enlarged plot of Fig. 1 allowed a rough quantitative estimate of their swelling behavior under anti-porin antibodies. Accordingly, 1 min after the hypotonic stimulus the cells showed dilatations of about 8% (relative increase factor: 1.05–1.11). After 15 min the relative increase was 1.14 –1.18. The application of several of eight monoclonal anti-porin antibody preparations (13–15) revealed differences in their effectiveness concerning RVD blocking, indicating a specific antibody effect on cell behavior. What is more, the inhibitory effect of the monoclonal antibodies on HeLa RVD could be

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FIG. 1. Anti-porin antibodies block the regulatory volume decrease (RVD) of HeLa cells. The series documents video camera monitoring over time of the microscopic inspection of adherent HeLa cells which were preincubated with a monoclonal mouse anti-human type-1 porin antibody (Calbiochem Cat. No. 529538, 30 ␮g/ml) and then hypotonically stimulated by 70 mM NaCl Ringer. (a) First photograph immediately after the hypotonic stimulus; (b) swelling of cells after about 1 min; (c) cells still remain swollen after 15 min, indicating the blocking effect of the antibody on the RVD of the cells. Objective magnification, 63⫻.

abolished by their preincubation with a 10-fold molar surplus of synthetic peptides of the N-terminus of human type-1 porin, which represents the epitope recognized by the antibodies (13–15). Our laboratory had already shown by the indirect immunofluorescence approach that the antibodies used here label the surface of HeLa cells (17). Preincubation of the cells with antibodies against the human epithelial membrane antigen (DAKO Diagnostika GmbH, Hamburg, Germany, Cat. No. M0613), an established epithelial surface marker, had no blocking effect on the cell function under study, while they were active in indirect surface labeling (data not shown). In quick-motion camera demonstrations of

the experiments the effect of the antibodies is even more evident. Examples will be available on the World Wide Web. (http://www.mpiem.gwdg.de). The anion channel inhibitor DIDS is well established as a blocker of chloride/anion channels and of the RVD reaction of hypotonically swollen cells (12,21). Furthermore, highly enriched human type-1 porin reconstituted to artificial planar lipid bilayers heavily changed main channel characteristics after preincubation with 100 ␮M DIDS (8). Figure 2 shows that preincubation of HeLa cells with the same amount of agonist induces corresponding effects on cell swelling and RVD as shown above for anti-human type-1 antibodies. While cells quickly

FIG. 2. The anion channel inhibitor DIDS blocks the regulatory volume decrease (RVD) of HeLa cells. Adherent Hela cells were preincubated in isotonic Ringer solution with 100 ␮M 4,4⬘-diisothiocyanato-stilbene-2,2⬘-disulfonic acid for 10 min and then hypotonically stimulated by 70 mM NaCl Ringer. The swelling behavior of the cells over time was documented by video camera monitoring of microscopic cell inspection. (a) First photograph immediately after hypotonic stimulation; (b) maximal swelling of cells after about 1 min; (c) cells are still swollen after 20 min, indicating a RVD block by DIDS. Objective magnification, 63⫻.

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FIG. 3. Regulatory volume decrease (RVD) of HeLa cells. Adherent HeLa cells were suddenly exposed to hypotonic 70 mM NaCl Ringer solution. Cell swelling followed by normal RVD were documented by video camera monitoring of microscopic cell inspection over time. (a) First photograph immediately after osmotic stimulation; (b) maximal cell swelling after about 1 min; (c) reduced swelling of cells after 15 min, indicating RVD reactions of the cells. Objective magnification, 63⫻.

swell in about a minute after hypotonic stimulation (Figs. 2a and 2b), they lose the ability to return to the configuration before the hypotonic stimulus by RVD (Figs. 2a and 2c). Figure 3, as a control, demonstrates normal swelling and regulatory volume decrease of hypotonically stimulated HeLa cells. Cells exposed to a 70 mM NaCl Ringer solution first enlarge their volume in about 1 min (Figs. 3a and 3b), a process which is in full agreement with the reactions shown above for HeLa cells after preincubation with anti-porin antibodies (Fig. 1) or DIDS (Fig. 2), respectively. Cells then start to decrease their volume, this RVD reaction being monitored in our experiments for about a quarter of an hour. According to the experimental approach used, RVD becomes evident by reduction of two-dimensional extensions of the cells as observed at maximal swelling (Fig. 3b). It proceeds slowly and can be observed directly in quick-motion camera demonstrations of the process (see above). Finally, cells do not fully return to their configuration before swelling (Figs. 3a and 3c), probably on the basis of a disturbance of cell substrate fixation during the swelling-induced dilatation process. DISCUSSION Numerous studies prove the relevance of chloride/ anion channels for cell volume regulation. However, while there are detailed physiological characterizations of the channel(s) involved, structural identity is still under debate (12,21–27). Meanwhile, several

groups of chloride channels have been formulated in molecular terms: the GABA and the glycine receptor channels (28,29), the product of the cystic fibrosis gene CFTR (30), the Ca 2⫹ activated chloride channel family (31), the ClC chloride channel family (32), and the porin channel or VDAC family (8,33– 41). Some of them are discussed in the context of cell volume regulation. However, two members of the ClC chloride channel family attracted high attention in recent studies (42– 44), and there is increasing evidence that plasmalemma integrated porin channels play a significant role in this important cell biological process (2,3,9,10,16,17,20,22,45). Here we demonstrate that adherent HeLa cells, preincubated with monoclonal mouse anti-human type-1 porin antibodies, show normal swelling whenever they are exposed to hypotonic surroundings (70 mM Ringer solution), but lose their ability to reestablish starting volume by regulatory volume decrease. Furthermore, the effect induced on the cells by the antibodies coincides with the one observed after the application of 100 ␮M DIDS. The agonist is known to block the RVD reaction of cells (12,23) and it had been shown to affect main channel traits of human type-1 porin after reconstitution to planar artificial lipid bilayers (8). Otherwise, channel active human type-1 porin had been reversibly fixed to the stilbene-disulfonate grouping of immobilized DIDS (46). Taken together, the present data strongly argue for an involvement of plasma membrane standing porin channels in the RVD of HeLa cells, thus contributing to endeavors toward the molecular definition of the chloride/anion channel(s)

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involved in cell volume regulation (7–12,20 –27, 42– 45). Video camera monitoring of microscopic cell inspection over time appears to be a rather physiologic approach to document cell swelling behavior and regulatory volume decrease. Moreover, the antiporin antibodies used (13–15) had already proved to be suitable for physiological measurements by several approaches: (a) they closed maxi-chloride channels in excised patches of mammalian endothelial cells and astrocytes, while this effect could be blocked by preincubation with the antigen (9,10); (b) they showed effects on the chloride or taurine efflux of Xenopus laevis oocytes and HeLa cells after hypotonic stimulation (17); and (c) most recently they blocked gadolinium-induced cell swelling of human B lymphocytes and HeLa cells in isotonic or hypotonic salt or taurine Ringer solutions. These data argue for a single channel pathway for either osmolyte (3,20). In topochemical studies on the expression of porin channels in the cytoplasmic membranes of different vertebrate cell lines, the antibodies always induced a dot-wise labeling pattern, thus indicating specific reactions (3,16). In a recent study, surface labeling of frog oocytes was prevented by preincubation of the antibodies with the epitope they recognize (6,15). Western blots and ELISA measurements proved the high specificity of the monoclonal antibodies against porin in vertebrates (5,13–15). A decade ago our endeavors to define the function(s) of plasmalemma integrated porin channels began with the proposal that they form part of a ubiquitous chloride channel complex putatively affected in cystic fibrosis (8). We then elaborated circumstantial evidence that porin molecules at this site may figure as maxi-chloride channels (3,9,10), midi-chloride channels [e.g., outwardly rectifying chloride channel (3,8,12,47–52)], or even mini-chloride channels (3,11), depending on the electrophysiological approach used. Corresponding data were recently reviewed (3,16). Each type of chloride channel is discussed in the context of cell volume regulation (12). However, we looked for the impact of anti-porin antibodies on this process, applying different methods on several vertebrate cell types, and reported effects on hypotonically induced chloride or taurine fluxes at the cell membranes (9,10,16 –20). The present paper, with its direct and noninvasive physiological approach, adds further data in support of our early proposals.

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Note added in proof. We recently demonstrated that gadolinium induced swelling on different human cell lines in isotonic and hypotonic surroundings. We now have data showing that fluoxetine counteracts these effects in black membrane measurements and also in physiological experiments.

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