ISSN 1990-7478, Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology, 2008, Vol. 2, No. 4, pp. 404–411. © Pleiades Publishing, Ltd., 2008. Original Russian Text © A.V. Sokolov, V.S. Sokolov, T.B. Feldman, M.A. Ostrovsky, 2008, published in Biologicheskie Membrany, 2008, Vol. 25, No. 6, pp. 499–507.
Interaction of All-trans-Retinal with Bilayer Lipid Membranes A. V. Sokolova, V. S. Sokolova, T. B. Feldmanb, and M. A. Ostrovskyb aFrumkin
Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Leninsky prosp., 31, build. 5, Moscow, 119071 Russia; e-mail:
[email protected] bEmmanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, Russia Received August 4, 2008
Abstract—The interaction of all-trans-retinal (hereinafter referred to as retinal) with planar bilayer lipid membranes has been studied. Addition of retinal into aqueous solutions on both sides of the membrane formed from diphytanoilphosphatidylcholine (DPhPC) or its mixture with diphytanoilphosphatidylethanolamine (DPhPC/DPhPE in w/w proportion of 3 : 5) led to a change of conductance induced by ionophores nonactin (increase of conductance) or pentachlorophenol (decrease). Increase of nonactin-induced conductance was dependent on the membrane lipid composition and was two times higher in the case of DPhPC/DPhPE mixture. The change of conductance caused by ionophores of different signs (plus or minus) had different direction suggesting the influence of the retinal on the dipole potential upon its incorporation into BLM. The boundary potentials difference measured by the intramembrane field compensation method (IFC) after the retinal addition on one side of the membrane did not exceed 2.5 mV suggesting that its distribution in the bilayer is almost symmetrical. The illumination of the retinal-containing BLM caused a decrease in its lifetime when the membranes were formed from unsaturated lipids. Retinal incorporated into BLM led also to photoinactivation of the gramicidin channels. The process was completely inhibited by a singlet oxygen quencher (sodium azide). These results indicate that retinal accumulated in the membrane can affect both membrane proteins and the unsaturated lipids by their oxidation by the singlet oxygen. DOI: 10.1134/S1990747808040156
The photolysis of the visual rhodopsin, specifically on its last stage, is followed by a release of all-trans-retinal (hereinafter referred to as retinal or ATR). Later the ATR is reduced by retinol dehydrogenase to all-transretinol, which is subsequently transferred from rod outer segments into retinal pigment epithelial cells. Besides, retinal molecules can be transferred through the photoreceptor membrane by ATP-binding cassette transporter (ABCR) [1]. However, in several pathologies such as different forms of degenerative retinal diseases, the mechanism of retinal removal from the photoreceptor membrane can be disrupted and retinal can accumulate in the lipid bilayer. In this case retinal binds with one of the bilayer phospholipids, phosphatidylethanolamine, resulting in the formation of N-retinylidene-phosphatidylethanolamine and then N-bisretinylidene-ethanolamine (A2E) [2, 3]. A2E is accumulated together with other retinal-containing by-products in lipofuscin granules, which tend to pile up in retinal pigment epithelial cells (Fig. 1) [1, 4]. Exposure of the lipofuscin granules to visible light is known to cause generation of free radicals and reactive oxygen species [5, 6]. Besides, even in dark A2E is able to cause disruption of artificial membranes [7]. Earlier we have studied destructive impacts of A2E upon bilayer lipid membranes (BLM) [8, 9]. We showed that A2E molecules can accumulate into BLM and decrease its lifetime both in darkness (so-called “detergent-like effect”) and during light exposure. In
addition to the destructive effect of A2E on BLM, a light-induced inactivation of gramicidin channels in the membrane was shown in these studies. During the exposure of gramicidin-containing BLM to visible light in the presence of A2E, a profound decrease of the membrane conductance was observed. It was explained by photodestruction of gramicidin channels incorporated in the bilayer. These experiments model the destruction of membrane-associated proteins [10, 11]. Presumably, similar destruction processes can occur in lipids and proteins of photoreceptor membranes. They may be caused by both A2E and other retinal-containing by-products including retinal itself. As was shown recently, all-trans-retinal, while being covalently bound to amide groups of opsin (socalled “A2-rhodopsin”), which are accessible to retinal in the photoreceptor membrane, tends to act as a photosensitizer. It can impair natural ability of rhodopsin to regenerate [12], implying to be not just a precursor of A2E. Retinal itself is able to affect stability of biological membranes when accumulated inside the lipid bilayer. In order to test this suggestion, a model of bilayer lipid membrane and gramicidin channel as a simple artificial model of membrane-incorporated polypeptide can be used in the same way as they were used earlier in A2E-related research. The main goal of this study was to investigate consequences of the ATR incorporation into BLM. In par-
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Fig. 1. The structure of all-trans-retinal (at the top) and the reactions of the A2E synthesis from two molecules of retinal and one molecule of phosphatidylethanolamine, according to review [1] (below).
ticular, we tried to find out if the processes of retinal accumulation and retinal-related impairment of membrane stability were dependent on the lipid composition of the membrane both in the dark and during irradiation. In a way similar to our earlier research of the A2E effects, we studied the impact of retinal accumulated in BLM on photodynamic destruction of the membranes containing unsaturated lipids and on photoinactivation of gramicidin channel. EXPERIMENTAL Bilayer lipid membranes were formed by MuellerRudin method in a Teflon cell. The cell was divided by
a septum into two compartments. The compartments of the cell had equal volumes (750 µl each) and were filled with a buffer solution. BLM was formed on a round hole of diameter 0.8-mm in the center of the septum. Electrical measurements were performed with the aid of silver-chloride electrodes with agar bridges. The bridges were made from standard 1-ml plastic pipette tips, the bottom part of which was closed by agar, and the remaining volume was filled with 0.1 M KCl solution. Total electrical resistance of the electrodes with bridges did not exceed 50 kΩ. Capacitance and conductance of BLM were determined by measuring the electric current evoked by voltage ramps applied to the membrane. The voltage was
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applied from the analog output of the ADC/DAC board (LCard L780, Russia) to one electrode in the cell. The other electrode was connected with the input of the current–voltage converter Keithley-427 (USA), the output voltage of which was sampled by ADC (LCard board L780) and recorded in a computer memory. The membrane capacitance value was calculated as a ration of the current step amplitude to the speed of the applied potential change. The conductance of BLM was estimated from the current–voltage plot by the slope at zero voltage. Capacitance increase to a steady-state value (about 1–3 nF) indicated that the membrane formation was completed. Aqueous solutions were prepared in bidistilled water with KCl (Reachim, Russia) and HEPES (Calbiochem, USA). The buffer solution composition was 10 mM KCl and 1 mM HEPES, pH 7.5. The pH of the solution was adjusted by KOH (Reachim). The lipids used to form the bilayer membranes were diphytanoylphosphatidylcholine (DPhPC, 15 mg/ml in decane), dioleoylphosphatidylcholine (DOPC, 40 mg/ml in decane) (Avanti Polar Lipids, USA), diphytanoylphosphatidylethanolamine (DPhPE) (Avanti Polar Lipids, USA), and mixture of DPhPC with DPhPE (w/w ratio 3:5, 40 mg/ml in decane in total). Retinal solution in ethanol (5 mM) was added to the cell after the formation of BLM. Retinal concentration in the cell was 50– 80 µM. Ethanol concentration in the cell did not exceed 2% and did not affect BLM stability. Retinal accumulation in the membrane was determined by either of two methods: by measurement of bilayer conductance change in the presence of ionophores or by detection of boundary potentials difference. In the first case the conductance of BLM was induced by nonactin (N) that carries cations or by pentachlorophenol that transfers anions through the membrane. In these experiments both ionophores and retinal were added symmetrically to both compartments of the cell. The concentration of nonactin or pentachlorophenol in the buffer solution of the cell was 50 µM. In the second case, boundary potentials difference was assessed with the aid of intramembrane field compensation method (IFC). The principle of IFC method is based on the ability of electric field to compress a membrane and thus to increase its capacitance [13–15]. Minimal value of capacitance is reached when the electric field inside the membrane equals zero. The voltage difference across the membrane corresponding to the minimum of the capacitance compensates the electric field inside the membrane and is equal to the difference of boundary potentials on two sides of the membrane. This voltage was determined using an experimental setup for the IFC measurements similar to that used in our previous studies [8, 9]. A sum of sinusoidal and DC voltages was applied across the membrane separating two solutions. The value of the second harmonic of capacitive current was used as an indicator of the intramembrane field. The value of DC component of the applied voltage which corresponds to zero point of second harmonic
(which is equal to the boundary potentials difference) was measured every 5 seconds and recorded on the hard disk of the computer. All experiments regarding photoeffects including gramicidin photoinactivation studies were performed using a special cell with a transparent window allowing membrane illumination. BLM was illuminated by visible light from a mercury lamp, the electric power of which was 250 W. The light beam passed through a water filter to remove infrared radiation (light), a glass filter to restrict the spectrum on the blue side (below 550 nm), and than focused by a lens on the hole of the septum in the cell. The intensity of the illumination measured by the RTN-31S calorimeter (Russia) was about 340 W/m2. In all experiments regarding photoeffects and photoinactivation, the retinal was added only to one compartment of the cell opposite to the source of light (further called the cis-side of BLM). Gramicidin photoinactivation experiments were performed with the same technique that was used in other photosensitizer related studies [8, 10, 11]. Gramicidin A (Sigma-Aldrich Corp., USA) was added to both compartments; its concentration in the buffer solution was 10–10 M. In the experiments with singlet oxygen quencher, the sodium azide dissolved in water was added to both compartments of the cell; final concentration of sodium azide in the buffer solution was 20 mM. RESULTS AND DISCUSSION Retinal incorporation into BLM. Incorporation of retinal into the membrane was verified by the changes of conductance caused by ionophores N and PCP. Figure 2a illustrates the kinetics of the induced conductance alteration in the nonactin-containing BLM. The conductance of the membranes formed from different lipids (DPhPC or DPhPC/DPhPE 3:5 w/w mixture) increased within 30–40 minutes after the retinal addition and subsequently reached a steady value. Relative increments of the conductance (the final steady conductance level divided to its initial level caused by ionophore in retinal-free BLM) were averaged over 4–5 measurements (Fig. 2b). The membranes conductance increased by a factor of 1.5 or 3 if BLM was formed from DPhPC or DPhPC/DPhPE mixture, respectively. A greater conductance increase in the presence of DPhPE can be explained by a better accumulation of retinal in these membranes. Furthermore, the dependence of the effect on the lipid composition implies that the effect is produced by the presence of retinal in the BLM rather than by its direct interaction with the ionophore. This result affiliates with the data obtained in papers [2, 3] that illustrated the formation of stable complexes of retinal with phosphatidylethanolamine of the retina cell membranes. As it is known, the conductance is determined by the concentration of the charge carriers (the complex of nonactin with potassium ions, for instance) and by their
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mobility inside the membrane [16]. Accumulation of retinal in a bilayer may affect both non-electrical properties of the membrane (thickness and microviscosity) and the electrical potential jumps on its boundaries as well. The latter can be due to the fact that the retinal molecule, although electroneutral and lipophilic, contains an aldehyde group that has a dipole moment, which can cause a shift in the boundary potential. According to [16–20], the contribution of electrical and non-electrical factors can be distinguished by comparison of the conductance alterations induced by the ionophores carrying positive and negative ions. Following this approach, we made a similar conductance measurement with PCP, a ionophore which carries ions of the opposite charge, i.e., anions. Introduction of retinal into the cell in presence of PCP induced a 4-fold decrease in the conductance (Fig. 2c). Opposite shifts of conductance induced by ionophores with different signs of charge of the complex transferring through the membrane, were observed earlier [16–20] and were explained by the electrical effect, specifically, by an alteration of the boundary potentials on both sides of the membrane. Using this interpretation we can suppose that in our system the boundary potentials on both sides of BLM decrease. This leads to a decrease of the potential barrier for cation-carrying ionophores and its increase for anions. Accordingly, we see an increase of conductance of the membrane modified by a cationic ionophore and a decrease, when BLM is modified by an anionic ionophore. We suggest that the effect of retinal on the membrane conductance is caused by an alteration of the dipole component of the boundary potential, which is also termed a dipole effect. The relative increments of conductance with nonactin and its decrement with pentachlorophenol were significantly different in magnitude. In particular, nonactin changed the conductance by a factor of 1.5, and PCP, by a factor of 4. If the effect of retinal were only electrical (a change either in surface charge or in dipole potential of the membrane), the magnitudes of relative conductance changes induced by nonactin and PCP would be equal. Their difference indicates that besides the changes of electrical properties of BLM, retinal induces some structural changes as well (microviscosity, for instance). Our suggestion about the dipole effect of retinal was tested by the IFC method, which measures the shift of boundary potential difference between two sides of BLM upon adding the retinal into the cis-compartment of the cell. However, alteration of the boundary potentials difference observed in the experiment was very low and did not exceed 2.5 mV (Fig. 3). For a reference, the adsorption of A2E on BLM which we studied earlier, led to an effect of about +80 mV [9]. An insignificant shift of the boundary potential difference detected here is presumably due to the lipophilicity of retinal that makes it able to penetrate through a membrane and
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Fig. 2. Influence of retinal on the conductance of BLM modified by several ionophores. (a) Kinetics of the conductance change of BLM formed from DPhPC (1) or DPhPC/DPhPE mixture (w/w ratio 3 : 5) (2) with incorporated nonactin, after the retinal addition at the moment marked with arrow. (b) Relative change in the conductance of BLM of different lipid compositions (see explanation to Fig. 2a) with nonactin, caused by retinal introduction (average of 5 measurements). (c) Relative change in the conductance of BLM (DPhPC) with PCP after retinal introduction into the cell at the moment marked by the arrow. Concentration of retinal in the cell is 80 µM.
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Fig. 3. Kinetics of the boundary potential difference measured by the IFC method. BLM is formed from DPhPC. An arrow indicates the moment of introduction of retinal (80 µM) into the cis-compartment of the cell.
to distribute almost symmetrically between two boundaries of BLM. By the way, this ability of retinal to penetrate through the membrane was also confirmed by significant staining of the solution not only in the ciscompartment where it was initially added to, but also in the trans-compartment of the cell. Because of this ability of the retinal molecules, the boundary potential difference measured by IFC turned to be much less than the shift of the boundary potential value on each side of BLM and this makes it undetectable by this method. We have also found that the addition of retinal into the cell in all range of the concentrations (up to 80 µM) in the absence of illumination did not affect BLM stability, suggesting that the detergent-like mechanism of the membrane disruption which had a place in the case of A2E [8] is not realized under these conditions, although the concentration of retinal used here was much higher than the A2E concentration used before (80 µM of retinal in this study and 4 µM of A2E in [8]). However, a profound detergent-like effect of A2E was observed in liposomes only at the concentration equal or higher than 300 µM [7]. Photodynamic disruption of BLM in the presence of retinal. To investigate a photodynamic effect of retinal on the membranes, we explored its impact on the BLM lifetime both in dark and when exposed to visible light (Fig. 4). The lifetime of membranes was measured from the moment of their formation to the moment of their rupture, which was detected as a steep avalanchelike conductance increase. The membranes composed of a saturated lipid (DPhPC) were not disrupted either upon illumination or in the dark, regardless of the presence of retinal. However, the membranes composed of unsaturated lipid DOPC were ruptured 3 times faster when exposed to the light than in darkness (lifetimes
0
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Fig. 4. Lifetime (average from 5 measurements) of BLM formed from DOPC in the dark (1) and upon illumination (2), determined after the retinal accumulation.
was 13 and 40 min, respectively). Similar results were obtained in our earlier studies with A2E, where the BLM lifetime upon illumination decreased in the case of DOPC membranes in contrast to DPhPC [8, 9]. We suppose that as in the case of A2E, a photodynamic breakdown of BLM with retinal is a result of the oxidation of unsaturated lipids by reactive oxygen species. The ability of retinal to generate the reactive oxygen species was detected by various methods earlier [21, 22]. Photoinactivation of gramicidin channel. We have shown that the retinal is capable to disrupt the BLM stability upon exposure to visible light, presumably due to oxidation of unsaturated lipids. It was determined recently that retinal can also affect membrane proteins in a similar way by impairing their functions [12]. Earlier, in [8], a simple membrane polypeptide gramicidin was used to investigate the photodynamic damage of membrane proteins in the presence of A2E. Disruption of gramicidin can be easily determined as a decrease in the conductance of BLM modified by the gramicidin channels. It was shown that photosensitizerlike action of A2E is partially inhibited by sodium azide. This result suggests that singlet oxygen generation contributes to the process of the A2E-induced gramicidin photoinactivation. We used the same approach to study similar effects in the presence of retinal. To assess the photoinactivation kinetics we recorded the kinetics of the change of the BLM conductance with time (Fig. 5a). After the addition of gramicidin to both compartments of the cell, the conductance grew up to several tens or hundreds nS due to the gramicidin incorporation into BLM and formation of the gramicidin channels. After the conductance reached a steady level, retinal was added to the cell and 40–
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Fig. 5. Photoinactivation of gramicidin channels. (a) Conductance of BLM formed from DPhPC with gramicidin, measured in presence of retinal. Light was switched on and off at the moments marked by arrows 1 and 2, respectively. (b) Influence of A2E and retinal on gramicidin channel photoinactivation rate in the presence and in the absence of sodium azide, calculated as the reciprocal time constant (average from 5 measurements) of the exponential fitting of the kinetics of the photoinduced decrease of the BLM conductance.
60 min later the light was turned on. The data shown earlier (Fig. 2a) suggest that this time is necessary for the retinal accumulation in the bilayer. As was predicted, the illumination led to a profound decrease in the conductance, and the effect was fully reversible: after terminating the exposure to light, a recovery of the conductance was observed, due to incorporation into the membrane of new molecules of undamaged gramicidin from the bulk water solution (Fig. 5a).
To evaluate the photoinactivation rate quantitatively, we calculated the reciprocate time constant of the exponent fitting the kinetics of conductance decrease. In the presence of retinal this value was 3 times less than in the presence of A2E (Fig. 5b). Taking into account these results and the known ability of retinal to generate reactive oxygen species and free radicals upon illumination, we suggest that the gramicidin photoinactivation can also be caused by
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these processes. To test this assumption, we studied the effect of a singlet oxygen quencher, sodium azide, on the photoinactivation of gramicidin channels in the presence of retinal. It was shown that sodium azide profoundly inhibited the retinal-induced photoinactivation of gramicidin channels. This result suggests that photodynamic effect of retinal on gramicidin can be primarily caused by singlet oxygen generated by excited retinal molecules. It is noteworthy that in the case of A2E its disruptive effect could be induced not only by singlet oxygen but by other reactive oxygen species (superoxide anion radical, hydroxonium, etc.), because sodium azide leads only to a partial inhibition of the A2E-induced gramicidin channels photoinactivation [8]. In conclusion, the results of this study provide evidence that retinal is able to accumulate in lipid bilayer and distributes symmetrically inside it, affecting the dipole potential of the membrane. Under illumination, retinal can induce the photooxydation of both lipid and protein components of membranes. These processes are apparently caused by the singlet oxygen generated by the excited retinal. This suggests that an abundant accumulation of all-trans-retinal during an impaired visual cycle may subsequently cause photodestruction of cell membranes of the retinal pigment epithelium. ACKNOWLEDGMENTS The authors are grateful to Drs. Yu.A. Chizmadzhev, Yu.A. Ermakov, and Yu.N. Antonenko for valuable advice and helpful discussion. The work was supported by the Russian Foundation for Basic Research (project no. 06-04-49500) and by the grant of the President of the Russian Federation for the State Support of Leading Scientific Schools (project no. 4181.2008.4). REFERENCES 1. Ostrovsky, M.A, Molecular Mechanisms of Light Damage to the Eye Structures and Photoprotective System of the Eye, Uspekhi Biologicheskoi Khimmii (Rus.), 2005, vol. 45, pp. 173–204. 2. Eldred, G.E. and Lasky, M.R., Retinal Age Pigments Generated by Self-Assembling Lysosomotropic Detergents, Nature, 1993, vol. 361, pp. 724–726. 3. Parish, C.A., Hashimoto, M., Nakanishi, K., Dillon, J., and Sparrow, J., Isolation and One-Step Preparation of A2E and Iso-A2E, Fluorophores from Human Retinal Pigment Epithelium, Proc. Natl. Acad. Sci. USA, 1998, vol. 95, pp. 14609–14613. 4. Ben Shabat, S., Parish, C.A., Vollmer, H.R., Itagaki, Y., Fishkin, N., Nakanishi, K., and Sparrow, J.R., Biosynthetic Studies of A2E, a Major Fluorophore of Retinal Pigment Epithelial Lipofuscin, J. Biol. Chem., 2002, vol. 277, pp.7183–7190. 5. Boulton, M., Dontsov, A., Jarvis-Evans, J., and Ostrovsky, M., Lipofuscin Is a Photoinducible Free Radical
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