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Rapid and improved reconstitution of bacterial mechanosensitive ion channel proteins MscS and MscL into liposomes using a modified sucrose method
FEBS Letters 583 (2009) 407–412
journal homepage: www.FEBSLetters.org
Rapid and improved reconstitution of bacterial mechanosensitive ion channel proteins MscS and MscL into liposomes using a modified sucrose method Andrew R. Battle, Evgeny Petrov, Prithwish Pal, Boris Martinac * Department of Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia
a r t i c l e
i n f o
Article history: Received 6 November 2008 Revised 8 December 2008 Accepted 10 December 2008 Available online 25 December 2008 Edited by Maurice Montal Keywords: Patch clamp Confocal microscopy Fluorescence Lipid Sugar
a b s t r a c t The bacterial mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance have functionally been reconstituted into giant unilamellar liposomes (GUVs) using an improved reconstitution method in the presence of sucrose. This method gives significant time savings (preparation times as little as 6 h) compared to the classical method of protein reconstitution which uses a dehydration/rehydration (D/R) procedure (minimum 2 days preparation time). Moreover, it represents the first highly reproducible method for functional reconstitution of MscS as well as MscS/MscL co-reconstitution. This novel procedure has the potential to be used for studies of other ion channels by liposome reconstitution. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Bacterial MS ion channel proteins act as emergency relief valves during osmotic shock in order to help bacterial cells survive sudden changes in external osmolarity [1–3]. Based on their conductance and pressure sensitivity three types of such channels have been identified in Escherichia coli: MS channel of Large conductance (MscL), MS channel of Small conductance (MscS/MscK) and MS channel of Mini conductance (MscM) [1,4]. MscS and MscL ion channels were first discovered in giant spheroplasts of E. coli [2,5,6]. Incorporation of MscL and MscS into artificial liposomal membranes has also been achieved and their functional channel activity has been recorded using the patch-clamp technique [2,6– 10]. In contrast to MscL functional studies, which to a large extent have been carried out using liposome reconstitution methods [3,7,11,12], studies to date of MscS have mostly been using giant spheroplasts [13,14] leading to a limitation in the conditions in which to study this protein. Very few studies of MscS in liposomes have been undertaken: Sukharev [8] studied MscS in azolectin, and more recently Vásquez et al . [9] successfully reconstituted the MscS protein into E. coli polar lipids. However, both procedures required time consuming dehydration/rehydration methods as well as high protein/lipid ratios for MscS channel reconstitution.
* Corresponding author. Address: The Victor Chang Cardiac Research Institute, Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia. Tel.: +61 2 9295 8600; fax: +61 2 9295 8734. E-mail address: [email protected] (B. Martinac).
An alternative method of preparing artificial liposomes is through the formation of giant unilamellar vesicles (GUV), which are becoming more attractive as models of membranes [15,16]. However, incorporation of proteins into such vesicles has been less successful. The vast majority of reports detailing protein reconstitutions into liposomes usually involve detergent-mediated procedures which results in small relative sizes of the liposomes (0.1–0.2 lm) [17,18]. Doeven and co-workers [19] incorporated MscL (at a protein-to-lipid ratio of 1:50 wt/wt) and three other less stable membrane proteins into GUVs using a rehydration-dehydration step in which sucrose was added to the buffer/GUV/protein solution during dehydration. More recently Krishnamurthy et al. [20] successfully incorporated bis-gramicidin A into GUVs suspended in sucrose solutions and recorded channel activity. They dissolved the peptide in ethanol and added it to the prehydrated lipid film, followed by 1 M sucrose addition and GUV formation. In this work we report a time-efficient incorporation of the bacterial MS channel protein MscS into GUVs prepared by rehydrating lipids in sucrose solutions followed by protein reconstitution. Electrophysiological recordings of membrane patches have shown that reconstituted MscS retains its conductance and mechanosensitivity, comparable with MscS in E. Coli membranes. 2. Materials and methods GST-MscL-WT and 6-His-MscS-WT proteins were purified according to a published procedure ((MscL) [7], (MscS) [9]).
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.12.033
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Chloroform, Azolectin (30% L-a-phosphatidylcholine from soyabean) Potassium chloride, magnesium chloride, calcium chloride, sucrose, HEPES (4-2-hydroxyethyl)-piperazine-1-ethanesulfonic acid), TRIS (2-amino-2-hydroxymethyl)-1,3-propanediol), tris(2carboxyethyl)phosphine (TCEP), n-dodecyl beta-D-maltoside (DDM) and potassium hydroxide were all purchased from Sigma and were of analytical grade. BioBeads were purchased from Bio-Rad whereas 1,2-dioleoyl-sn-glycero-3-phosphethanol amine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-1-glycerol)] (sodium salt) (POPG) were obtained as powders from Avanti Polar lipids Inc, and dissolved in CHCl3. Doubly-distilled water was used in all experiments. The dehydration/rehydration (D/R) method follows that described by Häse et al. [7]. 2.1. Sucrose method for incorporation of MscS Purified MscS was taken from a stock solution containing the protein dissolved in PBS and 10 mM DDM detergent and was incorporated into giant artificial liposomes, which were prepared using a modified procedure [15,16]. Two hundred microliters of a 10 mg/ mL solution of azolectin in chloroform was dried in a test tube under a stream of N2 whilst rotating the tube to make a homogeneous lipid film once dry. Once dry, 5 lL of purified water was added to the bottom (pre-hydration) followed by 1 mL of 0.4 M sucrose. The solution was incubated for 3 h at 45 °C. Temperatures lower than 45 °C caused much slower rates of liposome formation. The required amount of protein was then added to achieve a desired protein-to-lipid ratio, and the solution shaken on an orbital mixer for further three hours, after this time a cloud of liposomes was seen to float in the solution. Manual shaking of the tube is not recommended, as it will dissipate the liposome cloud. At this time the sample was ready for patch clamping. However, to ensure complete protein incorporation when using low protein-to-lipid ratios, shaking can be continued overnight. BioBeads (Bio-Rad) can also be added to remove traces of detergent (used to solublize the protein) if desired. In this case, after the overnight shaking step the BioBeads (approx spatula full) are added and shaking is continued for 3 h at RT. 2.2. Patch-clamp recording An aliquot of liposomes (2–4 lL) was taken from the liposome cloud and introduced to the recording bath. Channel activity was
examined in the excised liposome patches and was recorded using the patch-clamp method on liposomes that settled on the bottom of the recording chamber and formed unilamellar blisters [11]. Negative pressure (suction) recorded in mm Hg was applied to patch pipettes by using a syringe and was monitored using a piezoelectric pressure transducer (Omega Engineering, Stamford, USA). Borosilicate glass pipettes (Drummond Scientific Co., Broomall, PA) were pulled using a Flaming/Brown pipette puller (P-87, Sutter Instrument Co., Novato, CA) to a diameter which corresponded to a pipette resistance in the range of 3.0–6.0 MX. Ion currents arising from activation of either MscL or MscS using negative pipette pressure were recorded using an Axon 1D patch-clamp amplifier (Axon Instruments), filtered at 2 kHz and digitized at 5 kHz. Single channel analysis was done using pCLAMP9 software (Axon Instruments). Bath and pipette solutions were symmetrical and consisted of 200 mM KCl, 40 mM MgCl2, 5 mM HEPES (MscL, solution adjusted to pH 7.2 using KOH) or 200 mM KCl, 90 mM MgCl2, 10 mM CaCl2, 5 mM TRIS (MscS, solution adjusted to pH 7.2 using HCl). 2.3. Fluorescence labeling and confocal microscopy To observe the incorporation of MS channels into liposomes, single cysteine mutant versions of MscL (I49C) and MscS (S243C) were generated, labeled with AlexaFluor-488-maleimide (Invitrogen) and incorporated into azolectin liposomes as above at 1/100 or 1/200 protein/lipid ratios (by weight). Confocal images were obtained in recording buffers using an Olympus FluoView1400 Laser Scanning Microscope (Olympus) with Nikon 60 water-immersion objective, NA 1.00 (Nikon, Japan), with excitation at 488nm. 3. Results 3.1. Liposome formation and MscS incorporation Using the sucrose procedure, a cloud of liposomes formed in the sucrose solution, was stable at 4 °C and could be used for several days for experiments. MscS was effectively incorporated into azolectin using the sucrose method (Fig. 1). At a protein/lipid ratio of 1/1000 (wt/wt) more than 75% of examined patches exhibited channel activity. As a comparison, only 31% of patches of MscS incorporated into the liposomes using the D/R method at the same ratio showed activity, a significant improvement over the published methods for MscS reconstitution into liposomes [8,9], where
Fig. 1. Single channel recording from purified MscS reconstituted into azolectin liposomes using the sucrose method.
A.R. Battle et al. / FEBS Letters 583 (2009) 407–412
both authors report incorporations at 1/200 wt/wt. The unitary conductance in liposomes is about one-third that of MscL, which is also analogous to conductance seen in giant spheroplasts [5]. At positive voltages the channel conductance was 1.41 ± 0.03 nS (n = 7), while at negative voltages the conductance was 0.85 ± 0.02 nS (n = 7) (Fig. 2), i.e. two-thirds of that recorded at positive voltages, in agreement with those previously reported [2,5,8]. MscS has been reported to inactivate under prolonged exposure to membrane tension in liposomes [8,9,21], with channels becoming responsive again to applied negative pressure after some time. We did not observe this behavior in azolectin liposomes using the sucrose method, indeed the channels remained active over an extended time period, in some cases remaining open for periods greater than 20 min (Fig. 3A). However, a very gradual inactivation occurred and this is shown in a recording containing two channels, both of which immediately opened upon tension but the second channel closed after >100 s (at constant pressure (Fig. 3B)). We investigated channel behavior in other lipids. In mixtures of DOPC/DOPE/POPG (2:7:1), a ratio of 1/200 wt/wt was required for channel activity in 50% of patches. Channels did not incorporate in pure DOPC. 3.2. MscS and MscL co-reconstitution MscS and MscL were successfully co-reconstituted into azolectin using the sucrose method and in all cases MscS was reconstituted at a ratio of 1/1000 wt/wt. A higher success rate of channel reconstitution (at a given protein/lipid ratio) occurs when sucrose is present (60% of patches contained MscS compared to 46% in the D/R method). Both channels exhibited activity, MscS opening first at a lower pressure threshold, followed by MscL (Fig. 2). MscS inactivation over time also occurred on a slow time scale, analogous to that observed when MscS alone was reconstituted. After release and reapplication of tension (Fig. 3C), MscS often inactivated, while MscL remained responsive to tension. The inactivation of MscS did not affect the opening threshold of MscL, as clearly shown in the pressure trace in Fig. 3C. This observation was seen in all current traces we obtained, regardless of the number of MscS channels in the patch.
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The opening threshold of MscS relative to MscL was also measured, in all experimental conditions the ratio being approximately 1.9 ± 0.3 (n = 30). This ratio falls within the range reported in giant spheroplasts; three separate publications noting a range from 1.39 ± 0.02 to 1.64 ± 0.08 [22–24]. Two ratios of MscL were used (1/1000 and 1/5000 wt/wt) and did not affect the opening threshold ratio of the channels to one another. 3.3. Confocal imaging Using confocal imaging (Fig. 4), we can observe that the proteins are being incorporated into blisters as early as 2 h after addition into the lipid clouds (Fig. 4, left panels). Overnight incubation also produces blisters of various sizes containing fluorescent proteins on the edges as expected for both proteins (Fig. 4, centre and right panels). 4. Discussion This study demonstrates an improved and time-efficient procedure for MscS reconstitution into liposomes and importantly is the first demonstration of co-reconstitution of MscL and MscS in liposomes. It therefore represents a significant advantage over the classical D/R method for both channel proteins. The D/R method is more time consuming requiring a minimum of two days before electrophysiological recordings can be run [11]. Several steps are involved, including an hour of centrifugation, an overnight dehydration step and a rehydration step of at least 3 h. In contrast, the sucrose method allows for electrophysiological recordings in as little as 6 h. It is also a simpler procedure, without the need for centrifugation or dehydration/rehydration steps. These steps result in a minimum time saving of 24 h compared to the D/R method. Most interestingly, it represents the first reported method for efficient and reproducible MscS incorporation and functional analysis into liposomes. Sukharev [8] and Vásquez et al. [9] have described the reconstitution of MscS into azolectin and E. coli native lipids, respectively, albeit at high protein-to-lipid ratios (1/ 200 wt/wt). Using the D/R method [7,11], the incorporation of MscS was less successful than using the sucrose method, where channels were observed in 30% of patches. Interestingly we were
Fig. 2. MscS and MscL co-reconstitution into azolectin liposomes. MscL activates at approximately double the required pressure for MscS activation.
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A
B
C
Fig. 3. MscS reconstituted into azolectin liposomes using the sucrose method. The channels remain active for several minutes (A) and slowly inactivate (B). Inactivation also occurs in co-reconstituted MscS and MscL (C).
not the only research group to experience difficulties in MscS incorporation into azolectin using the D/R method with buffer rehydration (Vásquez and colleagues, personal communication). Although there are many differences between the two methods, it is known that sucrose and other sugars stabilize proteins [25– 27]. One suggested mechanism resulting in this stabilization [28] is through hydrogen bonding of sucrose with proteins. It has also been postulated that protein stabilization occurs through preferen-
tial exclusion of the sucrose from the protein domain (leading to protein stabilization as the unfolded state is thermodynamically less favourable in the presence of sucrose) [27]. Thirdly, sucrose does not influence lipid bilayer packing or phase transition temperatures [29,30]. Although best to our knowledge the precise mechanism is not known it is feasible that stabilization of MscS by sucrose by at least one of the aforementioned methods is an important factor in successful incorporation of the protein during
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Fig. 4. Representative confocal images demonstrating incorporation of fluorescently labelled single cysteine mutants of MscL (top panel) and MscS (bottom panel) into azolectin liposomes.
the preparation. Furthermore, it is also known that lipid films form GUVs in sucrose solutions [15,16,20] hence the large surface area of the lipid bilayer would increase the likelihood of more protein incorporation. Using our procedure highly reproducible results can be obtained at protein-to-lipid ratios as low as 1/3000 (wt/ wt). Consequently, this method should as well be very useful for recording of ion channel activities from eukaryotic ion channels, which are not readily available in quantities of hundreds of micrograms available for bacterial MS channels. The report by Doeven and co-workers [19] detailing sucrose mediated channel incorporation used MscL at a 1/50 wt/wt protein/lipid ratio. Not only have we shown incorporation of this protein at much lower ratios, but also MscS incorporation, which again has not previously been shown. In azolectin we observed very slow inactivation of MscS. Sukharev [8] reported that MscS quickly inactivated in azolectin, analogous to observations in spheroplasts. Using the sucrose method we were nevertheless able to obtain recordings of up to 15 min (or more) showing MscS remaining open over minutes-long periods under continuously applied negative pressure (Fig. 3A). This fortuitous observation opens up the possibility for studying in more detail the properties of this MS channel in liposome membranes. A recent publication by Perozo and co-workers [31] reported that MscS present in giant spheroplasts did not inactivate upon addition of the ampiphath lysophosphatidylcholine (LPC) and suggested that LPC may be exerting different bilayer perturbation forces than those of the better characterized transbilayer difference (see [32]). Indeed, they were unable to repeat this
observation in lipids, the seals not remaining stable for a long enough time frame for characterization under patch-clamp conditions (V. Vásquez, personal communication). Although sucrose is not expected to influence MscS behavior in the same way that LPC does, our observations nevertheless suggest that an intrinsic (and as yet undetermined) interaction of azolectin membranes (or sucrose) with MscS may encourage activation for prolonged time periods without rapid inactivation. Further investigations currently undertaken in our laboratory (including investigating channel behavior in other lipids) should help to elucidate this effect. The confocal images also demonstrate the usefulness of the modified sucrose method for fluorescence studies of ion channel proteins, such as FRET or FRAP, in lipid environments. In conclusion, we have described a novel time-efficient procedure for reconstitution and co-reconstitution of MscS into liposomes using a sucrose solution as the rehydrating medium. We have shown for the first time using the patch-clamp method that MscS can be reconstituted into liposomes made of azolectin or mixtures of pure lipids using the sucrose method at relatively low protein/lipid ratios (1/1000 wt/wt). MscS can also be incorporated into azolectin using the D/R method. We also report the first example of co-reconstitution of MscS and MscL into azolectin. Fluorescence labeling of the proteins and confocal imaging of them in azolectin further demonstrated the incorporation. We have also shown that, under these conditions (unlike in spheroplasts), MscS remains active for prolonged time periods. Furthermore, the improved sucrose method provides time-efficient method at much
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lower protein/lipid ratios than the D/R method, as well as preparations more suitable for prolonged patch-clamp recordings. Acknowledgements This work has been supported by the grants of the Australian Research Council (DP0769983) and National Health and Medical Research Council of Australia (455968). References [1] Martinac, B. (2007) 3.5 billion years of mechanosensory transduction: Structure and function of mechanosensitive channels in prokaryotes. Mechanosensitive Ion Channels, Part A 58, 25–57. [2] Martinac, B., Buechner, M., Delcour, A.H., Adler, J. and Kung, C. (1987) Pressuresensitive ion channel in Escherichia-coli. Proc. Natl. Acad. Sci. USA. 84, 2297– 2301. [3] Perozo, E. (2006) Gating prokaryotic mechanosensitive channels. Nat. Rev. Mol. Cell Biol. 7, 109–119. [4] Berrier, C., Besnard, M., Ajouz, B., Coulombe, A. and Ghazi, A. (1996) Multiple mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied pressure. J. Membr. Biol. 151, 175–187. [5] Sukharev, S.I., Martinac, B., Arshavsky, V.Y. and Kung, C. (1993) 2 Types of mechanosensitive channels in the Escherichia-coli cell-envelope – solubilization and functional reconstitution. Biophys. J. 65, 177–183. [6] Sukharev, S.I., Blount, P., Martinac, B., Blattner, F.R. and Kung, C. (1994) A largeconductance mechanosensitive channel in E. Coli encoded by mscL alone. Nature 368, 265–268. [7] Häse, C.C., Ledain, A.C. and Martinac, B. (1995) Purification and functional reconstitution of the recombinant large mechanosensitive ion-channel (Mscl) of Escherichia-Coli. J. Biol. Chem. 270, 18329–18334. [8] Sukharev, S. (2002) Purification of the small mechanosensitive channel of Escherichia coli (MscS): the subunit structure, conduction, and gating characteristics in liposomes. Biophys. J. 83, 290–298. [9] Vasquez, V., Cortes, D.M., Furukawa, H. and Perozo, E. (2007) An optimized purification and reconstitution method for the MscS channel: strategies for spectroscopical analysis. Biochemistry 46, 6766–6773. [10] Sukharev, S.I., Sigurdson, W.J., Kung, C. and Sachs, F. (1999) Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL. J. Gen. Physiol. 113, 525–539. [11] Delcour, A.H., Martinac, B., Adler, J. and Kung, C. (1989) Modified reconstitution method used in patch-clamp studies of Escherichia-Coli ion channels. Biophys. J. 56, 631–636. [12] Perozo, E., Cortes, D.M., Sompornpisut, P., Kloda, A. and Martinac, B. (2002) Open channel structure of MscL and the gating mechanism of mechanosensitive channels. Nature 418, 942–948. [13] Blount, P. and Moe, P.C. (1999) Bacterial mechanosensitive channels: integrating physiology, structure and function. Trends Microbiol. 7, 420–424. [14] Booth, I.R., Edwards, M.D., Black, S., Schumann, U. and Miller, S. (2007) Mechanosensitive channels in bacteria: signs of closure? Nat. Rev. Microbiol. 5, 431–440.
[15] Akashi, K., Miyata, H., Itoh, H. and Kinosita, K. (1996) Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope. Biophys. J. 71, 3242–3250. [16] Di Maio I.L. et al. (2006). Structural properties of liposomes from digital holographic microscopy. In: Proceedings of SPIE – The International Society for Optical Engineering (2006), 6036 (BioMEMS and Nanotechnology II), 60361R/1-60361R/9. [17] Girard, P., Pecreaux, J., Lenoir, G., Falson, P., Rigaud, J.L. and Bassereau, P. (2004) A new method for the reconstitution of membrane proteins into giant unilamellar vesicles. Biophys. J. 87, 419–429. [18] Yamashita, Y., Oka, M., Tanaka, T. and Yamazaki, M. (2002) A new method for the preparation of giant liposomes in high salt concentrations and growth of protein microcrystals in them. Biochim. Biophys. Acta –Biomembr. 1561, 129– 134. [19] Doeven, M.K., Folgering, J.H., Krasnikov, V., Geertsma, E.R., van den Bogaart, G. and Poolman, B. (2005) Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles. Biophys. J. 88, 1134–1142. [20] Krishnamurthy, V. et al. (2007) Gramicidin ion channel-based biosensors: Construction, stochastic dynamical models, and statistical detection algorithms. IEEE Sensors J. 7, 1281–1288. [21] Wang, W. et al. (2008) The structure of an open form of an E. coli mechanosensitive channel at 3.45 Å resolution. Science 321, 1179–1183. [22] Blount, P., Sukharev, S.I., Schroeder, M.J., Nagle, S.K. and Kung, C. (1996) Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli. Proc. Natl. Acad. Sci. USA 93, 11652–11657. [23] Iscla, I., Levin, G., Wray, R., Reynolds, R. and Blount, P. (2004) Defining the physical gate of a mechanosensitive channel, MscL, by engineering metalbinding sites. Biophys. J. 87, 3172–3180. [24] Yoshimura, K., Batiza, A., Schroeder, M., Blount, P. and Kung, C. (1999) Hydrophilicity of a single residue within MscL correlates with increased channel mechanosensitivity. Biophys. J. 77, 1960–1972. [25] Arakawa, T. and Timasheff, S.N. (1982) Stabilization of protein structure by sugars. Biochemistry 21, 6536–6544. [26] Back, J.F., Oakenfull, D. and Smith, M.B. (1979) Increased thermal-stability of proteins in the presence of sugars and polyols. Biochemistry 18, 5191–5196. [27] Lee, J.C. and Timasheff, S.N. (1981) The stabilization of proteins by sucrose. J. Biol. Chem. 256, 7193–7201. [28] Allison, S.D., Chang, B., Randolph, T.W. and Carpenter, J.F. (1999) Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding. Arch. Biochem. Biophys. 365, 289– 298. [29] Kiselev, M.A., Wartewig, S., Janich, M., Lesieur, P., Kiselev, A.M., Ollivon, M. and Neubert, R. (2003) Does sucrose influence the properties of DMPC vesicles? Chem. Phys. Lipids 123, 31–44. [30] Nagase, H., Ueda, H. and Nakagaki, M. (2004) (Effect of water on lamellar structure of DPPC/sugar systems for the application on biological samples), Proceedings of the International Conference Photonics Europe, 26–30 April 2004 SPIE 5457, 581–588. [31] Vasquez, V., Sotomayor, M., Cordero-Morales, J., Schulten, K. and Perozo, E. (2008) A structural mechanism for MscS gating in lipid bilayers. Science 321, 1210–1214. [32] Moe, P. and Blount, P. (2005) Assessment of potential stimuli for mechanodependent gating of MscL: effects of pressure, tension, and lipid headgroups. Biochemistry 44, 12239–12244.
journal homepage: www.FEBSLetters.org
Rapid and improved reconstitution of bacterial mechanosensitive ion channel proteins MscS and MscL into liposomes using a modified sucrose method Andrew R. Battle, Evgeny Petrov, Prithwish Pal, Boris Martinac * Department of Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia
a r t i c l e
i n f o
Article history: Received 6 November 2008 Revised 8 December 2008 Accepted 10 December 2008 Available online 25 December 2008 Edited by Maurice Montal Keywords: Patch clamp Confocal microscopy Fluorescence Lipid Sugar
a b s t r a c t The bacterial mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance have functionally been reconstituted into giant unilamellar liposomes (GUVs) using an improved reconstitution method in the presence of sucrose. This method gives significant time savings (preparation times as little as 6 h) compared to the classical method of protein reconstitution which uses a dehydration/rehydration (D/R) procedure (minimum 2 days preparation time). Moreover, it represents the first highly reproducible method for functional reconstitution of MscS as well as MscS/MscL co-reconstitution. This novel procedure has the potential to be used for studies of other ion channels by liposome reconstitution. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Bacterial MS ion channel proteins act as emergency relief valves during osmotic shock in order to help bacterial cells survive sudden changes in external osmolarity [1–3]. Based on their conductance and pressure sensitivity three types of such channels have been identified in Escherichia coli: MS channel of Large conductance (MscL), MS channel of Small conductance (MscS/MscK) and MS channel of Mini conductance (MscM) [1,4]. MscS and MscL ion channels were first discovered in giant spheroplasts of E. coli [2,5,6]. Incorporation of MscL and MscS into artificial liposomal membranes has also been achieved and their functional channel activity has been recorded using the patch-clamp technique [2,6– 10]. In contrast to MscL functional studies, which to a large extent have been carried out using liposome reconstitution methods [3,7,11,12], studies to date of MscS have mostly been using giant spheroplasts [13,14] leading to a limitation in the conditions in which to study this protein. Very few studies of MscS in liposomes have been undertaken: Sukharev [8] studied MscS in azolectin, and more recently Vásquez et al . [9] successfully reconstituted the MscS protein into E. coli polar lipids. However, both procedures required time consuming dehydration/rehydration methods as well as high protein/lipid ratios for MscS channel reconstitution.
* Corresponding author. Address: The Victor Chang Cardiac Research Institute, Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia. Tel.: +61 2 9295 8600; fax: +61 2 9295 8734. E-mail address: [email protected] (B. Martinac).
An alternative method of preparing artificial liposomes is through the formation of giant unilamellar vesicles (GUV), which are becoming more attractive as models of membranes [15,16]. However, incorporation of proteins into such vesicles has been less successful. The vast majority of reports detailing protein reconstitutions into liposomes usually involve detergent-mediated procedures which results in small relative sizes of the liposomes (0.1–0.2 lm) [17,18]. Doeven and co-workers [19] incorporated MscL (at a protein-to-lipid ratio of 1:50 wt/wt) and three other less stable membrane proteins into GUVs using a rehydration-dehydration step in which sucrose was added to the buffer/GUV/protein solution during dehydration. More recently Krishnamurthy et al. [20] successfully incorporated bis-gramicidin A into GUVs suspended in sucrose solutions and recorded channel activity. They dissolved the peptide in ethanol and added it to the prehydrated lipid film, followed by 1 M sucrose addition and GUV formation. In this work we report a time-efficient incorporation of the bacterial MS channel protein MscS into GUVs prepared by rehydrating lipids in sucrose solutions followed by protein reconstitution. Electrophysiological recordings of membrane patches have shown that reconstituted MscS retains its conductance and mechanosensitivity, comparable with MscS in E. Coli membranes. 2. Materials and methods GST-MscL-WT and 6-His-MscS-WT proteins were purified according to a published procedure ((MscL) [7], (MscS) [9]).
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.12.033
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Chloroform, Azolectin (30% L-a-phosphatidylcholine from soyabean) Potassium chloride, magnesium chloride, calcium chloride, sucrose, HEPES (4-2-hydroxyethyl)-piperazine-1-ethanesulfonic acid), TRIS (2-amino-2-hydroxymethyl)-1,3-propanediol), tris(2carboxyethyl)phosphine (TCEP), n-dodecyl beta-D-maltoside (DDM) and potassium hydroxide were all purchased from Sigma and were of analytical grade. BioBeads were purchased from Bio-Rad whereas 1,2-dioleoyl-sn-glycero-3-phosphethanol amine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-1-glycerol)] (sodium salt) (POPG) were obtained as powders from Avanti Polar lipids Inc, and dissolved in CHCl3. Doubly-distilled water was used in all experiments. The dehydration/rehydration (D/R) method follows that described by Häse et al. [7]. 2.1. Sucrose method for incorporation of MscS Purified MscS was taken from a stock solution containing the protein dissolved in PBS and 10 mM DDM detergent and was incorporated into giant artificial liposomes, which were prepared using a modified procedure [15,16]. Two hundred microliters of a 10 mg/ mL solution of azolectin in chloroform was dried in a test tube under a stream of N2 whilst rotating the tube to make a homogeneous lipid film once dry. Once dry, 5 lL of purified water was added to the bottom (pre-hydration) followed by 1 mL of 0.4 M sucrose. The solution was incubated for 3 h at 45 °C. Temperatures lower than 45 °C caused much slower rates of liposome formation. The required amount of protein was then added to achieve a desired protein-to-lipid ratio, and the solution shaken on an orbital mixer for further three hours, after this time a cloud of liposomes was seen to float in the solution. Manual shaking of the tube is not recommended, as it will dissipate the liposome cloud. At this time the sample was ready for patch clamping. However, to ensure complete protein incorporation when using low protein-to-lipid ratios, shaking can be continued overnight. BioBeads (Bio-Rad) can also be added to remove traces of detergent (used to solublize the protein) if desired. In this case, after the overnight shaking step the BioBeads (approx spatula full) are added and shaking is continued for 3 h at RT. 2.2. Patch-clamp recording An aliquot of liposomes (2–4 lL) was taken from the liposome cloud and introduced to the recording bath. Channel activity was
examined in the excised liposome patches and was recorded using the patch-clamp method on liposomes that settled on the bottom of the recording chamber and formed unilamellar blisters [11]. Negative pressure (suction) recorded in mm Hg was applied to patch pipettes by using a syringe and was monitored using a piezoelectric pressure transducer (Omega Engineering, Stamford, USA). Borosilicate glass pipettes (Drummond Scientific Co., Broomall, PA) were pulled using a Flaming/Brown pipette puller (P-87, Sutter Instrument Co., Novato, CA) to a diameter which corresponded to a pipette resistance in the range of 3.0–6.0 MX. Ion currents arising from activation of either MscL or MscS using negative pipette pressure were recorded using an Axon 1D patch-clamp amplifier (Axon Instruments), filtered at 2 kHz and digitized at 5 kHz. Single channel analysis was done using pCLAMP9 software (Axon Instruments). Bath and pipette solutions were symmetrical and consisted of 200 mM KCl, 40 mM MgCl2, 5 mM HEPES (MscL, solution adjusted to pH 7.2 using KOH) or 200 mM KCl, 90 mM MgCl2, 10 mM CaCl2, 5 mM TRIS (MscS, solution adjusted to pH 7.2 using HCl). 2.3. Fluorescence labeling and confocal microscopy To observe the incorporation of MS channels into liposomes, single cysteine mutant versions of MscL (I49C) and MscS (S243C) were generated, labeled with AlexaFluor-488-maleimide (Invitrogen) and incorporated into azolectin liposomes as above at 1/100 or 1/200 protein/lipid ratios (by weight). Confocal images were obtained in recording buffers using an Olympus FluoView1400 Laser Scanning Microscope (Olympus) with Nikon 60 water-immersion objective, NA 1.00 (Nikon, Japan), with excitation at 488nm. 3. Results 3.1. Liposome formation and MscS incorporation Using the sucrose procedure, a cloud of liposomes formed in the sucrose solution, was stable at 4 °C and could be used for several days for experiments. MscS was effectively incorporated into azolectin using the sucrose method (Fig. 1). At a protein/lipid ratio of 1/1000 (wt/wt) more than 75% of examined patches exhibited channel activity. As a comparison, only 31% of patches of MscS incorporated into the liposomes using the D/R method at the same ratio showed activity, a significant improvement over the published methods for MscS reconstitution into liposomes [8,9], where
Fig. 1. Single channel recording from purified MscS reconstituted into azolectin liposomes using the sucrose method.
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both authors report incorporations at 1/200 wt/wt. The unitary conductance in liposomes is about one-third that of MscL, which is also analogous to conductance seen in giant spheroplasts [5]. At positive voltages the channel conductance was 1.41 ± 0.03 nS (n = 7), while at negative voltages the conductance was 0.85 ± 0.02 nS (n = 7) (Fig. 2), i.e. two-thirds of that recorded at positive voltages, in agreement with those previously reported [2,5,8]. MscS has been reported to inactivate under prolonged exposure to membrane tension in liposomes [8,9,21], with channels becoming responsive again to applied negative pressure after some time. We did not observe this behavior in azolectin liposomes using the sucrose method, indeed the channels remained active over an extended time period, in some cases remaining open for periods greater than 20 min (Fig. 3A). However, a very gradual inactivation occurred and this is shown in a recording containing two channels, both of which immediately opened upon tension but the second channel closed after >100 s (at constant pressure (Fig. 3B)). We investigated channel behavior in other lipids. In mixtures of DOPC/DOPE/POPG (2:7:1), a ratio of 1/200 wt/wt was required for channel activity in 50% of patches. Channels did not incorporate in pure DOPC. 3.2. MscS and MscL co-reconstitution MscS and MscL were successfully co-reconstituted into azolectin using the sucrose method and in all cases MscS was reconstituted at a ratio of 1/1000 wt/wt. A higher success rate of channel reconstitution (at a given protein/lipid ratio) occurs when sucrose is present (60% of patches contained MscS compared to 46% in the D/R method). Both channels exhibited activity, MscS opening first at a lower pressure threshold, followed by MscL (Fig. 2). MscS inactivation over time also occurred on a slow time scale, analogous to that observed when MscS alone was reconstituted. After release and reapplication of tension (Fig. 3C), MscS often inactivated, while MscL remained responsive to tension. The inactivation of MscS did not affect the opening threshold of MscL, as clearly shown in the pressure trace in Fig. 3C. This observation was seen in all current traces we obtained, regardless of the number of MscS channels in the patch.
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The opening threshold of MscS relative to MscL was also measured, in all experimental conditions the ratio being approximately 1.9 ± 0.3 (n = 30). This ratio falls within the range reported in giant spheroplasts; three separate publications noting a range from 1.39 ± 0.02 to 1.64 ± 0.08 [22–24]. Two ratios of MscL were used (1/1000 and 1/5000 wt/wt) and did not affect the opening threshold ratio of the channels to one another. 3.3. Confocal imaging Using confocal imaging (Fig. 4), we can observe that the proteins are being incorporated into blisters as early as 2 h after addition into the lipid clouds (Fig. 4, left panels). Overnight incubation also produces blisters of various sizes containing fluorescent proteins on the edges as expected for both proteins (Fig. 4, centre and right panels). 4. Discussion This study demonstrates an improved and time-efficient procedure for MscS reconstitution into liposomes and importantly is the first demonstration of co-reconstitution of MscL and MscS in liposomes. It therefore represents a significant advantage over the classical D/R method for both channel proteins. The D/R method is more time consuming requiring a minimum of two days before electrophysiological recordings can be run [11]. Several steps are involved, including an hour of centrifugation, an overnight dehydration step and a rehydration step of at least 3 h. In contrast, the sucrose method allows for electrophysiological recordings in as little as 6 h. It is also a simpler procedure, without the need for centrifugation or dehydration/rehydration steps. These steps result in a minimum time saving of 24 h compared to the D/R method. Most interestingly, it represents the first reported method for efficient and reproducible MscS incorporation and functional analysis into liposomes. Sukharev [8] and Vásquez et al. [9] have described the reconstitution of MscS into azolectin and E. coli native lipids, respectively, albeit at high protein-to-lipid ratios (1/ 200 wt/wt). Using the D/R method [7,11], the incorporation of MscS was less successful than using the sucrose method, where channels were observed in 30% of patches. Interestingly we were
Fig. 2. MscS and MscL co-reconstitution into azolectin liposomes. MscL activates at approximately double the required pressure for MscS activation.
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A
B
C
Fig. 3. MscS reconstituted into azolectin liposomes using the sucrose method. The channels remain active for several minutes (A) and slowly inactivate (B). Inactivation also occurs in co-reconstituted MscS and MscL (C).
not the only research group to experience difficulties in MscS incorporation into azolectin using the D/R method with buffer rehydration (Vásquez and colleagues, personal communication). Although there are many differences between the two methods, it is known that sucrose and other sugars stabilize proteins [25– 27]. One suggested mechanism resulting in this stabilization [28] is through hydrogen bonding of sucrose with proteins. It has also been postulated that protein stabilization occurs through preferen-
tial exclusion of the sucrose from the protein domain (leading to protein stabilization as the unfolded state is thermodynamically less favourable in the presence of sucrose) [27]. Thirdly, sucrose does not influence lipid bilayer packing or phase transition temperatures [29,30]. Although best to our knowledge the precise mechanism is not known it is feasible that stabilization of MscS by sucrose by at least one of the aforementioned methods is an important factor in successful incorporation of the protein during
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Fig. 4. Representative confocal images demonstrating incorporation of fluorescently labelled single cysteine mutants of MscL (top panel) and MscS (bottom panel) into azolectin liposomes.
the preparation. Furthermore, it is also known that lipid films form GUVs in sucrose solutions [15,16,20] hence the large surface area of the lipid bilayer would increase the likelihood of more protein incorporation. Using our procedure highly reproducible results can be obtained at protein-to-lipid ratios as low as 1/3000 (wt/ wt). Consequently, this method should as well be very useful for recording of ion channel activities from eukaryotic ion channels, which are not readily available in quantities of hundreds of micrograms available for bacterial MS channels. The report by Doeven and co-workers [19] detailing sucrose mediated channel incorporation used MscL at a 1/50 wt/wt protein/lipid ratio. Not only have we shown incorporation of this protein at much lower ratios, but also MscS incorporation, which again has not previously been shown. In azolectin we observed very slow inactivation of MscS. Sukharev [8] reported that MscS quickly inactivated in azolectin, analogous to observations in spheroplasts. Using the sucrose method we were nevertheless able to obtain recordings of up to 15 min (or more) showing MscS remaining open over minutes-long periods under continuously applied negative pressure (Fig. 3A). This fortuitous observation opens up the possibility for studying in more detail the properties of this MS channel in liposome membranes. A recent publication by Perozo and co-workers [31] reported that MscS present in giant spheroplasts did not inactivate upon addition of the ampiphath lysophosphatidylcholine (LPC) and suggested that LPC may be exerting different bilayer perturbation forces than those of the better characterized transbilayer difference (see [32]). Indeed, they were unable to repeat this
observation in lipids, the seals not remaining stable for a long enough time frame for characterization under patch-clamp conditions (V. Vásquez, personal communication). Although sucrose is not expected to influence MscS behavior in the same way that LPC does, our observations nevertheless suggest that an intrinsic (and as yet undetermined) interaction of azolectin membranes (or sucrose) with MscS may encourage activation for prolonged time periods without rapid inactivation. Further investigations currently undertaken in our laboratory (including investigating channel behavior in other lipids) should help to elucidate this effect. The confocal images also demonstrate the usefulness of the modified sucrose method for fluorescence studies of ion channel proteins, such as FRET or FRAP, in lipid environments. In conclusion, we have described a novel time-efficient procedure for reconstitution and co-reconstitution of MscS into liposomes using a sucrose solution as the rehydrating medium. We have shown for the first time using the patch-clamp method that MscS can be reconstituted into liposomes made of azolectin or mixtures of pure lipids using the sucrose method at relatively low protein/lipid ratios (1/1000 wt/wt). MscS can also be incorporated into azolectin using the D/R method. We also report the first example of co-reconstitution of MscS and MscL into azolectin. Fluorescence labeling of the proteins and confocal imaging of them in azolectin further demonstrated the incorporation. We have also shown that, under these conditions (unlike in spheroplasts), MscS remains active for prolonged time periods. Furthermore, the improved sucrose method provides time-efficient method at much
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lower protein/lipid ratios than the D/R method, as well as preparations more suitable for prolonged patch-clamp recordings. Acknowledgements This work has been supported by the grants of the Australian Research Council (DP0769983) and National Health and Medical Research Council of Australia (455968). References [1] Martinac, B. (2007) 3.5 billion years of mechanosensory transduction: Structure and function of mechanosensitive channels in prokaryotes. Mechanosensitive Ion Channels, Part A 58, 25–57. [2] Martinac, B., Buechner, M., Delcour, A.H., Adler, J. and Kung, C. (1987) Pressuresensitive ion channel in Escherichia-coli. Proc. Natl. Acad. Sci. USA. 84, 2297– 2301. [3] Perozo, E. (2006) Gating prokaryotic mechanosensitive channels. Nat. Rev. Mol. Cell Biol. 7, 109–119. [4] Berrier, C., Besnard, M., Ajouz, B., Coulombe, A. and Ghazi, A. (1996) Multiple mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied pressure. J. Membr. Biol. 151, 175–187. [5] Sukharev, S.I., Martinac, B., Arshavsky, V.Y. and Kung, C. (1993) 2 Types of mechanosensitive channels in the Escherichia-coli cell-envelope – solubilization and functional reconstitution. Biophys. J. 65, 177–183. [6] Sukharev, S.I., Blount, P., Martinac, B., Blattner, F.R. and Kung, C. (1994) A largeconductance mechanosensitive channel in E. Coli encoded by mscL alone. Nature 368, 265–268. [7] Häse, C.C., Ledain, A.C. and Martinac, B. (1995) Purification and functional reconstitution of the recombinant large mechanosensitive ion-channel (Mscl) of Escherichia-Coli. J. Biol. Chem. 270, 18329–18334. [8] Sukharev, S. (2002) Purification of the small mechanosensitive channel of Escherichia coli (MscS): the subunit structure, conduction, and gating characteristics in liposomes. Biophys. J. 83, 290–298. [9] Vasquez, V., Cortes, D.M., Furukawa, H. and Perozo, E. (2007) An optimized purification and reconstitution method for the MscS channel: strategies for spectroscopical analysis. Biochemistry 46, 6766–6773. [10] Sukharev, S.I., Sigurdson, W.J., Kung, C. and Sachs, F. (1999) Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL. J. Gen. Physiol. 113, 525–539. [11] Delcour, A.H., Martinac, B., Adler, J. and Kung, C. (1989) Modified reconstitution method used in patch-clamp studies of Escherichia-Coli ion channels. Biophys. J. 56, 631–636. [12] Perozo, E., Cortes, D.M., Sompornpisut, P., Kloda, A. and Martinac, B. (2002) Open channel structure of MscL and the gating mechanism of mechanosensitive channels. Nature 418, 942–948. [13] Blount, P. and Moe, P.C. (1999) Bacterial mechanosensitive channels: integrating physiology, structure and function. Trends Microbiol. 7, 420–424. [14] Booth, I.R., Edwards, M.D., Black, S., Schumann, U. and Miller, S. (2007) Mechanosensitive channels in bacteria: signs of closure? Nat. Rev. Microbiol. 5, 431–440.
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