Article Cite This: J. Phys. Chem. C 2019, 123, 17899−17907
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Metal Ion Size-Dependent Effects on Lipid Transmembrane Flip-Flop Yong-Hao Ma,† Bolin Li,‡ Jingjing Yang,§ Xiaofeng Han,† Zhan Chen,*,‡ and Xiaolin Lu*,† †
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China ‡ Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States § Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, Nanjing 210093, China
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S Supporting Information *
ABSTRACT: Ion homeostasis regulated by diverse transport systems is important for cell functions, where the two major components of cell membranes, namely proteins and lipids, must play critical roles. The role of transport systems based on proteins, for example ion pumps and channels, has generally been acknowledged and studied in the past few decades, while the importance of lipid systems, for example lipid ion channels, has not been fully appreciated so far with the lack of detailed molecular-level information. Here, we applied sum frequency generation vibrational spectroscopy to investigate the lipid transmembrane movement, namely flip-flop, affected by metal ions for lipid bilayers, which is related to the cellular ion homeostasis. Metal ions in the same group (Ba2+, Sr2+, and Ca2+) but with different sizes were used; the distance between lipid molecules in a bilayer was also manipulated upon adjusting the surface pressure. We found that, on the one hand, there existed a match relationship between the metal ion size and the distance among the lipid molecules, which led to the significant decrease of the flip-flop rate for the lipid bilayer. On the other hand, the flip-flop process was accelerated when the metal ion size and the lipid distance were mismatched. This study highlights the ion size effects on the lipid transmembrane flip-flop rates, providing the inspiring clue for understanding the lipid function related to the cellular ion homeostasis.
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ical measurements such as patch-clamp techniques,4,7,10 so more elaborate detection techniques at the molecular and dynamic levels are highly needed. Flip-flop is the transmembrane movement of lipids between the two leaflets of a cell membrane in the protein-free and protein-assisted (flippase, floppase, and scramblase) manners, with the innate asymmetric lipid compositions for the two leaflets.11−13 In this respect, the ubiquitous lipid transmembrane asymmetry14,15 not only preserves the membrane mechanical stability11 but also regulates critical cellular events such as apoptosis,16 blood coagulation,17 and cell signaling.18 For example, disruption of lipid transmembrane asymmetry can result in the development of Alzheimer’s disease19 and Scott syndrome.20 Therefore, studying the flip-flop process is critical to understand structural and dynamic features in asymmetric cell membranes. Compared to label-free measurements using deuterated lipids, studies on the lipid flip-flop using spin-labeled or fluorescent lipids are indirect approaches.21 For electron spin resonance spectroscopy using spin-labeled lipids, the chemical reducing procedure is needed to make the probe molecules asymmetrically distributed in the two leaflets.22 This method is similar to the fluorescence approach in which only the movement of fluorescent lipids can
INTRODUCTION Ions, especially metal ions, play crucial and diverse roles in cell biology, including being regulators in membrane potentials, cofactors in protein functions, and stimuli in signal transduction.1 Maintaining metal ion homeostasis is thus essential to cells, which relies on various exquisitely controlled transport systems on cell membranes.2 Previous studies have been focused on versatile membrane protein-related transport systems, including pumps, channels, transporters, G-protein coupled receptors, and enzymes.3 However, albeit unappreciated, another main component of cell membrane−lipids, should also play an important role in preserving ion homeostasis across cell membranes.4 More than a simple passive building block in cell membranes, lipids perform as substrates for cellular signaling with a rapid response5 and regulate protein structures and functions.6 For example, in the lipid environment, the affinity between ions and proteins is very different from that in the aqueous media.3 More interestingly and importantly, pure lipid membranes themselves can display conduction activities, behaving like voltagegated, temperature-gated, and mechanosensitive protein channels, thus leading to the so-called lipid ion channels in cell membranes with the intrinsic biological significance.7−9 Previous studies on the ion−lipid interaction for lipid ion channels are mostly in the macroscopic and static levels, usually using a large number of vesicles, based on fluorescence spectroscopy, fluorescence microscopy, and electrophysiolog© 2019 American Chemical Society
Received: May 14, 2019 Revised: June 27, 2019 Published: June 27, 2019 17899
DOI: 10.1021/acs.jpcc.9b04557 J. Phys. Chem. C 2019, 123, 17899−17907
Article
The Journal of Physical Chemistry C be detected. Small-angle neutron scattering using deuterated lipids can also be employed to probe the inter-bilayer lipid exchange from one vesicle to another and the intra-bilayer lipid exchange (flip-flop) within the two leaflets in a vesicle. However, this approach is somehow complicated.21 Considering the current state-of-the-art techniques, sum frequency generation (SFG) vibrational spectroscopy, being surface/ interface selective and nondestructive, enables the direct measurements of the lipid flip-flop kinetics and energetics in model cell membranes when deuterated lipids are used.23 In the previous studies, a number of biological cues in connection to the lipid transmembrane asymmetry and the flip-flop have been studied via SFG, including lipid chain length, lipid headgroups, cholesterol, drugs, peptides, and polymers,21,24−29 suggesting that SFG is a powerful tool to investigate lipid transmembrane asymmetry. The lipid flip-flop rate decreases when the acyl chain length increases, the lipid packing becomes denser, or the membrane potential caused by lipid headgroups becomes higher. Addition of cholesterols and transmembrane peptides can facilitate the lipid transmembrane movement due to the introduced defects. With respect to the ion−lipid interaction, a number of SFG studies have been focused on lipid monolayers.30−33 Only a molecular dynamics simulation result indicates that the ion permeation across the lipid membrane can be modulated by the lipid flip-flop.34 To the best of our knowledge, there is yet no SFG experimental study related to the ion effect on the lipid flip-flop. Consequently, the ion effect on the flip-flop process needs to be investigated, which can deepen our understanding of the lipid structure−property relationship related to maintaining cellular ion homeostasis. In this work, we applied SFG to investigate the lipid flip-flop dynamics in model cell membranes using different metal ions. Divalent metal ionscalcium ions (Ca2+), strontium ions (Sr2+), and barium ions (Ba2+)in the same element group with different ion sizes were chosen as representatives in consideration of their nonspecific interaction with lipids. The common mammal membrane lipid, 1,2-dipalmitoyl-sn-glycero3-phosphocholine (DPPC), and its deuterated counterpart, 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (dDPPC), were used to construct the asymmetric supported lipid bilayers as model cell membranes where the averaged distance among the individual lipid molecules can be adjusted by changing the surface pressure. We discovered that the lipid flip-flop dynamics was in direct correlation to the match/mismatch between the ion size and average lipid distance, indicating its size-dependent behavior and the potential role of lipids in the ion homeostasis.
Figure 1. Schematic showing the SFG experimental geometry for the lipid asymmetry study and molecular structures of the corresponding lipid components.
(Aladdin, China). All chemicals were used as received. Ultrapure water (18.2 MΩ·cm) was obtained from a Millipore water purification system. Fused silica right angle prisms (Jinzhou Quartz Glass, China) of IR (JGS3) grade were soaked in toluene (SINOPHARM, China) over 24 h and then washed with ethanol and detergent solution followed by rinsing and drying. Afterward, the prisms were soaked in piranha solution (a mixture of sulfuric acid (H2SO4, SINOPHARM) and hydrogen peroxide (H2O2, Aladdin) at a volume ratio of 3 to 1) overnight followed by water rinsing. Before deposition of the lipid monolayer, the prisms were dried by nitrogen gas and cleaned by oxygen plasma (PDC-MG, Mingheng, China) for 4 min. All experiments were carried out at an ambient temperature of ∼22 °C. Asymmetric Lipid Bilayer Preparation. Lipid bilayers on fused silica prisms were prepared using the Langmuir− Blodgett (LB) and Langmuir−Schaefer (LS) method.26,35 In brief, a dDPPC lipid monolayer at a desired surface pressure was deposited on one prism using a KN 2003 LB system (KSV NIMA). This monolayer was then put into contact with the other DPPC monolayer spread on the water surface at the same surface pressure to form a lipid bilayer. The as-prepared asymmetric lipid bilayer was thus composed of the dDPPC proximal leaflet (attached to the prism) and the DPPC distal leaflet (in contact with the water). After the formation of the bilayer, the stock salt solution was injected immediately into the water to achieve a final concentration of 100 mM under stirring. SFG Measurement. SFG is a powerful analytical tool capable of probing molecular-level surface/interfacial structures and dynamics with submonolayer sensitivity and intrinsic surface/interface selectivity in situ in real time.36 Under the electric dipole approximation, SFG, as a second-order nonlinear optical process, is forbidden for materials with inversion symmetry but is allowed on the surfaces or at the interfaces where the inversion symmetry is necessarily broken.37 Accordingly, the asymmetric bilayers are suitable for the SFG experiment, and the lipid transmembrane movement (flip-flop) can be studied by monitoring the timedependent SFG signal change due to the symmetry change.23 Two pulsed laser beams, a frequency-tunable infrared (IR) beam and a frequency-fixed visible beam (∼532 nm), overlapped at the bilayer interface temporally and spatially to generate a sum frequency beam containing the interfacial molecular information. The SFG measurements were conducted using ssp (s polarized signal beam, s polarized visible
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EXPERIMENTAL METHODS Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (dDPPC) were purchased from Avanti Polar Lipids, United States, and their detailed molecular formulas are shown in Figure 1. DPPC and dDPPC were dissolved in chloroform (SINOPHARM, China) at a concentration of 3 mg/mL for further construction of lipid bilayers. Three kinds of salts, calcium chloride (CaCl 2·2H2 O, 99.99% metals basis), strontium chloride (SrCl2·6H2O, ACS certified), and barium chloride (BaCl2·2H2O, 99.99% metals basis), ordered from Aladdin, China, were used to prepare the aqueous stock solution at a concentration of 1 M. These stock ionic solutions were purified by syringe filters with 0.22 μm membranes 17900
DOI: 10.1021/acs.jpcc.9b04557 J. Phys. Chem. C 2019, 123, 17899−17907
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The Journal of Physical Chemistry C
Figure 2. Decay curves of the time-dependent SFG signals at 2875 cm−1 (methyl stretching vibrational mode) for the dDPPC−DPPC bilayers in contact with different subphases (water, 100 mM Ca2+ solution, 100 mM Sr2+ solution, and 100 mM Ba2+ solution) at different surface pressures (30, 20, 15, and 10 mN/m). Dots are measured data points, and lines are fitted results using eq 3.
ICH3(t ) = Imax e−4kt + Imin
beam, p polarized IR beam) and ppp polarization combinations. Data Analysis. The reflective SFG output intensity is proportional to the squared modulus of effective second-order 38,39 nonlinear susceptibility χ(2) eff I(ω) ∝
8π 3ω 2 sec 2 β |χ (2) |2 I1(ω1)I2(ω2) c 3n(ω1)n(ω2)n(ω) eff
Imax and Imin are the maximum and minimum SFG signal intensities of the methyl ss mode, respectively. The lipid flipflop rate constant k was deduced by fitting the time-dependent SFG signal data using eq 3. The decay rate constants kion(π) and kH2O(π) for ionic solution and pure water, respectively, at the surface pressure π were then obtained. The relative decay rate constant of each ion kion r (π) can thus be calculated.
(1)
Here, I1(ω1), I2(ω2), and I(ω) are the intensities of input visible and IR beams and output sum frequency beam with the frequencies of ω1, ω2, and ω, respectively. The reflective index n(ωi) corresponds to the beam i in the incident medium at the frequency ωi. The angle β is the reflected angle of the output beam. Additionally, the collected SFG spectra, plotted by normalized SFG signal intensity ISFG versus input IR beam frequency ω2, can be fitted using the following Lorentz equation39 ISFG ∝
(2) 2 |χeff |
=
(2) χNR
+
∑ q
Aq ω2 − ωq + i Γq
(3)
k rion(π ) =
k ion(π ) k H2O(π )
(4)
After this rescaling process, the flip-flop rates for the same ionic solution but at a different surface pressure can be compared. The half-life of the lipid flip-flop process can be expressed as: t1/2 =
ln 2 2k
(5)
The activation barrier to the lipid flip-flop process can be represented by the activation free energy (ΔG‡) in the framework of transition state theory, which can be determined from the measured decay rate constant k40,41
2
(2)
The nonresonant background χ(2) NR is typically a constant for a particular spectrum because of interfacial electron polarization contribution. Aq, ωq, and Γq are the signal amplitude, resonant frequency, and damping coefficient (or peak width) of the vibrational mode q, respectively. The time-dependent SFG signal of the methyl (CH3) symmetric stretching (ss) mode ICH3(t), monitored at 2875 cm−1, can be described using the following equation, which accounts for the flip-flop process of the lipid bilayer.23
k=
kBT −ΔG‡ / RT e h
(6)
where kB is the Boltzmann constant, T is the temperature in kelvin, h is the Planck constant, and R is the gas constant.
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RESULTS AND DISCUSSION As demonstrated in the Experimental Methods, the asymmetric dDPPC−DPPC bilayer was deposited on the fused silica prism 17901
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more fluid. The results suggested that the interaction between ions and lipids is ion-specific with respect to the distinct lipid bilayer and monolayer. Here, the different effects of the Ca2+, Sr2+, and Ba2+ ions on the flip-flop rates should be attributed to their intrinsic characteristics since they are in the same element group. When the surface pressure was 20 mN/m, the ion effects on the lipid flip-flop rates were not the same as those at 30 mN/ m, as shown in Figure 2A,B. Basically, the reduced surface pressure can increase the decay rate of the vibrational signal at 2875 cm−1, as evidenced by the larger flip-flop rate constant for pure water (Figure 2B), which can still serve as a reference. The decay rate constants for all the three ions decreased with respect to that for pure water. In other words, Ca2+, Sr2+, and Ba2+ slowed down the flip-flop process and somehow stabilized the asymmetric dDPPC−DPPC bilayer. The flip-flop rate
using the LB−LS method, with dDPPC as the proximal leaflet and DPPC as the distal leaflet. During the LB−LS preparation, four different surface pressures for the two leaflets of a bilayer were chosen, that is, 30, 20, 15, and 10 mN/m. These asprepared bilayers were kept in contact with the 100 mM ionic solutions or pure water at the ambient temperature (∼22 °C). The time-dependent SFG signal at 2875 cm−1 was monitored to trace the dynamic DPPC flip-flop process. Afterward, the SFG spectra at the end equilibrium state were collected. Therefore, the ion effects on the lipid transmembrane movements and the bilayer structures can be discussed. Ion Effects on Flip-Flop Dynamics. After the asymmetric bilayer was formed in direct contact with the desired ionic solution (or water), the SFG signal intensity at 2875 cm−1 (ICH3), corresponding to the symmetric stretching (ss) mode of the terminal hydrogenated methyl group, was monitored versus time, as shown in Figure 2. The intensity decay curves were fitted using eq 3 to determine the flip-flop rate constants. The decay rate constants with four different surface pressures (30, 20, 15, and 10 mN/m) under different ionic conditions (100 mM Ca2+, 100 mM Sr2+, 100 mM Ba2+, and water) are summarized in Table 1.
2+
constants at 20 mN/m in sequence were shown as kBa (20) < 2+
Table 1. Flip-Flop Kinetic Parameters for the dDPPC− DPPC Bilayera π (mN/m)
ion
30
(H2O) Ba2+ Sr2+ Ca2+ (H2O) Ba2+ Sr2+ Ca2+ (H2O) Ba2+ Sr2+ Ca2+ (H2O) Ba2+ Sr2+ Ca2+
20
15
10
k × 105 (s−1)b 8.8 18.6 7.5 6.4 46.0 22.7 41.6 37.6 43.0 21.3 35.6 41.9 58.7 41.0 46.7 63.3
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.2 0.5 0.7 0.7 0.6 0.7 0.6 0.5 0.6 0.8 0.5 0.6 0.8 0.5 0.5 0.8
ΔG‡ (kJ/mol)c 95.2 93.3 95.6 96.0 91.1 92.8 91.4 91.6 91.3 93.0 91.7 91.3 90.5 91.4 91.1 90.3
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.3 0.1 0.1 0.3 0.2 0.3 0.3 0.3 0.2 0.3 0.3 0.3 0.3 0.3 0.3
t1/2 (min)c 66 31 77 90 13 25 14 15 13 27 16 14 10 14 12 9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2+
kSr (20) < kCa (20) < kH2O(20). It is amazing to find that, for the Ba2+ ions, the flip-flop of the bilayer was changed from an accelerated process to a decelerated process upon the change of the surface pressure from 30 to 20 mN/m. Such a switch was unexpected since the DPPC lipids were still in the liquidcondensed phase at ∼20 mN/m (Figure S1). With the reduced surface pressure, the packing density of the DPPC lipids also decreased, no matter for a monolayer30 or a bilayer.42 The lipid structural change must alter interaction between the Ba2+ ions and the bilayer, resulting in decelerated flip-flop dynamics reversely. For the Sr2+ and Ca2+ ions, the flip-flop processes at 20 mN/m were still slower than that for pure water, similar to the case at a surface pressure of 30 mN/m. Attention should be paid, as the divalent cations in the same element group, the Ba2+, Sr2+ and Ca2+ ions, have the same charge and similar physicochemical properties. Even if there is still a disparity between the three ions, the changes of the flip-flop rates cannot simply be attributed to the ion−lipid binding affinity due to the nonsynchronous change of the flip-flop rates. We thus assume that the sizes of the cations play an important role here to affect the interaction between the cations and the lipid bilayer. When the surface pressure was 15 mN/m (Figure 2C), the decay rate constants for the three cations, Ba2+, Sr2+, and Ca2+, were all smaller than that for pure water. The sequence of the 2+ rate constants is the same as that at 20 mN/m, that is, kBa (15)
1 1 7 10 1 1 1 1 1 1 1 1 1 1 1 1
a Flip-flop kinetic parameters for the dDPPC−DPPC bilayers with various surface pressures (π=30, 20, 15, 10 mN/m) under different ionic conditions (100 mM Ca2+, 100 mM Sr2+, 100 mM Ba2+, and pure water). bThe flip-flop rate constants (k) were fitted from the decay curves in Figure 2 using eq 3. cThe half-lives (t1/2) and the activation free energies (ΔG‡) were calculated from k using eqs 5 and 6, respectively.
2+
2+
< kSr (15) < kCa (15) < kH2O(15). The only dissimilarity is that the flip-flop rate constant for the Ca2+ ions was extremely close to that for pure water, indicating the very weak effect on lipid flip-flop for the Ca2+ ions. It is interesting to see that, with the surface pressure decreasing from 20 to 15 mN/m, the ion effect on the lipid flip-flip remained almost unchanged, which is quite different from that with the surface pressure decreasing from 30 to 20 mN/m, especially for the Ba2+ ions. The isotherm curve of DPPC (Figure S1) indicates that a surface pressure of 15 mN/m corresponds to the coexistence region of the liquid-condensed and liquid-expanded phases, while surface pressures of 30 and 20 mN/m correspond to the liquid-condensed phase. Therefore, it is difficult to impute the ion effect to the phase state change of the lipid bilayer. Again, the sizes of the three ions are believed to be a key factor to affect the interaction between the ions and the lipid bilayer. When the surface pressure was 10 mN/m, as shown in Figure 2D, the Ca2+ ions increased the flip-flop rate referenced to that for pure water, while the Ba2+ and Sr2+ ions decreased
When the surface pressure was 30 mN/m (Figure 2A), the rate constants for the Ca2+ and Sr2+ ions were smaller but that for the Ba2+ solution was larger, compared with that for the pure water. These results indicate that the Ca2+ and Sr2+ ions have the capability to retard the flip-flop process of the asymmetric lipid bilayers while the Ba2+ ions can accelerate the process. The decay rate constants at 30 mN/m are in the 2+ 2+ 2+ sequence of kCa (30) < kSr (30) < kH2O(30) < kBa (30). The previous studies on the divalent ion−lipid monolayer interactions under similar conditions (surface pressure and ionic concentration) showed that Ca2+ ions30 could order the DPPC lipid chains but Sr2+ ions33 made the DPPC monolayer 17902
DOI: 10.1021/acs.jpcc.9b04557 J. Phys. Chem. C 2019, 123, 17899−17907
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Figure 3. Static SFG spectra for the dDPPC−DPPC bilayers in contact with the ionic solutions (100 mM Ca2+, 100 mM Sr2+, 100 mM Ba2+) and pure water at different surface pressures (30, 20, 15, and 10 mN/m). The ssp spectra were collected after the apparent flip-flop process.
the flip-flop rate. More specifically, upon the surface pressure reduction from 15 to 10 mN/m, for the Ca2+ ions, the flip-flop process changed from a slowing-down state to a speeding-up state, while for the Ba2+ and Sr2+ ions the processes still maintained their slowing-down state. The sequence of the 2+
ion−DPPC binding affinity. However, the variation trend of the affinity should be similar for all the three divalent cations in terms of the surface pressure. In consequence, the ion effect on the flip-flop behavior in terms of the surface pressure should also be similar for the three ions if the main influencing factor is the ion−DPPC binding affinity. Meanwhile, the phase transition should occur with the surface pressure decreasing from 20 to 15 mN/m. However, at the two surface pressures, the sequences of the rate constants for the three ions were the same, which indicates that the phase state is not the dominant factor. In this case, we believe that the ion size is a key factor to affect the flip-flop process, thus showing a match relationship with the lipid packing corresponding to the specific surface pressure. Static Structural Analysis. To gain more structural information, the SFG spectra (ssp, Figure 3) in the CH and OH stretching regions (2800−3700 cm−1) for the dDPPC− DPPC bilayers under the different ionic conditions were collected after the apparent flip-flop process of the bilayer (the signal at 2875 cm−1 leveling off). The corresponding ppp spectra are shown in Figure S2. The peaks centered at 2875, 2940, and 2960 cm−1 were assigned to the ss mode, Fermi resonance, and asymmetric stretching (as) mode of the terminal methyl (CH 3 ) groups of the DPPC lipids, respectively. The peaks centered at ∼3200 and ∼3400 cm−1 were assigned to the OH stretching vibrations from the strongly hydrogen-bonded and weakly hydrogen-bonded water molecules at the lipid/solution interface, respectively. The weak peaks centered at 2845 and 2905 cm−1 corresponding to the ss mode and Fermi resonance of the methylene (CH2) groups, respectively, can also be identified. Here, one issue needs to be clarified. The collected OH stretching vibrational (2) (3) signals (χ(2) eff ) should include both χS and χB as discussed in
2+
decay rate constants is kBa (10) < kSr (10) < kH2O(10) < 2+
kCa (10). Again, the ion effects on the flip-flop rates of the asymmetric bilayer were obviously different with the surface pressure decreasing from 15 to 10 mN/m, similar to the case with the surface pressure decreasing from 30 to 20 mN/m. Most notably, however, both upon the surface pressure reduction, the Ca2+ ions changed from a decelerated effect to an accelerated effect from 15 to 10 mN/m, while the Ba2+ ions changed inversely (from an accelerated effect to a decelerated effect) from 30 to 20 mN/m, indicating the critical role of the ion size in the flip-flop process. Up to now, it can be seen that the DPPC flip-flop rate seems to be dependent on the specific divalent cation in terms of the surface pressure. Referenced to the flip-flop rate for pure water, at the high surface pressure (30 mN/m), the Ba2+ ions accelerated the flip-flop process, but the Sr2+ and Ca2+ ions decelerated the flip-flop processes. At surface pressures of 20 and 15 mN/m, all the three cations slowed down the flip-flop processes. At the low surface pressure (10 mN/m), the Ca2+ ions accelerated the flip-flop process, while the Ba2+ and Sr2+ ions decelerated the flip-flop processes. The above experimental results indicate that the variation of the surface pressure changed the interaction between the cation and the lipid bilayer, resulting in the different flip-flop behaviors for the three ions. Upon the reduction of the surface pressure, the lipids in the bilayer should change to a more loosely and disordered packing state, leading to the different 17903
DOI: 10.1021/acs.jpcc.9b04557 J. Phys. Chem. C 2019, 123, 17899−17907
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Figure 4. Relative flip-flop rates of the dDPPC−DPPC bilayers for the Ba2+, Sr2+, and Ca2+ ions versus the surface pressure in the curve form (A) and in the heat map (C) or the average distance among lipids (D) and relative water signals of the dDPPC−DPPC bilayer for the Ba2+, Sr2+, and Ca2+ ions versus the surface pressure (B).
the recently published literature.43−47 The corresponding equation (eq S1) can be found in the Supporting Information in comparison to eq 2. In this concern, the following discussion related to the water OH stretching signals only dealt with χ(2) eff , which cannot affect the final conclusion on the metal ion sizedependent effects. When the surface pressure was 30 mN/m, the prominent spectral feature was the increased water OH stretching signals for the Ba2+ ions in the region of 3000−3700 cm−1. It could be attributed to the increased order of the water molecules surrounding the bilayer after the flip-flop process. On the contrary, the water signals for the Ca2+ and Sr2+ ions slightly decreased compared with that for pure water, which indicated that the water molecules became slightly disordered at the bilayer/ionic solution interface. It was worth noting that the measured SFG water signals can also match up with the extracted sequences for the flip-flop rates. In other words, the flip-flop process can be accelerated by the Ba2+ ions but decelerated by the Ca2+ and Sr2+ ions. The water signals can be increased by the Ba2+ ions but decreased by the Ca2+ and Sr2+ ions. When the surface pressure was 20 or 15 mN/m, the water OH vibrational signals for pure water were larger than those at 30 mN/m. Upon the reduction of the surface pressure, the packing of the lipid bilayer became more loosely, leading to more ordered interfacial water molecules, not only near the bilayer surface but also in the bilayer headgroup region. For the Ba2+, Sr2+, and Ca2+ ions, the water signals all significantly decreased compared with those for pure water at the same pressure. It was mainly caused by the ion shielding effect, giving rise to the reduced water vibrational signals at the
interface. The three ions had the same effect on the water vibrational signals and the flip-flop rates. When the surface pressure was 10 mN/m, the water vibrational signals for pure water further increased due to the increased order of the water molecules. Meanwhile, the water signals for the three ions still decreased compared with those for pure water. It should be noted that, however, for the Ca2+ ions, the effect on the water vibrational signals was not consistent with that on the flip-flop rate, which differed from the effects for the Ba2+ and Sr2+ ions. From the above results, the water OH vibrational signals varied in terms of the surface pressure for the Ba2+, Sr2+, and Ca2+ ions. The changing trends were the same when the surface pressure changed, except at a pressure of 10 mN/m for the Ca2+ ions. Size Dependence of Ion Effects. According to the timedependent SFG signals and the static SFG spectra, it was found that the ion effects on the flip-flop for the asymmetric lipid bilayer were dependent on the surface pressure. To directly compare the flip-flop rates at the different surface pressures for the three ions, the relative decay rate constant kion r (π) can be calculated semiquantitatively using eq 4, which was normalized by the decay rate for pure water at the corresponding surface pressure. The relative water signal intensity can be normalized by the water signal intensity for pure water using a similar method. Figure 4A,B shows the relative decay rates and the relative water signals in terms of surface pressure, respectively. The data for pure water was taken as a reference line, above or below which the flip-flop rates/water signals were increased or decreased, respectively. At the high surface pressure (30 mN/m), the Ba2+ ions can increase the dynamic flip-flop rate of the bilayer and the static 17904
DOI: 10.1021/acs.jpcc.9b04557 J. Phys. Chem. C 2019, 123, 17899−17907
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The Journal of Physical Chemistry C vibrational water signals at the lipid/solution interface, while the Sr2+ and Ca2+ ions decreased both the flip-flop rates and the water vibrational signals. At the middle pressure range (20 and 15 mN/m), all the three ions decreased the decay rates and the water vibrational signals. At the low surface pressure (10 mN/m), the Ba2+ and Sr2+ ions decreased the flip-flop rates and the water vibrational signals. The only exception was the Ca2+ ions that increased the flip-flop rate but decreased the water vibrational signals. The data of the relative flip-flop rate constant can also be visualized in the two-dimensional heat map (Figure 4C) where the warm colors stand for the accelerated flip-flop rates and the cold colors stand for the decelerated flip-flop rates. The warm colors appeared for the Ba2+ ions at a surface pressure of 30 mN/m and for the Ca2+ ions at a pressure of 10 mN/m. The effective ion radii of Ba2+, Sr2+, and Ca2+ are 1.35, 1.18, and 1.00 Å, respectively. Accordingly, the accelerated phenomenon of the flip-flop process occurred at the closely packed bilayer for the big-sized Ba2+ ions and at the loosely packed bilayer for the small-sized Ca2+ ions, which strongly suggested the existence of a match relationship, between the ion size and the lipid distance, with respect to the flip-flop process. Figure 4D shows the relative flip-flop rates of the dDPPC− DPPC bilayer for the Ba2+, Sr2+, and Ca2+ ions versus the average distance among lipids. The lipid distance can be estimated from the pressure−area (π−A) isotherm of DPPC (Figure S1) using the equation d = A mm , where d represents the average distance in a lipid bilayer and the mean molecular area (Amm) corresponds to a specific surface pressure. A similar tendency can be observed in Figure 4A,D. At the high surface pressure, the lipid distance corresponding to the compact packing can match up to the small Sr2+ and Ca2+ ions, which can stabilize the bilayer because of the 1:2 ion−lipid complex formation,30 leading to the decelerated flip-flip rate. However, the big-sized Ba2+ ions mismatched the small lipid distance so that they may form the 1:1 ion−lipid complexes with the lipids. The accelerated flip-flop rate for the Ba2+ ions may thus be due to the electrostatic repulsion of the ion−lipid complexes in the bilayer. At the middle pressure range, all the three ions can match with the increased lipid distance to stabilize the asymmetric bilayer and decelerate the flip-flop process. At the low surface pressure, the big Ba2+ and Sr2+ ions can still match up with the large lipid distance corresponding to the loosely packed bilayer. However, the small Ca2+ ions became mismatched. Therefore, the Ca2+ ions can accelerate the flipflop process. With above information in mind, it can be concluded that the match relationship between the ion size and the lipid distance dominated the ion effects on the lipid flipflop processes. This size match dependence can also explain the water signal changes, as shown in Figure 5. At the high surface pressure, the lipids packed closely and only the ordered water molecules around the lipid headgroups contributed to the SFG water signals.48 For the small-sized Sr2+ and Ca2+ ions, the stable ion−lipid complexes can be formed, leading to the electrostatic screening for the surrounding ordered water molecules and the decreased water vibrational signals. For the big-sized Ba2+ ions, however, the residue charges of the ion−lipid complexes induce ordering of the surrounding water molecules, resulting in the increased SFG water vibrational signals. At the middle surface pressure range, the looser packing of the bilayer allowed more water molecules penetrating into the bilayer,
Figure 5. Schematic showing the lipid bilayers with the surrounding ions and water molecules at different surface pressure ranges. A match relationship between the ion size and the lipid distance was thus presented.
leading to the stronger water vibrational signals for pure water. Nevertheless, for all the three ions, the water vibrational signals can be electrostatically screened because of the stable complex formation to neutralize the interfacial charges. At the low surface pressure, the big-sized Ba2+ and Sr2+ ions can still match the large lipid distance and then the water vibrational signals were electrostatically shielded as before. The smallsized Ca2+ ions, at this time, can penetrate into the bilayer and screen almost all the ordered water molecules in the lipid headgroup region, resulting in the further decrease of the SFG water vibrational signals. Here, the match/mismatch between the ion size and the average lipid distance can dominate the dynamic flip-flop rate and the water vibrational signals for the different ions when the surface pressure was changed. Therefore, the ion effects on the lipid transmembrane flip-flop rates were size-dependent. This size-dependent ion effects should be a general phenomenon regarding the biological process. There were ion sizedependence for ion-transport proteins,3 which indicates the relevance of our finding to the lipid ion channels in maintaining cellular ion homeostasis. Furthermore, the ion size-dependence also existed in ion sieving using graphene oxide membranes.49,50 As a consequence, the biological significance of the size-dependent ion effects should be appreciated, which can provide an inspiring clue for the biological studies, especially for biomembranes.
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CONCLUSIONS In this work, SFG was applied to investigate the metal ion effects on the lipid transmembrane movement (flip-flop process) for the asymmetric dDPPC−DPPC bilayer. The ion effects were size-dependent, presenting a match relationship between the ion size and the average lipid distance in the bilayer. When the match happened, the flip-flop process was decelerated and the surrounding ordered water molecules were electrostatically screened. Otherwise, the flip-flop process was accelerated. In brief, we conducted a comprehensive SFG study related to the ion effects on the flip-flop process of the lipid bilayer, revealing the size dependence of the ion effects. 17905
DOI: 10.1021/acs.jpcc.9b04557 J. Phys. Chem. C 2019, 123, 17899−17907
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The Journal of Physical Chemistry C
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This experimental study on the dynamic interaction between the metal ions and the lipid bilayer can provide inspiring clues for understanding the lipid ion channels. With respect to the lipids themselves, the lipids can not only be the basic building block for cell membranes but also regulate the cellular ion homeostasis by interacting with the ions, evidenced by this study.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.9b04557. SFG for a charged water interface; isotherm of DPPC at 22 °C; static SFG spectra, integrated water OH vibrational intensities, and relative water intensities of the dDPPC−DPPC bilayers in contact with the ionic solutions (100 mM Ba2+, 100 mM Sr2+, 100 mM Ca2+) and pure water at different surface pressures (30, 20, 15, and 10 mN/m); ion effects on the flip-flop process of the PS-containing bilayers (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: lxl@seu.edu.cn (X.L.). *E-mail: zhanc@umich.edu (Z.C.). ORCID
Yong-Hao Ma: 0000-0002-3545-9650 Xiaofeng Han: 0000-0002-2688-3425 Zhan Chen: 0000-0001-8687-8348 Xiaolin Lu: 0000-0002-0932-8489 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by the State Key Development Program for Basic Research of China (2017YFA0700500), the National Natural Science Foundation of China (21574020), the National Demonstration Center for Experimental Biomedical Engineering Education (Southeast University), the Fundamental Research Funds for the Central University, and the Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions. Z.C. thanks the University of Michigan to support his sabbatical leave.
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