13664
2007, 111, 13664-13667 Published on Web 11/15/2007
Controlling the Charge and Organization of Anionic Lipid Bilayers: Effect of Monovalent and Divalent Ions Emily R. Lamberson, Lee R. Cambrea, and Jennifer S. Hovis* Department of Chemistry, Purdue UniVersity, West Lafayette, Indiana 47907 ReceiVed: August 6, 2007; In Final Form: October 26, 2007
It is shown that the organization of lipid bilayers containing phosphatidic acid (PA) and phosphatidlycholine (PC) can be controlled by altering the monovalent and divalent ion concentrations. At high pH and/or calcium concentration, 1:1 Ca2+-PA2- complexes form; these complexes demix, and PA-rich and PC-rich regions are observable with epifluorescence microscopy. The results are compared with predictions from electrostatic theory. It is noted that the complex formation correlates in a roughly linear fashion with the monovalent/ divalent ion ratio, a parameter that cells adjust.
Introduction There is currently great debate about the role that lipids play in cell function. In particular, there is interest in whether lipids can be used to organize and activate signaling proteins.1,2 One crucial element missing from many of these discussions is control; for a function to be turned on, there must be a control parameter. In most biophysical studies of membranes, the parameter is temperaturesa parameter that does not significantly change in most cells. Herein, we will show that the organization of anionic/zwitterionic membranes can be controlled by varying the monovalent/divalent ion ratiosa parameter that cells do control. Anionic lipids comprise a significant fraction of cellular lipids. Lipids are tightly coupled to their environment, anionic lipids especially. The charge on a lipid (and thus its mixing properties) can be controlled by varying a wide range of highly interdependent parameters: lipid chemistry, charge density, ionic strength, pH, and adsorption of charged objects. Several examples are given: The pKa is sensitive to the lipid chemistry; for example, the pKa2 of DOPA (1,2-dioleoyl-sn-glycero-3phosphate) is ∼7.9 when DOPC (1,2-dioleoyl-sn-glycero-3phosphocholine) is present and ∼6.9 when DOPE (1,2-dioleoylsn-glycero-3-phosphoethanolamine) is present.3 The reported numbers are for 10 mol % DOPA, but if the amount of DOPA is increased, it will become more difficult to charge the membrane, and the pKa will shift upward. The charge on a lipid is also sensitive to the ionic strength; at high ionic strength, it is easier to maintain a charged surface.4 The relationship between lipid charge and ionic strength is relatively straightforward when the ions are monovalent. More highly charged objects cannot be treated simply as point charges; they typically have high binding affinities for lipids and may adsorb, and in doing so alter the lipid charge.5 In prior work from our lab, we showed that the organization of DOPA/DOPC bilayers could be controlled by adjusting the * Corresponding author.
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10.1021/jp076306a CCC: $37.00
monovalent ion concentration.6 Herein, a divalent ion is added, which as will be shown significantly increases the complexity of modeling the electrostatic interactions. The lipids DOPA and DOPC were chosen because the pKa’s of DOPA in DOPC have been measured3 and because the lipids are fluid at room temperature. In the monovalent work, we have replaced the lipid tails with egg derivatives and see essentially the same behavior (unpublished work), indicating that the observed effects are headgroup-mediated. Results and Discussion The experiments discussed below were all performed using supported bilayers composed of 30 mol % DOPA/69 mol % DOPC/1 mol % tail-labeled NBD PC. The bilayers were formed by vesicle fusion in a 250 mM KCl, 50 mM MES, pH 5, buffer. After excess vesicles were rinsed away, the external solution was exchanged for either a pH 7 or 8 buffer containing 250 mM KCl and 50 mM HEPES. The bilayers were imaged using epifluorescence microscopy, and fluidity was checked using fluorescence recovery after photobleaching (FRAP). Further detailed experimental information is available in the Supporting Information. Previously, we showed that when DOPA1- is converted to DOPA° it demixes from DOPC.6 With epifluorescence microscopy, large-scale separation into DOPC-rich and DOPA-rich regions was observed. The lipids in both regions were determined to be in the fluid phase by FRAP and attenuated total reflection-Fourier transform infrared spectroscopy. The DOPA was protonated by reducing the monovalent ion concentration (only monovalent ions were present). The experiments were conducted at pH 5; the pKa1 of DOPA in DOPC is ∼3.2,3 nearly two full units away. However, from Poisson-Boltzmann theory it is known that the charge on a lipid is sensitive to the ionic strength as well as the pKa and the surface charge density: The lower the ionic strength, the higher the probability the lipid will be protonated.4 If we begin at pH 8 and raise the ionic strength, we should create more DOPA2-; the pKa2 is ∼7.9.3 We have done this, going as high as 1 M KCl, and the bilayers continue © 2007 American Chemical Society
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Figure 1. Epifluorescence images of 30 mol % DOPA/69 mol % DOPC/1 mol % tail-labeled NBD PC bilayers. The bilayers were initially uniformly fluorescent, and the solution contained 250 mM KCl, 50 mM HEPES, buffered to pH 7 (a) or 8 (b). The images were acquired after the solution was exchanged for one containing 100 mM KCl, 50 mM HEPES, and the stated amount of CaCl2 at the given pH: (a) 5 mM, pH 7; (b) 0.5 mM, pH 8. The images are 160 × 160 µm.
to be uniformly mixed; due to the fact that DOPA2- is highly charged, which promotes mixing with DOPC, we would not expect to be able to detect the formation of DOPA2- by epifluorescence microscopy. As will be shown below, 1:1 Ca2+-DOPA2- complexes demix from DOPC, providing a way to examine the effect of ionic strength on the formation of DOPA2-. To observe demixing, it is necessary to add ∼10-fold more CaCl2 at pH 7 than pH 8 (Figure 1): (a) 5 mM CaCl2, 100 mM KCl; (b) 0.5 mM CaCl2, 100 mM KCl. The solution ionic strength was initially 250 mM KCl (and the pH as indicated), the bilayers appeared uniformly mixed, and upon the addition of the indicated amounts of CaCl2, rapid (within 1 s) separation was observed. As the (2-) species can be created by increasing either the pH or the ionic strength, we conclude that the dark regions (∼20% depletion in the fluorescent probe) are rich in 1:1 Ca2+-DOPA2- complexes while the gray regions are rich in DOPC (the fluorescent label is on the tail of a PC lipid). Calcium has a high affinity for PA,7 and there is strong evidence in the literature for the formation of 1:2 Ca2+-PA1- complexes.8 At lower amounts of calcium, we expect that 1:2 Ca2+-DOPA1complexes form, but they do not appear to separate from the DOPC. Further evidence that the dark regions are rich in 1:1 Ca2+DOPA2- complexes comes from protein binding studies. R-Synuclein, which avidly binds to membranes containing charged PA,9,10 does not bind to the dark regions (data not shown). The dark regions could be lower in intensity not because of reorganization, but because lipids have left the surface. This has been ruled out by acquiring images under very low light conditions; in doing so, we quantitatively determined that there is conservation of fluorophore, the gray regions are commensurately brighter than the dark regions relative to the initially mixed bilayers (Supporting Information). FRAP experiments (Supporting Information) show that the lipids in the dark regions recover on a time scale consistent with fluid bilayers and there is exchange between the two regions. Previous studies suggested that calcium promotes the formation of gel-phase regions.8,11,12 When the effect on the transition temperature was quantified, it was observed that calcium shifted the melting temperature up by a few degrees at most.13 The melting temperature of DOPA is -8 °C;14 thus, we would expect it to remain fluid at room temperature. The regions rich in Ca2+-DOPA2- complexes have an extended/branched structure. In our previous work protonating DOPA, we observed very similar structures.6 There we speculated that DOPA1- is a line-active lipid and due
J. Phys. Chem. B, Vol. 111, No. 49, 2007 13665 to charge-charge repulsions promotes branching. That could be the case here, or DOPA2- may be line-active. Using supported bilayers, we have looked at a variety of anonic/ zwitterionic and zwitterionic/zwitterionic mixtures, and only when PA is present do we observe the extended/branched structures. Consequently, we hypothesize that the structures arise due to the presence of PA and not as a result of the solid support. To further explore the electrostatic landscape, we have varied the concentration of both monovalent and divalent ions simultaneously. Shown in Figure 2 are representative images acquired after adjusting the bulk ion concentration from 250 mM KCl (where the lipids are uniformly mixed) to the indicated concentration. The solutions contain 50 mM HEPES and were adjusted to pH 8. For each concentration, many samples (2030) were examined and the percent area coverage of the dark regions determined; those values are shown on the images. In many of the images, bright spots appear. For many of the images, if a higher magnification objective is used the bright spots resolve into rings. Due to the relatively long working distances of the objectives used, caps can appear as rings. Previously, we showed that, when a PA bilayer is exposed to an asymmetric screening environment, the resulting asymmetry in the monolayer surface area causes the bilayers to bulge out of the plane and form caps.15 When the ionic strength is less on the proximal side than the distal side, the bilayer will want to bend away from the surface; for 100 mM, 50 mM, and 0 mM KCl columns, this is the case. In the 250 mM KCl column, the reverse is true; however, all of the white dots in these images are too small to be able to determine if they are rings, and thus three-dimensional objects, or if they arise for another reason. At the present, we are not sure why the bright spots appear in these images. The DOPA was incorporated at 30 mol %, and yet, area fractions of nearly 50% are reported. There are several possible explanations: One, there is a higher concentration of PA in the upper leaflet, and separation only occurs in that leaflet. To address this, we replaced the tail-labeled NBD PC with headlabeled Texas Red DHPE, which can be quenched using iodine.16,17 Changing the probe did not alter the separation behavior. Addition of 500 mM KI resulted in a loss of ∼50% of the fluorescence in both regions, i.e., the pattern remained visible (Supporting Information). This suggests that the separation occurs in both leaflets. This would not be unexpected: it has been suggested that when PA is chelated with calcium it rapidly flips across the membrane;7,18 also, coupling between leaflets has been observed.19,20 Two, the addition of calcium increases the headgroup area. It has been reported that calcium decreases the headgroup area, but in those experiments, the gel phase formed.8,11,12 To our knowledge, there is nothing known about calcium’s effect on the headgroup area when PA remains fluid. A third possibility is that the PA-rich regions contain an appreciable amount of PC, which leads to an area coverage of >30%. In the absence of a phase diagram for this system, it is difficult to predict the relative proportions of each component in each phase. However, fluorophore partitioning between the two phases should also depend on the tie lines. We therefore determined the percent depletions of fluorophore for each concentration shown in Figure 2. The percent depletions varied from 15 ( 2% to 22 ( 3% with no obvious trend (Supporting Information). On the basis of these results, we were unable to extract any information about the relative composition of each phase. In Figure 2, it is observed that the area coverage increases as the calcium concentration increases. This appears straightfor-
13666 J. Phys. Chem. B, Vol. 111, No. 49, 2007
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Figure 2. Epifluorescence images of 30 mol % DOPA/69 mol % DOPC/1 mol % tail-labeled NBD PC bilayers. The bilayers were initially uniformly fluorescent, and the solution contained 250 mM KCl, 50 mM HEPES, buffered to pH 8. The images were acquired after the solution was exchanged for one containing 50 mM HEPES, pH 8, and the stated amount of KCl and CaCl2. The percent area coverage of the dark region is given on the images; these were determined by examining 20-30 samples for each composition. The images are 160 × 160 µm.
ward: the more calcium present, the more DOPA2- created, and the more ions available for complex formation. It is also observed that as the monovalent concentration decreases the area coverage increases. From Poisson-Boltzmann theory, this would appear to be contradictory, as decreasing the monovalent concentration decreases the ionic strength, and this should decrease the charge on the membrane, resulting in fewer DOPA2-. Poisson-Boltzmann theory does not, however, take into account that ions can modify the surface charge by adsorbing. The binding constant for divalent cations can be written as
KMe ) KMe° exp(2eψo/kT)
(1)
where KMe° is the intrinsic binding constant and ψo is the surface potential.13 From Gouy-Chapman theory, it is known that, as the concentration of ions in solution decreases, the surface potential increases21sand therefore the binding constant increases, increasing the formation of Ca2+-DOPA2- complexes. Figure 2 can be explained if one ion is held constant. What about moving diagonallysdoes the area change as expected? Specific predictions are beyond the scope of this letter, as many of the relevant equations cannot be solved analytically when both monovalent and divalent ions are present. To further complicate matters, all of the parameterssconcentration of complexes, surface potential, surface charge density, and ion
concentrationsare highly interdependent. We end then with a phenomenological observation: there is a roughly linear relationship between the area coverage and the K+/Ca2+ ratio (Figure 3). These data are represented in two ways, with the plot on the top indicating which points belong to sets of constant potassium concentration, and the plot on the bottom indicating which points belong to set of constant calcium concentration. The observed linear trend appears when the monovalent concentration is in the millimolar range, the divalent concentration in the micromolar range, and the amount of the divalent ion is e1% of the monovalent ion; this is likely to be critical, as the calcium concentration will dominate the surface potential when it is in the millimolar range or g3% of the monovalent ion.21 The interest in the K+/Ca2+ ratio arises because organisms are known to regulate the ratio of monovalent/divalent ions. One mechanism is through the use of sodium/calcium exchangers,22 which import three sodium ions for every one calcium ion exported. As organisms typically maintain a constant monovalent ion concentration, another mechanism for changing the K+/Ca2+ ratio is simply varying calcium concentrations. In general, there is little known about the biophysical effect of varying this ratio. Here, we show that regulation of the ratio is an effective way to control the clustering of the lipid phosphatidic acid. Since PA is a second messenger in a variety of
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J. Phys. Chem. B, Vol. 111, No. 49, 2007 13667 affinity and increases the probability that DOPA will be in the (2-) charge state; (B) decreasing the ionic strength, which increases the calcium affinity, promoting the formation of the Ca2+-DOPA2- complex. The competition results in a roughly linear relationship between the monovalent/divalent ion ratio and the amount of complex formation. Acknowledgment. This work was supported in part by NIH grant R01 NS049221 Supporting Information Available: Experimental details, FRAP recoveries and quenching experiments. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes
Figure 3. Plots of percent area coverage vs K+/Ca2+ ratio. Both plots use the same data, which comes from Figure 2, and contain dashed lines to guide the eye. The plot on the top indicates which data points belong to sets of constant potassium concentration, while the plot on the bottom indicates which data points belong to sets of constant calcium concentration.
signaling pathways,23 anything that clusters and thus sequesters PA may have broad physiological implications. There is one other biological implication to note: Returning to the thought of varying one ion at a time, it can be observed in Figure 2 that, in order to effect the largest change in the area coverage with the smallest variation (and the least energy expenditure for an organism), one should hold at a low divalent concentration and a high monovalent concentrationswhich is exactly what cells do. In summary, we have shown that the organization of PA/PC bilayers can be controlled by varying the monovalent and divalent concentrations. The species that separates is a 1:1 Ca2+-DOPA2- complex. The gross features of the obtained results can be explained by invoking two competing factors: (A) increasing the ionic strength, which decreases the proton
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