Calcium Assists Dopamine Release by Preventing Aggregation on the

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Calcium Assists Dopamine Release by Preventing Aggregation on the Inner Leaflet of Presynaptic Vesicles Sini Mokkila, Pekka A. Postila, Sami Rissanen, Hanna Juhola, Ilpo Vattulainen, and Tomasz Rog ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00395 • Publication Date (Web): 06 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017

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ACS Chemical Neuroscience

Calcium Assists Dopamine Release by Preventing Aggregation on the Inner Leaflet of Presynaptic Vesicles

Sini Mokkila1, Pekka A. Postila2, Sami Rissanen1, Hanna Juhola1, Ilpo Vattulainen1,3,4, Tomasz Róg1,3

AUTHOR EMAIL ADDRESS [email protected], [email protected] CORRESPONDING AUTHOR FOOTNOTE Tel.: +358 40 198 1010. E-mail: [email protected] (T.R.).

1

Department of Physics, Tampere University of Technology, P.O. Box 692, FI-33101

Tampere, Finland. 2

Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering,

Åbo Akademi University, Turku, Finland. 3

Department of Physics, University of Helsinki, P.O. Box 64, FI-00014, Helsinki, Finland.

4

MEMPHYS – Center for Biomembrane Physics, University of Southern Denmark, Odense,

Denmark.

ACS Paragon 1 Plus Environment


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ABSTRACT In this study, the dopamine-lipid bilayer interactions were probed with three physiologically relevant ion compositions using atomistic molecular dynamics simulations and free energy calculations. The in silico results indicate that calcium is able to decrease significantly the binding of dopamine to a neutral (zwitterionic) phosphatidylcholine lipid bilayer model mimicking the inner leaflet of a presynaptic vesicle. We argue that the observed calciuminduced effect is likely in crucial role in the neurotransmitter release from the presynaptic vesicles docked in the active zone of nerve terminals. The inner leaflets of presynaptic vesicles, which are responsible for releasing neurotransmitters into the synaptic cleft, are mainly composed of neutral lipids such as phosphatidylcholine and phosphatidylethanolamine. The neutrality of the lipid head group region, enhanced by a low pH level, should limit membrane aggregation of transmitters. In addition, the simulations suggest that the high calcium levels inside presynaptic vesicles prevent even the most lipophilic transmitters such as dopamine from adhering to the inner leaflet surface, thus rendering unhindered neurotransmitter release feasible.

Keywords: synaptic neurotransmission, neurotransmitter release, presynaptic vesicle, phosphatidylcholine (PC), dopamine, molecular dynamics (MD) simulations, binding free energy.

Graphical Abstract:


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1. INTRODUCTION Synaptic neurotransmission is traditionally described to follow a seemingly simple path in three stages (Figure 1a). First, neurotransmitters (NTs) are released from the presynaptic neuron via exocytosis of vesicles into the synaptic cleft. Second, the NTs diffuse across the synaptic cleft towards the adjacent postsynaptic neuron. Third, the NTs bind to their specific membrane-embedded receptors, which either directly or indirectly causes influx of cations into the postsynaptic neuron.1

Figure 1. Synaptic neurotransmission models and a presynaptic vesicle. a) Membraneindependent model:8 (1) hydrophilic neurotransmitters (NTs) are released from the presynaptic vesicle, (2) diffuse in 3D across the synaptic cleft without aggregating on the membrane-water interface, and 3) bind into their receptors’ extracellular binding sites. b) Membrane-dependent model:8 (1) hydrophobic NTs are released from the presynaptic vesicle, (2) undergo 3D diffusion across the cleft, (3) bind onto the postsynaptic membrane surface, where they carry out lateral 2D diffusion on the membrane plane, (4) and finally bind into their receptors’ membrane-buried binding sites. c) The inner leaflet of the presynaptic vesicle lipid bilayer is mainly composed of neutral phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), while the highly anionic phosphatidylinositol (PIP) lipid is found in the outer leaflet.18,19 Divalent cations Ca2+ and Zn2+ and protons (H+) are actively pumped inside the vesicle.20 The cations aggregate on the membrane head group region and prevent even the most hydrophobic NTs such as dopamine from binding onto the inner leaflet. The vesicular pH remains low at 5.6 due to H+ pumps.20 This pH is close to the pKa value of the carboxylic groups of phosphatidylserine (PS), thus, keeping the lipid species in mostly neutral state. While this overall description of the synapse operation during transmission is accurate, it does not account for non-specific interactions that the NTs experience with macromolecules inside the synaptic cleft. These non-specific interactions such as NT-membrane binding deserve a closer look, because they affect the diffusion of NTs by coordinating their



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movements (Figure 1b) and, thus, likely slow or speed up their receptor binding rates as well. The synaptic cleft is a relatively narrow space between the cell membranes of two adjacent neurons (20-40 nm; Figure 1a and b).1 In addition to the synaptic receptors and NT transporters embedded in the lipid bilayers, proteins such as neurolignins2 and carbohydrates forming the glycocalyx3 span the cleft and link the two sides together. Accordingly, the NTs are released into a crowded space where numerous non-specific interactions affect their dynamics and the overall diffusion. These non-specific effects have been mostly overlooked in the past. However, a growing number of experimental and molecular dynamics (MD) simulation studies have addressed the issue in part by probing the NT-lipid bilayer interactions. The simulations have indicated that dopamine partitions to the membrane surface4 and this affinity towards the lipid head group region is enhanced by negatively charged or anionic lipids such as phosphatidylserine (PS).4 Monolayer experiments have confirmed the binding of dopamine to the anionic bilayer seen in the MD simulations with the same exact composition.4 Moreover, calorimetric studies have demonstrated dopamine binding with anionic lipid bilayers composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG).5 Recently, nuclear magnetic resonance (NMR) measurements have showed dopamine binding to both neutral and anionic lipid bilayers, although the adherence is stronger for membranes with an anionic composition.6 Similarly, serotonin and melatonin, which are also highly lipophilic NTs, have been shown to bind to membrane surfaces. According to dialysis equilibrium measurements and MD simulations, serotonin has a high affinity towards a neutral lipid bilayer composed of PC.7 The simulation studies have also showed that serotonin binding occurs with a neutral bilayer composed of cholesterol and sphingomyelin (SPM) and with an anionic lipid bilayer composed of PC, phosphatidylethanolamine (PE), and PS.8 In addition, melatonin binds to neutral lipid bilayers composed of PC and cholesterol in various concentrations, highlighted by a number of studies based on monolayer compression isotherms, small-angle neutron diffraction, small-angle neutron scattering, and MD simulations.9,10 In contrast to lipophilic NTs, the more hydrophilic transmitters containing polar groups such as γ-aminobutyrate (GABA), glycine, acetylcholine, and glutamate interact weakly with lipid bilayers. These interactions are dependent on the presence of anionic lipids in a bilayer.11,12 For example, positively charged acetylcholine has been shown to interact with lipid bilayers where anionic lipids, PS8 or phosphatidic acid (PA),13 are present; however, these interactions were decreased by the presence of Ca2+ ions.13 Interestingly,


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calcium in combination with anionic lipids has been shown to induce interactions of GABA and glutamate with lipids.13 In our recent study, 13 non-peptidic NTs were simulated with membrane models to probe their interactions with the postsynaptic membrane. The NTs generally fall into two distinct categories: 1) membrane-binding (dopamine, serotonin, epinephrine, norepinephrine, melatonin, and adenosine) and 2) membrane-nonbinding molecules (GABA, acetylcholine, serine, aspartate, and glutamate).8 Moreover, this classification matches the NT-binding site locations of the synaptic receptors in relation to the postsynaptic membrane: if a NT is bound to the postsynaptic membrane, then its receptor was observed to be a G protein-coupled receptor (GPCR) with a membrane-buried ligand-binding site (Figure 1b); in contrast, the membrane-nonbinding NTs were observed to have extracellular ligand-binding sites positioned above the cell membrane surface (Figure 1a). Given this, the synaptic neurotransmission can be divided into membrane-independent and membrane-dependent mechanisms (Figure 1a vs. b).8 In the present study, the aim is to examine the role of physiologically relevant cations +

(Na , K+, Ca2+) for dopamine-lipid bilayer interactions using atomistic MD simulations. Clions were used as the neutralizing anionic counter ions in the simulations. Ions are crucial components of the NT-mediated signal transduction in the synapse, hence they need to be included in the discussion involving NT-membrane interactions inside the synapses as well. The influx of Ca2+ ions into the presynaptic neuron is an integral process for triggering the NT release via the complex protein machinery embedded at the presynaptic membranes.14 The effect of different ion compositions is examined with dopamine – a major NT that is known to bind strongly onto lipid membranes. Dopamine signaling deficiency has been linked to several neuropathologies such as attention deficit hyperactivity disorder (ADHD), Tourette syndrome, schizophrenia, psychosis, depression, Parkinson’s disease, Huntington disease, and multiple sclerosis.15,16,17 Following our previous study,4,8 we here employ three lipid bilayer models composed of the most common lipid types (see Table 1): (1) a neutral bilayer composed of PC (PC bilayer); (2) an anionic bilayer composed of PC, PE, and PS (PC-PE-PS bilayer), and a neutral highly ordered bilayer composed of SPM, PC, and cholesterol (SPM-PC-Chol bilayer). The simulation results acquired in this work show that high calcium levels can effectively decrease dopamine binding to a PC lipid bilayer. Because the inner leaflet of presynaptic vesicles is mainly composed of neutral lipids such as PC and PE (Figure 1c),18,19



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the in silico results can be directly linked to the presynaptic NT release process. Moreover, the intravesicular space of presynaptic vesicle is kept at a pH of 5.6,20 which assures that the PS present in minute amounts in the inner leaflet is effectively rendered neutral. We argue based on the here presented results and previous experimental observations that the lipid content, together with the low pH inside the vesicles and the active inward pumping of divalent cations, prevent NT accumulation to the inner leaflet surfaces of the presynaptic vesicles, thereby, assuring unhindered release of the NTs from the presynaptic vesicles into the synaptic cleft.

Table 1. Composition of systems explored in this study together with simulation times for each model system. B i l a ye r s

PC-PE-PS K+ Ca2+

SPM-PC-Chol Na K+ Ca2+

+

PC K+

(1)

(2)

(3)

DLiPC

128

128

128

44

44

44

-

-

-

DLiPE

-

-

-

60

60

60

-

-

-

DLiPS

-

-

-

24

24

24

-

-

-

SPM

-

-

-

-

-

-

48

48

48

DOPC

-

-

-

-

-

-

48

48

48

Chol

-

-

-

-

-

-

32

32

32

Na+

33

Na

K+

Ca

2+

Na

+

(4)

(5)

(6)

30 33

Ca2+

+

(7)

(8)

(9)

26 30

11

26 12

9

Cl-

53

53

42

26

26

20

46

46

38

Water

11,75

11,75

11,79

10,88

10,88

10,89

9,247

9,247

9,272

9

9

2

0

0

5

Dopamine

20

20

20

20

20

20

20

20

20

Simulation

500

500

500

300

300

300

300

300

300

Time [ns]

2. RESULTS 2.1. Dopamine molecules bind onto the lipid bilayer surfaces In system preparation, the dopamine molecules were placed randomly in the water phase from which they quickly moved into the head group regions of the membrane models. Figure 2


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shows snapshots of the final configurations of all simulated systems, depicting the preferential placements of dopamine molecules and each cation species. Figure 3 shows the density profiles of dopamine, cations, and the phosphate atoms of PC lipids. In all of the cases, the maximum of dopamine density is observed at the region below the lipid head group phosphate atoms and in the corresponding position of the carbon groups of PC. With the neutral PC bilayer, a fraction of dopamine is found to stay in the water phase, however, in the SPM-PCCHOL and PC-PE-PS bilayers all of the dopamine molecules are bound to the membranewater interface.

Figure 2. Lipids and lipid bilayers used in the atomistic simulations. On the left are drawn the 2D structures of the lipids and dopamine used in the study. On the right are shown the



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snapshots of final configurations of all simulated lipid bilayer systems excluding water molecules and Cl- ions. Color code: dopamine (red CPK models); Na+ (white spheres); K+ (yellow spheres); Ca2+ (orange spheres); and lipids (cyan licorice models).

Figure 3. Density profiles for dopamine and each simulated cation species (a and b) in the PC bilayer, (c and d) the PC-PE-PS bilayer, and (e and f) in the SPM-PC-Chol membrane. Also shown is the average position of the phosphorous (P) atoms of PCs (gray line). The membrane depth of zero corresponds to the position of the center of the lipid bilayer. Cations are shown in panels b, d, and f and dopamine in panels a, c, and e. Color code: black (bilayers with Na+); red (bilayers with K+); blue (bilayers with Ca2+); green (bilayers without cations; referred as NONE). 2.2. Cations bind to the lipid bilayer and reduce dopamine adherence


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Interactions of cations with the lipid head groups observed in this study do not differ markedly from the previously published results.21,22,23,24,25,26 Majority of Na+ ions present in the simulated systems bind to lipid head groups (Figure 3) of the PC and PC-PE-PS bilayers, while with the SPM-PC-Chol membrane model the binding is weaker and, thus, a larger number of the cations remain unbound in the water phase. This is consistent with our previous simulation studies with neutral,21 anionic,22 and cholesterol-rich bilayers.23 Binding of Na+ ions onto lipid head groups as observed in the MD simulations is, however, a somewhat controversial issue, involving possible force field-related problems.27 K+ ions do not show affinity toward the membrane-water interface and remain in the water phase in agreement with previous studies.24,25,26 Cl- ions do not bind to the membrane surface and interact only with the choline groups (see Figure S1 in the Supporting Information). These anionic interactions are highly dynamic and similar to results found elsewhere.21 Most importantly, however, Ca2+ ions bind strongly to lipid head groups, which is also in agreement with previous experimental and theoretical studies.28,29,30,31

2.3. Free energy calculations indicate that calcium ions have the highest capacity for weakening the dopamine-membrane binding Figure 4 shows the free energy profiles of dopamine for moving from the water phase towards the hydrophobic core of the membrane models. The plotted profiles indicate that the optimal position of dopamine in terms of its free energy is at the membrane-water interface – a result that is in line with the representative density plots (Figure 3). Given this, the free energies of transfer of dopamine from the water phase to the membrane-water interface (∆F) are listed in Table 2. In agreement with our prior results, dopamine-membrane binding is weaker for the neutral PC bilayer than for the other two membrane models.4,8 Moreover, all cation types decrease the strength of the binding with the PC bilayer, but the effect of Ca2+ is the strongest as it decreases the ∆F from -15 to -5 kJ/mol. In the case of the PC-PE-PS bilayer, the ∆F values (Table 2) are identical and within the statistical error range for all of the simulated systems (Table 1). For the SPM-PC-Chol system, the presence of cations decreases the binding free energy barrier by 2-5 kJ/mol, but overall the ∆F values remain relatively high at 17 to -20 kJ/mol. The results obtained from the free energy calculations (Table 2; Figure 4) give a



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markedly different result from the density profile analysis acquired from unbiased MD simulations regarding the effect of Na+ ions (Figure 3). The free energy calculations focusing on one dopamine molecule moving across the lipid bilayer model indicates that the binding of dopamine to a PC bilayer is weaker with Na+ ions than with the divalent Ca2+ ions. Meanwhile, in unbiased MD simulations dopamine binding is somewhat more prominent together with the monovalent ions (Figure 3). Given that the simulation model (force field) is identical in the two approaches, the cause of the observed difference has to be based on sampling. The difference is therefore expected to arise from the limited length of the MD simulations used for the density profile analysis in comparison to the neurotransmitter residence time at the bilayer surface (Figure 3). In free energy calculations based on the umbrella sampling methodology, the times considered in the analysis were substantially longer than in the unbiased simulations, thus the sampling is expected to be more extensive than in unbiased simulations. .

Table 2. Free energy of translating dopamine from the water phase to the membrane-water interface. Error (± ) is given as a standard deviation obtained from the boot strap analysis. Cation PC PC-PE-PS SPM-PC-Chol [kJ/mol] [kJ/mol] [kJ/mol] + Na -8 ± 1.6 -20 ± 1.3 -18± 0.8 K+

-10 ± 1.4

-18± 1.6

-17± 1.3

Ca2+

-5 ± 1.3

-19 ± 1.6

-20 ± 0.9

-15 ± 1.0

-20 ± 1.0*

-22 ± 1.0

*Na+ counter ions added.


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Figure 4. Free energy profiles of dopamine translocation from the water phase to the center of the PC bilayer (a), the PC-PE-PS bilayer (b), and the SPM-PC-CHOL bilayer (c). At the membrane depth of zero is the center of the lipid bilayer and the bulk water phase begins at 2.5-3.0 nm. The color code: black (bilayers with Na+); red (bilayers with K+); blue (bilayers with Ca2+); green (bilayers without cations). The positions of the vertical gray-dashed lines, indicating the locations of the membrane-water interfaces, differ between membrane models due to lipid composition differences. Error bars are based on the boot strap analysis method and have been calculated in relation to the point located in the water phase. Curves starting from bilayer center are shown in the Supporting Information (Figure S2).

3. DISCUSSION This in silico study suggests that calcium could play a crucial and previously unrecognized role in the NT release process (Figure 1). It is an established fact that the influx of Ca2+ ions into the presynaptic neuron triggers the protein-mediated pore formation between the vesicular and presynaptic membranes.32 However, the simulations put forward an idea that



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these divalent cations, when pumped inside the vesicle could also be required for preventing the aggregation of NTs on the presynaptic vesicle’s inner leaflet. This calcium-induced effect, which is clearly visible in the dopamine simulations performed with the neutral PC lipid bilayer, should make the release of lipophilic NTs from the presynaptic vesicles efficient. Lipidomics studies have indicated that PC is the main component of presynaptic vesicles, constituting 36-41 mol% of their phospholipid content (Figure 1c).18,19 In fact, the inner leaflets of the presynaptic vesicles, confining the pools of NTs inside the vesicles, are enriched in PC (~50 mol%).19 The second most abundant lipid species in these vesicles are PEs and ether PEs constituting 23-25 and 19-12 mol% of phospholipids, respectively.18,19 The total amount of PE enriched in the inner leaflet of synaptic vesicles is high, constituting ~36 mol%, while the ether PE is slightly more enriched in the outer leaflet.19 SPM content is relatively small (7-12 mol%) and its distribution between the inner and outer leaflets is not known.18,19 Similarly, PS content is relatively small (7-12 mol%) and its distribution between the leaflets is unknown.18,19 The anionic phosphatidylinositol (PIP) content is only a few mol% and it is entirely located in the outer leaflet.18,19 Finally, cholesterol is also an important component of vesicular membranes as its molar content of all lipids is around 38 mol%. Summarizing, these data indicate that the majority of lipids in the inner leaflet of presynaptic vesicles belong to the neutral lipid species PC and PE (~86 mol% of phospholipids) and, therefore, the neutral PC lipid bilayer used in the present simulations mimics well the lipid content of a vesicular inner leaflet. The presynaptic vesicle membrane host a multitude of proteins such as vATPase and calcium pumps that adjust the H+ and the Ca2+ content inside them.20 The vATPase pumps protons inside presynaptic vesicles and, thereby, lowers the pH of the vesicular space down to 5.6, which is roughly the pKa value of the PS carboxyl group.33 Accordingly, at least half of the PS molecules present in the inner leaflet of the vesicles are in neutral form and, thus, are unlikely to bind NTs such as dopamine. Furthermore, the concentration of Ca2+ within the vesicles is upkept by specific calcium pumps. Although the exact concentration of Ca2+ ions inside the vesicles is not known, high calcium levels are certainly important for the NT release process in general. The presence of Zn2+ pumps in the presynaptic vesicles has also been reported.20 Because this divalent cation binds with lipids even stronger than Ca2+, it likely decreases NT binding at the inner leaflet of the vesicle in the same manner as calcium.Error! Bookmark not defined. The 75 mM Ca2+ concentration used in the dopamine-lipid bilayer simulations (Table 4) is admittedly high, if it is directly compared against the experimentally derived numbers


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acquired from synapses (3 mM). It is, however, important to note that the experimental values are only ballpark estimates of the physical reality, because the NT pre-release concentrations of Ca2+ ions are measured indirectly using a calcium-dependent photo protein34. Thus, the potentially significant fraction of the Ca2+ ions strongly bound at the lipid head group region is ignored in the measurements. In fact, the concentration of free Ca2+ ions moving freely in the water phase, as observed in our MD simulations, could be very low indeed. Based on the density plots, the concentration of Ca2+ ions that remain in the bulk water is estimated to be 4-7 mM with the PC bilayer, 0 mM with the PC-PE-PS bilayer, and 5 mM with the SPM-PC-Chol bilayer. This is due to the high capability of lipid head groups to bind Ca2+ ions. It has been shown for a POPC bilayer that it becomes saturated with calcium when one Ca2+ ion is bound per three lipid molecules, and the same saturation effect has been observed for the POPC-POPS bilayer with one calcium ion per two lipids.35 Given that the free energy minimum of Ca2+ is at the membrane-water interface, increasing its partitioning to a membrane, and given the high capacity of Ca2+ to bind multiple lipids, the calcium concentration in the vicinity of membrane surfaces is expected to be high. It is also worth noticing that in our simulations the bilayer surfaces are not fully saturated with calcium ions (if all Ca2+ present in the simulations were bonded to lipids, the calcium:lipid ratio would be 1:12) and therefore even more Ca2+ ions could potentially accumulate at the membrane surface for example inside the presynaptic vesicles. Moreover, it is reasonable to assume that with higher Ca2+ levels, the amount of dopamine-lipid binding would be reduced further for the PC-PE-PS and SPM-PC-Chol bilayers the same way as observed with the neutral PC-only bilayer. What is the cause for the observed calcium-induced effect lowering the amount of dopamine binding to the PC membrane? The obvious key factor is electrostatic repulsion. Ca2+ binds to the membrane surface; thereby, nulling in part its negative charge and increasing its surface charge density, and given that dopamine is also positively charged, this gives rise to repulsive effect that keeps dopamine molecules partitioned in the water phase. This view is consistent with the observations for Na+ ion species. The strong Ca2+lipid bilayer interactions are coordinated by 5-8 lipid oxygen atoms. In contrast, the Na+ binding is much less coordinated; involving 2-3 oxygen atoms.35 This difference is likely to explain the weaker effects of the Na+ ions in comparison to the Ca2+ ions on the dopamine binding in the free energy calculations (Figure 4); i.e. calcium ions neutralize the membrane head group region in a more permanent or effective manner than the physically smaller and less charged monovalent cations.



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Previous calorimetric studies investigating dopamine-lipid binding pointed out the importance of anionic lipids for its binding strength.5 This is in line with the simulation results for the PC-PE-PS bilayer (Figures 3 and 4); however, any oversimplification of this phenomenon should be avoided. Other properties of the membrane-water interface such as the overall head group region rigidity due to strong Ca+2 ion binding likely play a role in the process. Ca2+ also influences other properties of lipid bilayers. Average surface area per lipid molecule in the PC bilayer drops from 0.64 (system without cations) to 0.62 nm2 in response to the addition of Ca2+ ions. This effect can make the head group region denser and at least slightly contribute to the decrease in the membrane permeability.36 The dopamine-lipid bilayer simulations described in this study promote the idea that both the inner leaflet phospholipid composition and the divalent cation content of the presynaptic vesicles are optimized for the NT release into the synaptic cleft. The binding of lipophilic NTs onto the postsynaptic membrane surface should speed up their binding into the receptors’ membrane-buried binding sites (Figure 1b).8,37 However, strong membrane adherence inside the presynaptic vesicles could, in theory, harmfully slow down the NT release process. In conclusion, we argue that the NTs are physically obstructed from adhering to the inner leaflets of the vesicles by three safeguards (Figure 1c): 1) the lipid head group region is kept neutral with a high PC and PE content to prevent their well-documented attraction between the NTs and the anionic lipids;4,5,6 2) those few anionic lipids present at the inner leaflet (and charged NTs) are rendered neutral by lowering the intravesicular pH level; and 3) finally, the vesicular calcium levels are maintained at a sufficiently high level to saturate the membrane-water interface with divalent cations (Ca2+ and Zn2+) to prevent even the most lipophilic NTs (e.g. dopamine) from adhering onto the surface.

4. METHODS The atomistic molecular dynamics (MD) simulations were performed with nine lipid bilayer systems (Table 1). Three membrane models were composed of (1) 128 molecules of dilineoylphosphatidylcholine (DLiPC, di-18:2-DLiPC; Figure 2); (2) 48 sphingomyelin 16:0 (SPM; Figure 2), 48 dioleoylphosphatidylcholine (DOPC; Figure 2), and 32 cholesterol molecules; and (3) 44 DLiPC, 60 dilineoylphosphatidylethanolamine (DLiPE, di-18:2-PE; Figure 2) and 24 dilineoylphosphatidylserine (DLiPS, di-18:2-PS, anionic; Figure 2) molecules. Each bilayer was simulated using 150 mM NaCl, 150 mM KCl, or 75 mM CaCl concentrations. The initial configurations of all bilayers were equilibrated in our previous


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studies.4 The systems were hydrated with ~11,500 water molecules and 20 dopamine molecules (~100 mM) in ionized form were randomly inserted into the water phase. The NT concentrations in the presynaptic vesicles are reportedly even higher (~250 mM).38 The used interbilayer distance of ~8 nm was three times larger than the thickness of a layer of water commonly observed in multilamellar liposomes (up to 30 water per lipids compared to 90 used here) and provided a ~4 nm thick slab of water not affected/ordered by the interface. The OPLS all-atom force field was used to parameterize all molecules.39 For lipids, additional parameters specifically derived for saturated and unsaturated hydrocarbons, glycerol backbone, sphingosine, and head group were used.40,41,42,43 Partial charges for dopamine and serotonin were calculated in our previous studies.8 For water, we employed the TIP3P model that is compatible with the OPLS parameterization.44 Topologies and corresponding structure files for dopamine, DLiPC, DLiPE, and DLiPS are provided in the Supporting Information. Topology and structure files for DOPC are given in the Supporting Information of ref. 43, and for cholesterol and SPM as well as force field parameters file in the Supporting Information of ref. 42. MD simulations were performed using the GROMACS software package.45,46 Periodic boundary conditions with the usual minimum image convention were used in all three directions. The LINCS algorithm was used to preserve the length of each hydrogen atom covalent bond.47 The time step was set to 2 fs and the simulations were carried out at a constant pressure (1 bar) and temperature (310 K). The temperature and pressure were controlled by the V-rescale and Parrinello-Rahman methods, respectively.48,49 The temperatures of the solute and solvent were controlled independently. For pressure, we used a semi-isotropic control. The Lennard-Jones interactions were cut off at 1.0 nm, and for the electrostatic interactions we employed the particle mesh Ewald method with a real space cutoff of 1.0 nm, beta-spline interpolation (order of 6), and direct sum tolerance of 10-6.50 Equilibration of the bilayer systems was reached after 50-250 ns of MD simulation. This time was determined by observing dopamine adsorption to the membrane interface, reflected in the number of hydrogen bonds that these molecules established with the lipids. The averaged (equilibriated production) results were acquired from the subsequent 250 ns trajectories (Table 1). GROMACS input file (md.mdp) containing all simulation parameters is provided in the Supporting Information. The free energy profile for the potential of mean force (PMF) was calculated for the dopamine molecule by the umbrella sampling method51 using the GROMACS tool g_wham.52 The PMF was obtained using 45 sampling windows with distances of 0.1 nm between each



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window in the direction of the bilayer normal. The distances shown in the profiles are between the center of mass (COM) of the bilayer and the COM of dopamine. Equilibration times of 20 ns were used with respective 80-ns production simulations. Comprehensive review of free energy calculations of small molecules partitioning into lipid bilayers is given in ref. 53.


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AUTHOR INFORMATION Corresponding author: Email: [email protected] Author Contributions Simulations were prepared by SM, SR, and PAP; simulations were performed by SM; analysis was performed by SM, SR, and HJ; manuscript was written by TR, PAP, and IV; the study was designed and supervised by IV and TR, with TR leading. Funding This work was funded by the Academy of Finland (Center of Excellence in Biomembrane Research), European Research Council (Advanced Grant project CROWDED-PROLIPIDS), and the Paulo Foundation. Notes Authors declared no conflict of interest. ACKNOWLEDGMENTS. For computational resources, we wish to thank the CSC–IT Center for Science (Espoo, Finland). ABBREVIATIONS COM, center of mass; DLiPE, dilineoylphosphatidylethanolamine; DLiPC, dilineoylphosphatidylcholine; DLiPS, dilineoylphosphatidylserine; DOPC, dioleoylphosphatidylcholine; GABA, γ-aminobutyrate; MD, molecular dynamics; NTs, neurotransmitters; NMR, nuclear magnetic resonance; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol, PS, phosphatidylserine; PIP, phosphatidylinositol; Chol, cholesterol; PMF, potential of mean force; SPM, sphingomyelin.



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Figure 1. Synaptic neurotransmission models and a presynaptic vesicle. a) Membrane-independent model:8 1) hydrophilic neurotransmitters (NTs) move out of the presynaptic vesicle; 2) NTs carry out 3D diffusion across the synaptic cleft without aggregation on the membrane-water interface; and 3) they bind into their receptors’ extracellular binding sites. b) Membrane-dependent model:8 1) hydrophobic NTs move out of the presynaptic vesicle; 2) they undergo 3D diffusion across the cleft; 3) NTs bind to the postsynaptic membrane surface, where they carry out lateral 2D diffusion in the membrane plane; 4) NTs bind into their receptors’ membrane-buried binding sites. c) The inner leaflet of the presynaptic vesicle lipid bilayer is mainly composed of neutral phospholipids (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)), while the highly anionic phosphatidylinositol (PIP) lipid is solely found in the outer leaflet.18,19 Divalent cations (Ca2+ and Zn2+) and protons (H+) are actively pumped inside the vesicle.20 The cations aggregate on the membrane head group region and prevent even the most hydrophobic NTs such as dopamine from binding to the inner leaflet. The vesicular pH remains low at 5.6 due to H+ pumps.20 This pH is close to the pKa value of the carboxylic groups of phosphatidylserine (PS), thus PS remains mostly neutral. 70x30mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Figure 2. Lipids and lipid bilayers used in the simulations. On the left are shown the structures of the lipids and dopamine simulated in this work. On the right are depicted snapshots of final configurations of all simulated lipid bilayers. Color code: dopamine (red CPK models); Na+ (white spheres); K+ (yellow spheres); Ca2+ (orange spheres); and lipids (cyan licorice models).


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Figure 3. Density profiles for dopamine and each simulated cation species (a and b) in the PC bilayer, (c and d) the PC-PE-PS bilayer, and (e and f) in the SPM-PC-Chol membrane. Also shown is the average position of the phosphorous (P) atoms of PCs (gray line). The membrane depth of zero corresponds to the position of the center of the lipid bilayer. Cations are shown in panels b, d, and f and dopamine in panels a, c, and e. Color code: black (bilayers with Na+); red (bilayers with K+); blue (bilayers with Ca2+); green (bilayers without cations; referred as NONE). 295x319mm (300 x 300 DPI)



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Figure 4. Free energy profiles of dopamine translocation from the water phase to the center of the PC bilayer (a), the PC-PE-PS bilayer (b), and the SPM-PC-CHOL bilayer (c). At the membrane depth of zero is the center of the lipid bilayer and the bulk water phase begins at 2.5-3.0 nm. The color code: black (bilayers with Na+); red (bilayers with K+); blue (bilayers with Ca2+); green (bilayers without cations). The positions of the vertical gray-dashed lines, indicating the locations of the membrane-water interfaces, differ between membrane models due to lipid composition differences. Error bars are based on the boot strap analysis method and have been calculated in relation to the point located in the water phase. Curves starting from bilayer center are shown in the Supporting Information (Figure S2). 158x312mm (300 x 300 DPI)


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Calcium Assists Dopamine Release by Preventing Aggregation on the

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