Evidence for ATP Interaction with Phosphatidylcholine Bilayers

Jul 10, 2019 - ATP is a fundamental intracellular molecule and is thought to diffuse freely throughout the cytosol. Evidence obtained from nucleotide-...
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Evidence for ATP Interaction with Phosphatidylcholine Bilayers Alvaro Garcia,*,† Simon Pochinda,‡ Paninnguaq N. Elgaard-Jørgensen,‡ Himanshu Khandelia,‡ and Ronald J. Clarke§,∥ †

School of Life Sciences, University of Technology Sydney, Ultimo, NSW 2007, Australia PHYLIFE: Physical Life Sciences at SDU, Department of Physics, Chemistry and Pharmacy and MEMPHYS: Center for Biomembrane Physics, University of Southern Denmark, DK-5230 Odense M, Denmark § School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia ∥ The University of Sydney Nano Institute, Sydney, NSW 2006, Australia Downloaded via UNIV AUTONOMA DE COAHUILA on July 22, 2019 at 08:12:13 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.



S Supporting Information *

ABSTRACT: ATP is a fundamental intracellular molecule and is thought to diffuse freely throughout the cytosol. Evidence obtained from nucleotide-sensing sarcolemmal ion channels and red blood cells, however, suggest that ATP is compartmentalized or buffered, especially beneath the sarcolemma, but no definitive mechanism for restricted diffusion or potential buffering system has been postulated. In this study, we provide evidence from alterations to membrane dipole potential, membrane conductance, changes in enthalpy of phospholipid phase transition, and from free energy calculations that ATP associates with phospholipid bilayers. Furthermore, all-atom molecular dynamics simulations show that ATP can form aggregates in the aqueous phase at high concentrations. ATP interaction with membranes provides a new model to understand the diffusion of ATP through the cell. Coupled with previous reports of diffusion restriction in the subsarcolemmal space, these findings support the existence of compartmentalized or buffered pools of ATP.



INTRODUCTION Adenosine triphosphate (ATP) is fundamental to essential processes in all cells. These include its role as an integral building block in DNA and RNA synthesis and as the energy source for a multitude of enzymatic processes. Since, in animal cells, ATP is mainly produced in the mitochondria, it is generally assumed that once ATP is liberated out of the mitochondria it freely diffuses throughout the cell1 to maintain a cytosolic concentration ranging between 2 and 10 mM. Investigations into the homogeneity of the ATP concentration in the cytosol have led to the postulation of some form of cytosolic diffusion barrier limiting the free diffusion of ATP.2−5 Circumstantial evidence for a cytoplasmic barrier has been reported from functional studies of nucleotide-sensing ion channels.3 As an example, the ATP-dependent K+ (KATP) ion channel at the plasma membrane is significantly inhibited by millimolar concentrations of ATP with a measured IC50 of between 20 and 200 μM.6 Thus, it is predicted that under the presumed cytosolic concentration of ATP of 2−10 mM, this channel would very rarely be observed in the open state. However, not only has it been reported to open but also has © XXXX American Chemical Society

been reported to sense changes in ATP concentration originating from altered cellular metabolism.7 These observations are difficult to reconcile with the concept of a homogeneous cytosolic ATP concentration and free diffusion. To account for these observations based purely on the hypothesis that the subsarcolemmal space restricts diffusion of ATP, one would need to assume a reduction in the ATP diffusion coefficient relative to its value in water by 5 orders of magnitude.2,8 Since this seems very unlikely, some physical barriers have been postulated to reduce free diffusion of adenine nucleotides through the cytosol.8−17 These include macromolecular crowding of the cytosolic compartment, tortuosity of cytoskeletal structures, and ATP binding to cytoskeletal proteins in a manner that does not induce hydrolysis. Although plausible, the physical barrier hypothesis cannot explain results reported from giant excised patches, which demonstrate coupling of Na+, K+-ATPase activity with Received: April 29, 2019 Revised: July 7, 2019 Published: July 10, 2019 A

DOI: 10.1021/acs.langmuir.9b01240 Langmuir XXXX, XXX, XXX−XXX

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Langmuir alterations in KATP channel activity.18 In these experiments, the use of giant excised patches essentially removes any effective compartmentalization by cytoskeletal structures or macromolecular crowding. This report indicates that a component within the membrane proteins concerned or their associated phospholipid bilayer has the ability to modulate the apparent local concentration of ATP. Molecular interactions between phospholipid bilayers and RNA have been reported previously,19−21 and nucleic acidphospholipid complexes are used to improve DNA/RNA transfection efficiency, with the complexation driven by interaction between positively charged residues of lipid headgroups and the negatively charged phosphate backbone.22 Given the negative charge of the ATP phosphate groups at physiological pH, we decided to investigate the possibility that ATP itself may associate with phospholipid bilayers.



Na2HPO4/NaCl were prepared in the Tris.EDTA buffer and pH was adjusted to 7.2 with NaOH or HCl. 100 μL of the DOPC or DOPC/ POPS liposomal preparation was added to 900 μL of buffer. RH421 was added for a final concentration of 250 nM. The temperature of the cuvette holder was thermostatically controlled to 24 °C. Electrical Impedence Spectroscopy of Tethered Membranes. Tethered Membrane Formation. Tethered bilayer lipid membranes (tBLMs) were made using preprepared tethered benzyldisulfide ethylene glycol T10 coated gold slides including 10% eightoxygen-ethylene-glycol reservoir linkers with a C20 phytanyl group as tethers and 90% four-oxygen-ethylene-glycol reservoir linkers with a terminal OH group as spacers (SDx Tethered Membranes, Sydney, Australia). A lipid bilayer was formed over the monolayer using a solvent-exchange technique that employs ethanol and aqueous buffer. Briefly, 3 mM solution of a mobile lipid phase (70% C20 diphytanylether-glycero-phosphatidylcholine lipid/30% C20 diphytanyl-diglyceride ether as provided by SDx Tethered Membranes or DOPC or DOPC/POPS mixture) dissolved in ethanol/methanol was added to the flow-cell chamber. After ∼2 min of incubation at room temperature, the flow-cell chamber was rinsed with 300 μL of Tris.EDTA buffer. For a more detailed description of this method, refer to Cranfield et al.23 The formation of the tBLMs was tested using a tethaPod (SDx Tethered Membranes) operating as described in the AC impedance spectroscopy section below. Typical results obtained for a lipid membrane were 0.8−1.2 μF cm−2 and a conduction of 0.3−0.5 μS for a 2.1 mm2 electrode at room temperature with a pH 7.2. AC Impedance Spectroscopy. The conductance and capacitance of the tethered membrane were determined using a tethaPod conductance reader (SDx Tethered Membranes), employing realtime modeling of the tBLM while operating as a swept-frequency ratiometric impedance spectrometer. A sequential 25 mV excitation was applied over frequencies from 10000 to 0.125 Hz. Buffers containing ATP/adenosine/NaH2PO4 were added in increasing concentrations. All buffers were maintained at a pH of 7.2 with either NaOH or HCl. All buffers were maintained at room temperature to minimize temperature dependent effects on the phospholipid bilayer properties. Each buffer solution was allowed to equilibrate with the membrane for 10 min before subsequent buffers were added. For a more detailed description of AC impedance spectroscopy methods used, refer to Cranfield et al.23 Differential Scanning Calorimetry. The samples used for the DSC experiments were prepared by dissolving the appropriate amount of DMPC in 2 mL of chloroform. Chloroform was then removed using a rotary evaporator (Büchi, Flawil, Switzerland), initially at 474 mbar at 40 °C until sample became viscous (approximately 1 h) and then for 2 h at 10 mbar at 40 °C to produce a lipid film. The film was rehydrated with Tris.EDTA buffer (150 mM NaCl, 30 mM Tris, 1 mM EDTA at pH 7.2) by sonication. In experiments using ATP, Na2.ATP was added to the Tris.EDTA and pH was titrated to 7.2 using NaOH. The dispersion of the DMPC multilamellar vesicles was then degassed, and 700 μl aliquots were withdrawn for DSC analyses. DSC heating and cooling thermograms were obtained at a scan rate of 10 °C/h using a high-sensitivity MicroCal VP-DSC (Malvern Instruments, Malvern, U.K.). The data acquired were analyzed with the Origin 7.5 software package (OriginLab Corporation, Northampton, MA) and plotted with Graphpad Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, U.S.A.). Molecular Dynamics Simulations. Simulations of ATP Binding to DMPC. We performed three simulations: a pure 128-lipid DMPC bilayer, a DMPC bilayer with 16 ATP molecules and 64 Na+ counterions, and a DMPC bilayer with 32 ATP molecules and 128 Na+ counterions. The simulations will henceforth be referred to as DMPC, DMPC-Na+-16ATP, and DMPC-Na+- 32ATP. The simulations were carried out for 80, 1000, and 600 ns, respectively. The DMPC lipid bilayer was constructed using the CHARMM-GUI Membrane Builder,24 and the coordinates of the ATP molecule (adenosine-5′-triphosphate) were downloaded and ready for use from the RCSB Protein Data Bank.25 Each setup was solvated with water with a hydration number of 70, approximately 8900 water molecules.

EXPERIMENTAL SECTION

Reagents. N-(4-Sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)pyridinium salt (RH421) was obtained from Molecular Probes (Eugene, OR) and was used without further purification. The origins of the various reagents used were as follows: Tris-base (≥99%; Sigma, Castle Hill, Australia), NaCl (suprapure; Merck, Kilsyth, Australia), EDTA (99%; Sigma), Na2ATP·3H2O (special quality; Boehringer-Mannheim, Mannheim, Germany, or Roche, Castle Hill, Australia), Adenosine (≥99%; Sigma, Castle Hill, Australia), NaH2PO4 (≥99%; Sigma, Castle Hill, Australia), 1,2dioleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (DOPC, DMPC and POPS both ≥99%; Avanti Lipids, Alabama, U.S.A.), 70% C20 diphytanyl-ether-glycero-phosphatidylcholine lipid/ 30% C20 diphytanyl-diglyceride ether mix (SDx Tethered membranes, Roseville, Australia), NaOH (analytical grade; Merck), and HCl (0.1 N Titrisol solution; Merck). Liposomes. DOPC liposomes were produced via the ethanol injection method. Briefly, 1,2-dioleoyl-sn-glycero-3-phosphocholine was dissolved in ethanol and slowly injected into an excess of Tris.EDTA buffer (NaCl 130 mM, Tris 30 mM, EDTA 1 mM, pH 7.2) to obtain a final concentration of 3 mM DOPC. The ethanol concentration was reduced by dialysing against the Tris.EDTA buffer for 72 h. Mixed liposomes (80% DOPC/20% POPS mol %) were produced using the extrusion method. Briefly, DOPC and POPS were dissolved in chloroform and mixed to produce an 80% DOPC/20% POPS mixture. The chloroform was removed from the sample via rotary evaporation under a vacuum for 1 h at maximum rotation speed using an R-114 Rotavapor (Büchi, Flawil, Switzerland) with the thermal bath at a temperature of approximately 40 °C and the vacuum maintained at 474 mbar using a V-850 vacuum controller (Büchi). This formed a thin lipid film on the walls of a round-bottom flask. After no visible traces of chloroform could be detected in the flask, the resulting film was dried for a further 2 h at 10 mbar. Multilamellar liposomes were then formed at 3 mM by resuspending the lipid film in 5 mL of buffer containing 30 mM Tris, 130 mM NaCl, and 1 mM EDTA (adjusted to pH 7.2 using HCl). This multilamellar preparation was then extruded through a polycarbonate membrane with a pore size of 0.1 μm to produce a unilamellar liposome preparation (mini-extruder kit; Avanti Lipids, Alabama, U.S.A.). RH421 Steady State Fluorescent Measurements. Steady state fluorescence measurements were recorded with a Shimadzu RF5301PC fluorescence spectrophotometer. To minimize contributions from scattering of the exciting light and higher order wavelengths, a glass cutoff filter was used in front of the emission monochromator. The fluorescence emission was measured at an emission wavelength of 670 nm (+RG645 glass cutoff filter, Schott, Mainz, Germany). The fluorescence excitation ratio, R, is defined as the ratio of the fluorescence intensity at an excitation wavelength of 445 nm divided by that at 570 nm. Varying concentrations of ATP/adenosine/ B

DOI: 10.1021/acs.langmuir.9b01240 Langmuir XXXX, XXX, XXX−XXX

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Langmuir

Figure 1. RH421 excitation spectra shifts in unilamellar liposomes. (a) The relative change in F445/570 ratio for ATP (●; N = 23 or more for each group) and adenosine (○; N = 23 or more for each group) for DOPC unilamellar liposomes. (b) The relative change in F445/570 ratio for ATP (N = 5) for 80% DOPC/20% POPS (mol %) unilamellar liposomes. (c) The relative change in F445/570 ratio with the addition of NaH2PO4 (N = 7 or more for each group). (d) The relative change in F445/570 ratio with the addition of extra NaCl (N = 7 or more for each group). Errors represent ± s.e.m. from replicates (parts a, b, c, d). * indicates a P value of 500 ns) without binding the surface of the DMPC bilayer. The area per lipid, thickness, and the order parameters of the bilayer are thus not affected (data not shown). It is possible that the aggregate binds to the bilayer surface in longer simulations, and individual molecules of ATP can diffuse into the headgroup region. However, this possibility cannot be tested in the current set of simulations. To further characterize the ATP aggregate, we ran a simulation of 30 molecules of ATP with Na+ counterions and water, in the absence of a membrane. We find that all ATP molecules self-assemble in a single aggregate on a time scale of 100 ns (Figure S4A,C), driven by the coordination of ATP phosphate groups by Na+ ions (Figure S4A,D) and by the stacking of bases reminiscent of DNA (Figure S4B,E). We quantify the interactions between different molecular species using radial distribution functions, which are normalized histograms of distances between a pair of atom selections. The strong interactions between the triphosphate and Na+ are apparent in the sharp peak between 2 and 2.5 Å. The packing between nucleotide bases is quantified in Figure S4E, while the phosphate−phosphate interactions are quantified in Figure S4F. To calculate the G

DOI: 10.1021/acs.langmuir.9b01240 Langmuir XXXX, XXX, XXX−XXX

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Langmuir conditions, this may further enhance the apparent diffusion restriction of ATP. The evidence that ATP interacts with zwitterionic phospholipid bilayers could lead to the erroneous assumption that the phospholipid bilayer has the capacity to cause large concentration gradients in the subsarcolemmal space by sequestering “free” ATP to the bilayer surface. The MD simulations indicate that ATP has the same affinity for the bilayer interface as it does for the bulk. Under these conditions, membrane binding of ATP would simply be equivalent to a small increase in the available volume through which ATP can diffuse (i.e., an increase given by the membrane interfacial volume within which ATP can bind). This clearly would be unable to account for the apparent concentration that is estimated from the activity of the KATP ion channel in cells.6,7 However, this interaction becomes significant if the association of ATP with the membrane surface exacerbates the diffusion restriction previously observed in the cytosol.3,5 Compartmentalisation of the cytoplasm is considered an important component of the functional biochemistry of the cell,50−52 which is associated with the restricted diffusion of biological macromolecules.53,54 The large phospholipid bilayer surface area associated with intracellular organelles could greatly enhance diffusion restriction and compartmentalization if the kinetics of ATP dissociation from the membrane surface are either slower than or on a similar time scale to diffusion of ATP within the cytoplasm. These results demonstrate a new interaction, which has significant implications in understanding ATP diffusion in the cytosol. The complexity of the composition of the membranes, both of plasma and that of the intracellular organelles, means that further investigation is required to assess whether the endogenous heterogeneity of the membrane could modulate the strength of the interaction reported. If we consider the orientation of the ATP in the bilayer with the nucleobase close to the acyl chains and the triphosphate in between the phospholipid headgroups, the inclusion of a negatively charged lipid could repel ATP from the interface. We report here that the ATP interaction with the bilayer is still measurable with 20% POPS, which provides a comparable surface charge to the one attributed to the inner leaflet of the plasma membrane.55 This provides evidence that the presence of negatively charged lipids does not negate this interaction. Finally, with regard to the reported compartmentalization of biological macromolecules,51 which demonstrates a significant concentration gradient in the cytosol, one should consider the potential of association to intracellular membrane surfaces as a factor in their diffusion restriction. The large surface area of intracellular organelles provides ample opportunity for these interactions to occur, and the endogenous heterogeneity of these membranes could in turn selectively restrict the diffusion of specific macromolecules.





of umbrella sampling windows, ATP aggregation in the aqueous phase (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Alvaro Garcia: 0000-0002-1159-4567 Himanshu Khandelia: 0000-0001-9913-6394 Ronald J. Clarke: 0000-0002-0950-8017 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We would like to acknowledge Dr. Paul Fitzgerald and Dr. Paul Duckworth for their helpful discussions regarding the analysis of the electrical impedance spectroscopy data. A.G. acknowledges with gratitude financial support from the UTS Chancellors Postdoctoral Research Fellowship Scheme. R.J.C. acknowledges with gratitude financial support from the Australian Research Council (Discovery Grants DP121003548, DP-150101112, and DP170101732). H.K. is supported by Lundbeckfonden. The simulations were carried out on the Danish e-Infrastructure Cooperation (DeiC) National HPC Center, ABACUS 2.0 at the University of Southern Denmark, SDU as well as on computing resources on the Swiss cluster Piz Daint as part of the PRACE grant number 2016153468.



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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.langmuir.9b01240. RH421 Excitation spectra in DOPC vesicles, measurements of conductance of tethered diphytanyletherphosphatidylcholine/glycerodiphytanylether bilayers using electrical impedance spectroscopy, histogram overlap H

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DOI: 10.1021/acs.langmuir.9b01240 Langmuir XXXX, XXX, XXX−XXX