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A Fluorescence Lifetime Imaging Microscopy (FLIM) Supported Investigation on Temperature Dependent Penetration of Dopamine in DMPG Lipid Bilayer Shrabanti Das, and Pradipta Purkayastha Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01173 • Publication Date (Web): 29 Jun 2017 Downloaded from http://pubs.acs.org on June 30, 2017
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A Fluorescence Lifetime Imaging Microscopy (FLIM) Supported Investigation on Temperature Dependent Penetration of Dopamine in DMPG Lipid Bilayer Shrabanti Das, and Pradipta Purkayastha* Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur 741246, India.
ABSTRACT
Distribution of dopamine, an essential neurotransmitter in mammalian central and peripheral nervous system, in lipid bilayer and at the surface of DMPG vesicles has been studied herein. To track the progress of dopamine through different regions of the lipid vesicle, the vesicles were synthesized using 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) labeled phospholipid molecules either tagged to the head group (NBDPE) or the acyl chain (NBDPG). Dopamine induced quenching of NBD fluorescence in the lipid vesicles demonstrates that dopamine has a preference to diffuse into the lipid bilayer. Change in the excited state lifetime obtained for
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NBDPG clearly indicates the preference in dopamine binding. The propositions were supported by fluorescence lifetime imaging microscopy (FLIM).
1. INTRODUCTION Dopamine is a naturally occurring neurotransmitter in sympathetic nervous system and transports signals between cells.1 Even a slight abnormality in dopamine signaling may lead to several neurological diseases, such as, Parkinson’s disease and schizophrenia.2,3 Proper functioning of neurotransmitter at the synapse of two neurons plays crucial role in signal transduction. Thus, it is extremely important to understand the behavior of bio-organic neurotransmitters in biological fluid for proper diagnosis of hypertension, multiple sclerosis, Perkinson’s disease and other neuro-disorders.4 Although, lot of studies on vesicle fusion at the presynaptic membrane,5,6 exocytosis7 and signal transduction were performed, only a few reports are concerned to interaction of neurotransmitters with other macromolecules present in the synaptic cleft such as biomembranes. A series of neurotransmitters are found in biological media in the form of amino acids (glutamate, aspartate, GABA, D-serine, glysine), peptides (somatostatin, cocaine and amphetamine regulated transcript, opioid peptides), monoamines (dopamine, epinephrine, nor epinephrine, serotonin, histamine) and other small molecules (adenosine, acetylcholine, anadamide), etc. In this particular work we have chosen dopamine and its precursor L-dopa (as control) to study interaction with negatively charged lipid bilayer. The dopamine molecule, with a net positive charge (–NH3+) functions at biological pH ~7 and L-dopa contains a negatively charged carboxyl group.8
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Although anionic phosphatidylglycerol (PG) constitutes a minor part of the total lipid matrix of the eukaryotic biomembrane, it can act as both H-bond acceptor and donor. PG also plays an important role in stabilizing the membrane, controlling membrane peptide/protein interactions and maintaining the physiological pH of outer membrane surface.9,10 Hence, we chose 1,2ditetradecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) to prepare liposomes to study their interaction with dopamine. Moreover different parts of the lipid molecules (namely, head or tail) were tagged with a special marker called 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). This compound is applied in biology since its discovery. Fluorescence of NBD is highly sensitive to its environment. It is moderately fluorescent in aprotic solvents but almost non-fluorescent in aqueous medium.11 The 6-carbon and 12-carbon fatty acid analogs of NBD and the phospholipids derived from these probes tend to sense the lipid–water interfacial region of membranes instead of the hydrophobic interior.12 Hence, the environmental sensitivity of NBDlabeled fatty acids is exploited to probe the ligand-binding sites of fatty acid.13 Although NBD is non-fluorescent in water but can act as an excellent fluorescent probe while attached to lipid molecules as its fluorescence lifetime is highly sensitive to environmental polarity.14-16 The location of the NBD group in acyl chain-labeled lipids is very important as it can loop up to the membrane interface above the phase transition temperature of the host matrix due to its polarity sensitivity.17,18 This looping up can be explained by the hydrogen bonding affinity of the amino group and the oxygen atoms with interfacial water molecules or polar head groups.17 NBD is also known to be a selective probe for dopamine receptors.19 In this report, we have utilized the fluorescence property of NBD tagged to the head group (NBDPE) or end of the acyl chain (NBDPG) of DMPG (Scheme 1). The looping up of the NBD group in fluid phase of the host DMPG matrix acts as a marker to study the temperature
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dependent binding affinity of dopamine to anionic phosphatidylglycerol membrane. Earlier, theoretical and experimental studies showed that dopamine interacts superficially with anionic lipid membranes without penetrating into the bilayer.20,21 However, very recently, Matam et al. have shown by NMR spectroscopy that dopamine can bind as well as penetrate through lipid bilayer composed of Phosphatidylcholine (PC) and Phosphatidylserine (PS).22 This work is a seminal discovery on binding of dopamine within the hydrophobic layer as well as at the interfacial region of DMPG liposome. Fluorescence lifetime imaging microscopy (FLIM), applied to the NBD tagged giant unilamellar vesicles (GUV) has provided more evidence for the dopamine affinity inside the bilayer. To our knowledge, this is the first report on temperature dependent tracking of dopamine penetration in liposomes using fluorescence spectroscopy and lifetime imaging.
2. EXPERIMENTAL SECTION 2.1. Materials. Dopamine hydrochloride, L-dopa and 1,2-dimyristoyl-sn-glycero-3-phosphorac-(1-glycerol) (DMPG) sodium salt were purchased from Sigma Aldrich. 1,2-dimyristoyl-snglycero-3-phosphoethanolamine-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBDPE) (ammonium salt) and 1-myristoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero3-[phosphor-rac-(1-glycerol)] (NBDPG) (ammonium salt) were bought from Avanti Polar Lipids. Structure of NBDPE and NBDPG differs in the position of the NBD group as tagged on the head and the acyl chain of the lipids, respectively (Scheme1). Structures of dopamine hydrochloride and L-dopa are also shown in the same scheme. Stock solution of NBDCl was prepared in DMSO (1 mM). HPLC water was used in all experiments.
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Scheme 1. Upper panel: Molecular structures of NBDPE (top) and NBDPG (bottom) and lower panel: molecular structures of dopamine hydrochloride (left) and L-dopa (right). 2.2. Synthesis of Lipid Vesicles. Giant lipid vesicles (GUV) were prepared as reported elsewhere.23 Briefly, weighed amount of DMPG containing 1 mol% of NBDPE or NBDPG as required, was dissolved in chloroform (0.1 M) and 20 µL of this solution was added to a solution of 980 µL chloroform and 100-200 µL of methanol. The aqueous phase (5 mL of Tris buffer) was then carefully added along the walls of the flask. The organic solvent was removed by rotary evaporator under reduced pressure (60 mm Hg) at 40oC and 40 rpm. Due to different boiling points of Chloroform (61oC) and methanol (64oC) we observed two major boiling events. After evaporation for about 2 mins an opalescent solution was obtained containing high concentration of GUV. The NBDPE and NBDPG lipid vesicles were unilamellar and the size distribution is between 70 to 100 µm. Images of the synthesized lipid vesicles are provided in Figure S1. For spectroscopic measurements the samples were diluted 10 times in deionized water in order to stop light scattering. The concentration of NBD tagged liposomes used in all experiments was 0.05 mM.
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2.3. Instrumentation. The absorption and emission spectra were collected using U-2900 spectrometer from Hitachi and QM-40 spectrofluorimeter from PTI, respectively. All slit widths used for measurements with U-2900 and QM-40 spectrometers are 5 nm. The excitation wavelength was 470 nm. For absorption spectral measurements the optical wavelength regime was ~400 to ~550 nm and for fluorescence spectral measurements the optical wavelength regime was ~480 nm to ~700 nm. Time resolved fluorescence decay measurements were performed by the time correlated single photon counting (TCSPC) technique in a life time measurement system from Jobin Yvon using NanoLED-03 at 450 nm (temporal pulse width
