Synthesis
We synthesized ANNINE-6plus from a precursor of ANNINE-6 (Hübener et al. 2003). 11-Di-n-butylamino-3-aza-benzo[m] picene (0.2 g, 0.44 mM) and (3-bromopropyl)-trimethyl-ammonium bromide (Aldrich, 0.57 g, 2.2 mM) were dissolved in 3 ml DMF and heated for 9 h at 120°C. Upon cooling, Et2O (50 ml) was added to precipitate 11-Di-n-butylamino-3-(3-trimethyl-ammonium-propyl)-3-azonia-benzo[m] picene dibromide. After decanting the supernatant, the product (MW 717.6 Da) was purified by column chromatography (SiO2, 42–105 μm, YMC, with CHCl3:MeOH:H2O, 50:20:4) with a yield of 40 mg (12.6%, red solid, m.p. > 220°C). It was identified by 1H NMR and mass spectroscopy.
Solubility
To evaluate the saturation concentration of ANNINE-6 and ANNINE-6plus an excess of dye powder in dioxane/water mixtures was stirred for 6 h at room temperature. After centrifugation, the dye concentration was determined by light absorption at a maximum around 440 nm (Cary 3E, Varian). An extinction coefficient of 25,000 M−1 cm−1 was determined from a defined concentration in ethanol.
Membrane binding
We studied the membrane binding of ANNINE-6plus by fluorescence titration with lipid vesicles (Bashford et al. 1979; Fromherz and Röcker 1994; Hinner et al. 2004). The vesicles were made from 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC, Lipoid KG, Ludwigshafen, Germany) by extrusion (MacDonald et al. 1991) through a polycarbonate filter with 100 nm pore size (Avestin Europe, Mannheim, Germany) in Tris–NaCl buffer (20 mM Tris, 100 mM NaCl, pH 8.1). The lipid concentration was determined by a chromogenic enzyme assay (Biomerieux, Marcy l´Etoile, France). Vesicle size was checked by quasielastic light scattering. Suspensions of vesicles were prepared with lipid concentrations from 40 nM to 2 mM. They were mixed with an equal volume of 1 μM ANNINE-6plus in Tris–NaCl in 2 ml plastic tubes (Eppendorf, Hamburg, Germany). After sonication for 30 min (Sonorex RK 510H, Bandelin, Berlin, Germany), the fluorescence was recorded at 600 nm with an excitation at 488 nm (SLM Amino 8100, Acton Research, Acton, MA) at 25°C). The increase of fluorescence intensity with increasing lipid concentration was evaluated in terms of a binding isotherm
\( {{c_{\text{M}} } \mathord{\left/ {\vphantom {{c_{\text{M}} } {c_{\text{D}} }}} \right. \kern-\nulldelimiterspace} {c_{\text{D}} }} = \left[ {1 + \left( {{1 \mathord{\left/ {\vphantom {1 {K_{{\text{WM}}} c_{\text{L}}^{} }}} \right. \kern-\nulldelimiterspace} {K_{{\text{WM}}} c_{\text{L}}^{} }}} \right)} \right]^{ - 1} \) with the concentration c
M of dye bound to the membrane, with a total dye concentration c
D, with a lipid concentration c
L and a binding constant K
WM (Fromherz and Röcker 1994; Hinner et al. 2004). That relation has the form of a mass action law, but does not imply a 1:1 molecular association. It reflects a partitioning equilibrium of the dye between water and membrane (Fromherz and Röcker 1994). We assumed that only the outer monolayer of the vesicles was effective in dye binding.
Staining
The staining solutions for giant lipid vesicles and for HEK293 cells with a dye concentration of 5 μg/ml were prepared from stock solutions of 0.5 mg/ml either with ANNINE-6plus in pure water (Millipore) or with ANNINE-6 in DMSO containing 20% Pluronic F127 (Molecular Probes). Giant vesicles were made by electroswelling from 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) in 300 mM aqueous saccharose (Dimitrov and Angelova 1988; Hinner et al. 2004), and transferred to polypropylene culture dishes (BD Biosciences) containing ANNINE-6 or ANNINE-6plus at a concentration of 5 μg/ml in 300 mM glucose. After sedimentation for around 10 min, fluorescence images were taken with a microscope (Axioskop, 63× water immersion objective, Zeiss) equipped with a CCD camera (Sony ICX 285, Theta Systems, Germany). Excitation was at 450 nm with a bandwidth of 50 nm and detection was beyond 515 nm. HEK293 cells (DSMZ, Braunschweig, Germany) were cultured at 37°C and 5% CO2 in Dulbecco’s modified eagle medium with 10% fetal calf serum and l-glutamine (Gibco). Before imaging, cells were washed with physiological buffer (5.4 mM KCl, 135 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 5 mM HEPES, pH 7.3), incubated for 5 min in buffer containing the dye at a concentration of 5 μg/ml, washed twice in buffer and transferred to the microscope.
Voltage sensitivity
The measurements of voltage sensitivity followed the procedure described by Kuhn and Fromherz (2003). In short, Retzius neurons were dissociated from the ganglia of Hirudo medicinalis and held in defined culture medium on the stage of a fluorescence microscope. After staining, defined intracellular voltages were applied with a whole-cell patch pipette. Both the wavelength of excitation and of emission was varied. The normalized fluorescence intensity F/F
MAX at an intracellular voltage of −50 mV were measured as well as the relative change of fluorescence in response to a voltage step of 100 mV, i.e. the voltage sensitivity defined by S
V
= (ΔF/F)/100 mV. The data are plotted versus the wavenumber of excitation and emission that reflect the energy difference between excited state and ground state that is affected by the electrical field.