Di-8-ANEPPS

Di-8-ANEPPS emission spectra in phospholipid/cholesterol membranes: a theoretical study

We have investigated the effects of explicit molecular interactions and the membrane dipole potential on the absorption and emission spectra of a widely used fluorescent probe, di-8-ANEPPS, in a dipalmitoylphosphatidylcholine (DPPC) and a mixed DPPC/cholesterol membrane bilayer. Ground-state and excited-state geometries were calculated with the complete active space self-consistent field (CASSCF) method. Interactions with up to 260 atoms of the membrane bilayer were explicitly incorporated using a decoupled quantum mechanics/molecular mechanics (QM/MM) approach, utilizing recent advances in time-dependent density functional theory (TDDFT). We find that no specific molecular interactions affect the fluorescence of di-8-ANEPPS; rather, the magnitude of the membrane dipole potential is key to the shifts observed in both of the two lowest excited states.

Stimulatory actions of di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), voltage-sensitive dye, on the BKCa channel in pituitary tumor (GH3) cells

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH(3) cells. In whole-cell configuration, di-8-ANEPPS (10 microM) reversibly increased the amplitude of Ca(2+)-activated K(+) current. In inside-out configuration, di-8-ANEPPS (10 microM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels with an EC(50) value of 7.5 microM. This compound caused a left shift in the activation curve of BK(Ca) channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BK(Ca) channels. However, neither voltage-gated K(+) nor ether-角-go-go-related gene (erg)-mediated K(+) currents in GH(3) cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 microM) decreased the firing of action potentials in GH(3) cells. In pancreatic betaTC-6 cells, di-8-APPNES (10 microM) also increased BK(Ca)-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BK(Ca) channels could be one of potential mechanisms through which it may affect cell excitability.

Unravelling the Effects of Cholesterol on the Second-Order Nonlinear Optical Responses of Di-8-ANEPPS Dye Embedded in Phosphatidylcholine Lipid Bilayers

Cholesterol is known for its role in maintaining the correct fluidity and rigidity of the animals cell membranes and thus their functions. Assessing the content and the role of cholesterol in lipid bilayers is therefore of crucial importance for a deeper understanding and control of membrane functioning. In this computational work, we investigate bilayers built from three types of glycerophospholipid phosphatidylcholine (PC) lipids, namely dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and dioleoylphosphatidylcholine (DOPC), and containing different amounts of cholesterol by analyzing the second-harmonic generation (SHG) nonlinear optical (NLO) response of a probe molecule, di-8-ANEPPS, inserted into the membranes. This molecular property presents the advantage to be specific to interfacial regions such as lipid bilayers. To unravel these effects, Molecular Dynamics (MD) simulations have been performed on both DPPC and DOPC lipids by varying the cholesterol mole fraction (from 0 to 0.66), while POPC was only considered as a pure bilayer. In the case of the structural properties of the bilayers, all the analyses converge toward the same conclusion: as the mole fraction of cholesterol increases, the systems become more rigid, confirming the condensing effect of cholesterol. In addition, the chromophore is progressively more aligned with respect to the normal to the bilayer. On the contrary, addition of unsaturation disorders the lipid bilayers, with barely no impact on the alignment of the chromophore. Then, using the frames obtained from the MD simulations, the first hyperpolarizability 汕 of the dye in its environment has been computed at the TDDFT level. On the one hand, the addition of cholesterol induces a progressive increase of the diagonal component the 汕 tensor parallel to the bilayer normal. On the other hand, larger 汕 values have been calculated for the unsaturated than for the saturated lipid systems. In summary, this study illustrates the relationship between the composition and structure of the bilayers and the NLO responses of the embedded dye.

Measuring the induced membrane voltage with Di-8-ANEPPS

Placement of a cell into an external electric field causes a local charge redistribution inside and outside of the cell in the vicinity of the cell membrane, resulting in a voltage across the membrane. This voltage, termed the induced membrane voltage (also induced transmembrane voltage, or induced transmembrane potential difference) and denoted by DeltaPhi, exists only as long as the external field is present. If the resting voltage is present on the membrane, the induced voltage superimposes (adds) onto it. By using one of the potentiometric fluorescent dyes, such as di-8-ANEPPS, it is possible to observe the variations of DeltaPhi on the cell membrane and to measure its value noninvasively. di-8-ANEPPS becomes strongly fluorescent when bound to the lipid bilayer of the cell membrane, with the change of the fluorescence intensity proportional to the change of DeltaPhi. This video shows the protocol for measuring DeltaPhi using di-8-ANEPPS and also demonstrates the influence of cell shape on the amplitude and spatial distribution of DeltaPhi.

Confocal imaging of transmembrane voltage by SEER of di-8-ANEPPS

Imaging, optical mapping, and optical multisite recording of transmembrane potential (V(m)) are essential for studying excitable cells and systems. The naphthylstyryl voltage-sensitive dyes, including di-8-ANEPPS, shift both their fluorescence excitation and emission spectra upon changes in V(m). Accordingly, they have been used for monitoring V(m) in nonratioing and both emission and excitation ratioing modes. Their changes in fluorescence are usually much less than 10% per 100 mV. Conventional ratioing increases sensitivity to between 3 and 15% per 100 mV. Low sensitivity limits the value of these dyes, especially when imaged with low light systems like confocal scanners. Here we demonstrate the improvement afforded by shifted excitation and emission ratioing (SEER) as applied to imaging membrane potential in flexor digitorum brevis muscle fibers of adult mice. SEER--the ratioing of two images of fluorescence, obtained with different excitation wavelengths in different emission bands-was implemented in two commercial confocal systems. A conventional pinhole scanner, affording optimal setting of emission bands but less than ideal excitation wavelengths, achieved a sensitivity of up to 27% per 100 mV, nearly doubling the value found by conventional ratioing of the same data. A better pair of excitation lights should increase the sensitivity further, to 35% per 100 mV. The maximum acquisition rate with this system was 1 kHz. A fast "slit scanner" increased the effective rate to 8 kHz, but sensitivity was lower. In its high-sensitivity implementation, the technique demonstrated progressive deterioration of action potentials upon fatiguing tetani induced by stimulation patterns at >40 Hz, thereby identifying action potential decay as a contributor to fatigue onset. Using the fast implementation, we could image for the first time an action potential simultaneously at multiple locations along the t-tubule system. These images resolved the radially varying lag associated with propagation at a finite velocity.