Spectral Properties and
Orientation of Voltage-Sensitive
Dyes in Lipid Membranes
- Publication date
- Publisher
Abstract
Voltage-sensitive dyes are frequently used for probing
variations
in the electric potential across cell membranes. The dyes respond
by changing their spectral properties: measured as shifts of wavelength
of absorption or emission maxima or as changes of absorption or fluorescence
intensity. Although such probes have been studied and used for decades,
the mechanism behind their voltage sensitivity is still obscure. We
ask whether the voltage response is due to electrochromism as a result
of direct field interaction on the chromophore or to solvatochromism,
which is the focus of this study, as result of changed environment
or molecular alignment in the membrane. The spectral properties of
three styryl dyes, di-4-ANEPPS, di-8-ANEPPS, and RH421, were investigated
in solvents of varying polarity and in model membranes using spectroscopy.
Using quantum mechanical calculations, the spectral dependence of
monomer and dimer ANEPPS on solvent properties was modeled. Also,
the kinetics of binding to lipid membranes and the binding geometry
of the probe molecules were found relevant to address. The spectral
properties of all three probes were found to be highly sensitive to
the local environment, and the probes are oriented nearly parallel
with the membrane normal. Slow binding kinetics and scattering in
absorption spectra indicate, especially for di-8-ANEPPS, involvement
of aggregation. On the basis of the experimental spectra and time-dependent
density functional theory calculations, we find that aggregate formation
may contribute to the blue-shifts seen for the dyes in decanol and
when bound to membrane models. In conclusion, solvatochromic and other
intermolecular interactions effects also need to be included when
considering electrochromic response voltage-sensitive dyes