Ratiometric imaging of styryl potentiometric
dyes can be used to measure the potential gradient inside the membrane (intramembrane potential), which is the sum of contributions from transmembrane potential, dipole potential, and the difference in the surface potentials at both sides of the membrane. Here changes in intramembrane potential of the bilayer membranes in two different preparations,
lipid vesicles and individual N1E-115
neuroblastoma cells, are calculated from the fluorescence ratios of
di-4-ANEPPS and
di-8-ANEPPS as a function of divalent
cation concentration. In
lipid vesicles formed from the zwitterionic
lipid phosphatidylcholine (PC) or from a mixture of the negatively charged
lipid phosphatidylserine (PS) and PC,
di-4-ANEPPS produces similar spectral changes in response to both divalent
cation-induced changes in intramembrane potential and transmembrane potential. The changes in potential on addition of
divalent cations measured by the fluorescence ratios of
di-4-ANEPPS are consistent with a change in surface potential that can be modeled with the Gouy-Chapman-Stern theory. The derived intrinsic 1:1 association constants of Ba and Mg with PC are 1.0 and 0.4 M(-1); the intrinsic 1:1 association constants of Ba and Mg with PS are 1.9 and 1.8 M(-1). Ratiometric measurements of voltage sensitive
dyes also allow monitoring of intramembrane potentials in living cells. In
neuroblastoma cells, a tenfold increase of concentration of Ba, Mg, and Ca gives a decrease in intramembrane potential of 22 to 24 mV. The observed changes in potential could also be described by Gouy-Chapman theory. A surface charge density of 1 e(-)/115 A(2) provides the best fit and the intrinsic 1:1 association constants of Ba, Mg, and Ca with acidic group in the surface are 1.7, 6.1, and 25.3 M(-1).