Currents | Issue 4 • Fall 1996

Fluorescence Resonance Energy Transfer

A Method for the Optical Sensing of Membrane Depolarization in Single Cells

by Kirk M. Maxey, M.D.

In a normal, resting cell with an active Na⁺/K⁺ ATPase, the selective uptake of K⁺ ions into the cytoplasm rapidly establishes an intracellular K⁺ concentration of about 145 mM, compared to an extracellular concentration of about 4 mM. This difference drives the diffusion of K⁺ ions back down the concentration gradient to the outside of the cell. The plasma membrane is usually impermeable to the negative counter ions, which accumulate on the inner leaflet, creating a polarized membrane. In a cell permeable only to K⁺, this membrane potential is about -90 mV (negative on the inside). During depolarization, rapid changes in the permeability of the plasma membrane to K⁺ and Na⁺ reverse this concentration gradient and leads to a transient +60 mV membrane potential.

In theory, amphipathic anions will accumulate in the positively charged outer leaflet of the membrane of a resting, polarized cell. Upon depolarization, these anions rapidly translocate to the inner membrane, following the migration of positive charge from the exterior to the interior of the cell. The symmetrical bis(thiobarbiturate) oxonols are fluorescent anions which have been shown to undergo this translocation in about 2 milliseconds during depolarization. DISBAC₁₀ (Figure 1) is a member of this group of compounds.

Figure 1

Fluorescein-labeled lectins such as Fluorescein-Wheat Germ Agglutinin (FLA) can be used to permanently label the extracellular surface. Fluorescence Resonance Energy Transfer (FRET) occurs when a blue (490 nm) photon is absorbed by FLA, and then re-emitted as a green (540 nm) photon which is captured by a DISBAC molecule floating adjacent to it in the outer membrane. The DISBAC molecule in turn re-emits the photon, but again shifts it to a longer, orange (560 nm) wavelength. As long as the cell remains at its resting membrane potential of -90 mV, the DISBAC anions will remain in the membrane outer leaflet and FRET will occur. However, upon depolarization of the cell, the DISBAC anions will rapidly translocate to the suddenly positive inner membrane, and FRET will drop off as the fluorescent pair of molecules are separated from each other (Figure 2).

Figure 2

In recent years, many novel photometric systems have taken advantage of the fact that energy transfer between two adjacent molecules increases by a factor of 10⁶ as the distance between them decreases linearly. Transfer of a photon from one molecule to another drops to near zero if the two molecules are separated by more than 100 Å.

There is an increase in 540 nm (uncaptured) fluorescence on depolarization, a decrease in FRET 560 nm fluorescence, and an increase in the 540/560 ratio. Any one of these three data functions can be monitored in a cellular preparation loaded with these two dye labels. In a mixed cell population, fluorescein-labeled monoclonal antibodies or specific receptor ligands can be used in place of the FLA, allowing functional visualization of specific cell classes.