68 M. Priest and F. Bezanilla
gating current might not be is because the fluorescence tends to follow the gating
charge, which is proportional to the time integral of the gating current. Thus, as a
very slow gating process spreads the charge over a long period of time, it makes the
current difficult to resolve. However, its integral might remain resolvable by fluo-
rescence. In a similar strategy, fluorescence has also been used to measure Shaker
S4 movement during omega current conduction (Tombola et al. 2005 ).
Another novel application of site-directed fluorometry has been the use of elec-
trochromic dyes to detect local electric field strength in different locations of the
protein. By modifying the electrochromic ANEPPS dye with an iodoacetamide
moiety, the electric field was measured in different places of the Shaker channel. It
was found that the electric field is about three times stronger near the center of the
S4 segment as compared to the field in the hydrophobic core of the bilayer (Asa-
moah et al. 2003 ). Some dyes that are sensitive to the environment, such as TMR,
also exhibit electrochromic properties and have been used to infer the electric field
strength in other preparations (Dekel et al. 2012 ).
Furthermore, in spite of its great power as a corroborative technique once a flu-
orescence change has been found that correlates well with channel activity, it is
important to remember that fluorescence changes are the result of local conforma-
tional changes while the gating or ionic currents are the result of global changes.
Therefore there is no reason why a given fluorescence should be identical to the
charge movement. Rather, fluorescence changes provide new information on the
conformational changes occurring near the probe. These fluorescence changes may
occur during electrically silent steps, thus revealing new information that was not
visible by electrophysiology.
3.6 Limitations of Functional Site-Directed Fluorometry
One of the main limitations of functional site-directed fluorometry is in the interpre-
tation of the observed fluorescence changes. Fluorescence changes in a dye occur
due to changes in the dye’s environment; for example, if a fluorescence change is
observed during membrane depolarization, the environment surrounding the dye
must be different between the hyperpolarized and depolarized states. Different fluo-
rophores respond differently to their environment but typical factors that influence
fluorescence include whether the fluorophore is in an aqueous, proteinaceous or
lipid environment, whether the fluorophore is near particular amino acid residues
that can act as quenchers, the anisotropy of the dye in a particular conformational
state, and the pH of the environment. Identical fluorophores placed at different sites
in the protein can undergo fluorescence changes through wholly different mecha-
nisms, including shifts in the absorption spectra (Cha and Bezanilla 1998 ), and mul-
tiple quenching groups and environmental shifts can and do simultaneously alter