62 M. Priest and F. Bezanilla
significantly in the red region of the spectrum so red fluorophores may provide a
better signal-to-background ratio. However, their intrinsically larger volume makes
them less frequently used.
Typically, the optical detection consists of using a photodiode to measure the
time course of the change in fluorescence with respect to the background fluores-
cence (the ∆F/F 0 ) mainly because it is the simplest measurement possible. However,
fluorescence intensity changes do not allow for a direct determination of the mecha-
nism involved in the fluorescence change. For mechanistic studies other measure-
ments are necessary, such as spectral determinations of the emission and absorption
(Cha et al. 1998 ) and fluorescence lifetime measurements (Semenova et al. 2009 ).
Additionally, the equipment used for site-directed fluorometry in mammalian cells,
such as HCK cells, is different from that used for oocytes. Because many dyes tend
to penetrate mammalian cells and the area imaged is smaller in mammalian cells
than in oocytes, higher gain detectors are needed, such as avalanche photodiodes or
photomultiplers. For details, see Blunck et al. 2004.
3 Applications
Functional site-directed fluorometry in ion channels was initially used to directly
test the hypothesis that the putative voltage sensor in the S4 transmembrane domain
undergoes a physical motion during the activation of the channel. Prior to site-
directed fluorometry, the only direct experimental evidence for S4 movement was
more rapid accessibility at depolarized potentials of a methanethiosulfonate reagent
to a binding site near the extracellular extreme of the S4 in Nav1.4 (Yang and Horn
1995 ). While useful, this evidence was difficult to directly correlate temporally with
a single conformational change of the channel, such as activation or inactivation.
Furthermore, these temporal correlations could only be measured using a pertur-
bation of the channel activity produced by channel modification by the reagent.
Fluorophores attached to cysteines substituted near the extracellular end of the S4
segment, specifically at M356 and A359, displayed changes in fluorescence in re-
sponse to changes in membrane potential with kinetics and voltage-dependence that
closely followed the kinetics and voltage-dependence of the gating current (Man-
nuzzu et al. 1996 ; Cha and Bezanilla 1997 ). These results were interpreted as direct
evidence that the S4 segment undergoes physical movement during activation and
deactivation of ion channels, further supporting the now accepted hypothesis that
the S4 acts as the primary voltage sensor in voltage-gated ion channels. However,
we should remember that the changes in fluorescence indicated by the probe only
tell us that there is relative motion with respect to the probe; it is also possible that
the region around the probe is moving and that the probe itself is static. Verifica-
tion that S4 moves came from the study of fluorometry of many sites and by the
evidence provided by cysteine scanning studies of the S4 segment (Cha and Beza-
nilla 1997 ; Gandhi et al. 2000 ; Pathak et al. 2007 ). Additional confirmation of S4
movement was provided by fluorescence and lanthanide resonance energy transfer