Functional Site-Directed Fluorometry 65
more frequently, inter-molecular interactions. An excellent example is how site-
directed fluorometry has been extensively used to examine cooperativity between
voltage-sensing domains. By perturbing voltage sensor activity in one domain and
measuring fluorometry in a different wild-type domain, cooperativity was demon-
strated to be absent from Ci-VSP (Kohout et al. 2008 ) and absent from Shaker
gating until a final concerted step that was responsible for opening the channel
(Mannuzzu and Isacoff 2000 ; Pathak et al. 2005 ). On the other hand, cooperativity
was found among all domains in the muscle sodium channel Nav1.4, especially
between domains I and IV (Chanda et al. 2004 ). Cooperativity was also present in
an Hv channel (Tombola et al. 2010 ). Comparison of S4 fluorescence changes with
gating current further established that Hv channel cooperativity mirrored Shaker
cooperativity, only acting on a second, smaller component of the gating charge that
was responsible for channel opening (Qiu et al. 2013 ).
Site-directed fluorometry has also been used to study the effects on channel
movement of molecules ranging from modulatory subunits and toxins to small mol-
ecules such as anesthetics and lipids. Regarding auxiliary subunits, site-directed
fluorometry has been used to study the effect of Kvβ1 on Kv1.2 (Peters et al. 2009 )
and the effect of β 2 and RCK1 and RCK2 on the BK channel (Savalli et al. 2007 ,
2012 ). Additionally, fluorometry has suggested that the beta subunit KCNE1 slows
the kinetics of the voltage sensor movement directly (Ruscic et al. 2013 ) and that
KCNE1 separates the movement of the KCNQ1 voltage sensor into two compo-
nents, the latter of which is responsible for complete channel opening (Barro-Soria
et al. 2014 ; Osteen et al. 2010 ).
Mechanisms of toxin action can be tested in a similar fashion. The mechanism by
which the scorpion toxin Ts3 opens sodium channels was validated by fluorometry
from the four S4s of Nav1.4, demonstrating that Ts3 eliminated a fluorescent com-
ponent in domain IV that correlated with the loss of fast inactivation induced by the
toxin (Campos et al. 2008 ). This mechanism was very different than the mechanism
observed using similar site-directed fluorometry experiments with the Ts1 scorpion
toxin, which was shown to eliminate the movement of the voltage sensor of domain
II by holding it in an activated state (Campos et al. 2007 ).
Similar experiments showed that local anesthetics shifted fluorescence changes
from the S4 of DIII to hyperpolarized potentials (Muroi and Chanda 2009 ). In a
promising development, this insight was later used to develop a site-directed fluo-
rometry screening assay for molecular mediators of lidocaine on Nav1.4 (Arcisio-
Miranda et al. 2010 ). Intracellular calcium was also shown to induce conformational
changes in the S4 of BK (Savalli et al. 2012 ), and external protons made S4 move-
ment in hERG channels begin at more depolarized potentials (Shi et al. 2014 ). Lipid
effects on ion channels have also been studied using fluorometry, helping support
the hypothesis that KCNQ1 requires PIP 2 to couple the voltage sensor movement
to the pore (Zaydman et al. 2013 ) and to confirm that PIP 2 acts on the voltage sen-
sor gating currents rather than the ionic currents of Shaker (Abderemane-Ali et al.
2013 ). Conversely, ATP has been shown to be required for pore opening in KCNQ1
but to not affect voltage sensor moving and coupling (Li et al. 2013 ).