64 M. Priest and F. Bezanilla
behave fundamentally similarly to those in more typical channels (Sesti et al. 2003 ).
Other work has shown that the slow gating current of the hERG channel is an in-
trinsic property of the voltage-sensing domain and not due to the pore (Thouta et al.
2014 ). Finally, in voltage-gated sodium channels, fluorometry confirmed that do-
main IV was crucial for fast inactivation (Cha et al. 1999a; Chanda and Bezanilla
2002 ), as had been predicted (McPhee et al. 1995 , 1998 ; Sheets et al. 1999 , 2000 ;
West et al. 1992 ).
Usefully, fluorescence signals also indicated movements that remain incomplete-
ly understood, providing new hypotheses for research. One outstanding question is
whether the S2 segment with its highly conserved negative charges undergoes a
conformational change. While BK fluorometry has more definitively shown that the
S2 is undergoing conformational changes separable from those in S4 (Pantazis et al.
2010 ), fluorometry from Shaker and Kv1.2 remains only suggestive, with potential
S2 motion displaying dramatically different kinetics in the two seemingly similar
channels (Cha and Bezanilla 1997 ; Horne et al. 2010 ).
Another example is the interaction of the pore and voltage sensor on each other
during channel activation and deactivation. This is a distinct phenomenon from the
coupling of the voltage sensor to pore opening, which has been examined in Nav1.4
where mutations in the DIII S4-S5 linker uncoupled fluorescence changes from
ionic current, strongly suggesting these residues are involved in coupling the volt-
age sensor to the pore (Muroi et al. 2010 ). Other fluorometry studies suggest that
in addition to this conventional coupling, Shaker pore opening appears to induce
a conformational change in the voltage sensor, as measured by fluorescence from
both the S4 and the pore (Cha and Bezanilla 1998 ; Gandhi et al. 2000 ; Vaid et al.
2008 ). Alternatively, fluorescence from S4 sites in Shaker (Cha and Bezanilla 1997 ;
Gandhi et al. 2000 ; Loots and Isacoff 2000 ) and hERG (Es-Salah-Lamoureux et al.
2010 ) may simply follow conformational properties of the pore such as ionic deac-
tivation and inactivation. More recently, fluorometry has suggested that the mode-
shift, or relaxed state, of the voltage sensor is dependent on pore coupling (Batulan
et al. 2010 ; Haddad and Blunck 2011 ; Tan et al. 2012 ) while other fluorometry stud-
ies showed that this phenomenon was intrinsic to the voltage sensor (Labro et al.
2012 ; Villalba-Galea et al. 2008 , 2009 ). By improving the interpretive power of
site-directed fluorometry in these studies it should be possible to better understand
the nature of these interactions, as well as many other conformational changes.
3.3 Functional Site-Directed Fluorometry Provides Insight Into
Molecular Interactions
Typically, functional site-directed fluorometry is used in a corroborative role, in
which changes produced in fluorescence signals previously shown to correlate
with gating or ionic current are mirrored in the simultaneously recorded gating or
ionic current itself. Once such a system is established, numerous questions can be
asked regarding how the activity of the labeled site of interest is altered by intra- or,