Functional Site-Directed Fluorometry 63
(Cha et al. 1999b; Chanda et al. 2005 ; Glauner et al. 1999 ; Hyde et al. 2012 ; Posson
et al. 2005 )
3.1 Diversity of Membrane Proteins Explored with Functional
Site-Directed Fluorometry
Since then, site-directed fluorometry has been applied to a diverse set of ion chan-
nels to answer the basic question of whether a particular part of the protein under-
goes a conformational change in relationship to the rest of the protein in response
to an activating stimulus such as a pulse of voltage or ligand application. Examples
of ion channels on which site-directed fluorometry has been reported include Kv1.2
(Peters et al. 2009 ), Kv1.5 (Vaid et al. 2008 ), Kv7.1/KCNQ1 (Osteen et al. 2010 ),
kv11.1/hERG and eag (Schönherr et al. 2002 ; Smith and Yellen 2002 ), KCa1.1/BK
(Savalli et al. 2006 ), KcsA (Blunck et al. 2006 ), Nav1.4 (Cha et al. 1999a), NaCh-
Bac (Blunck et al. 2004 ), Hv (Gonzalez et al. 2010 ), HCN (Bruening-Wright and
Larsson 2007 ), CNG (Zheng and Zagotta 2000 ), ASIC1a (Passero et al. 2009 ),GA-
BAaR (Chang and Weiss 2002 ), nAChR (Dahan et al. 2004 ), GlyR (Pless et al.
2007 ), and ELIC (Ulens et al. 2014 ). Other membrane proteins have also been stud-
ied by site-directed fluorometry, including the SERT serotonin transporter (Li and
Lester 2002 ), the EAAT glutamate transporter (Larsson et al. 2004 ), the sodium/
glucose cotransporter SGLT1 (Loo et al. 1998 ), the sucrose transporter SUT1 (Der-
rer et al. 2013 ), the sodium/phosphate contransporter NaPi (Virkki et al. 2006 ), the
GABA transporter GAT (Li et al. 2000 ), the organic cation transporter Oct1 (Egen-
berger et al. 2012 ), the Na+/K+-ATPase (Geibel et al. 2003 ), the Ciona intestinalis
voltage-sensitive phosphatase (Kohout et al. 2008 ), and a muscarinic acetylcholine
G protein-coupled receptor (Dekel et al. 2012 ).
3.2 Functional Site-Directed Fluorometry Elucidates Voltage-
Induced Conformational Changes
At its most basic level, fluorometry provides insight into the physical movements of
the ion channel that are occurring during various conformational changes. For ex-
ample, it had been demonstrated that C-type inactivation in Shaker was a property
of the selectivity filter (Choi et al. 1991 ; Hoshi et al. 1991 ). Fluorescence changes
from sites near the extracellular side of S5 and S6 correlated kinetically with this
slow inactivation (Cha and Bezanilla 1997 ; Loots and Isacoff 1998 ), confirming
that conformational changes near the pore correlated with inactivation. In hyper-
polarization-activated cyclic nucleotide-gated channels, fluorescent probes pro-
duced fluorescent signals that correlated well with the gating charge movement that
occurred during activating hyperpolarizing pulses (Bruening-Wright et al. 2007 ),
further demonstrating that voltage sensors in hyperpolarization-activated channels