Functional Site-Directed Fluorometry 67
Like the investigations of cooperativity between subunits in voltage-gated ion
channels, the interactions between subunits in ligand-gated ion channels have also
been studied using site-directed fluorometry. For example, labeled residues in dif-
ferent subunits of heteromeric GABA receptors showed different responses to ap-
plication of the same ligand, suggesting that the presence of different subunits may
induce asymmetry in the conformational changes of these subunits and that the β
subunit may be particularly important in the activity of barbiturates on the recep-
tor (Muroi et al. 2006 , 2009 ). Additional studies showed that ligands binding at a
known interface between two subunits can produce conformational changes that
extend to the interface with an adjacent subunit (Wang et al. 2010 ).
The conformational changes induced by more exotic ligands have also been ex-
amined by site-directed fluorometry. In addition to the effect of barbiturates (Muroi
et al. 2009 ), other work has looked at the response of the GABA receptor to steroids
(Eaton et al. 2014 ; Li et al. 2010 ). In the prokaryotic ELIC channel, site-directed
fluorometry has been used to show that the Alzheimer’s disease drug memantine in-
duces conformational changes in the receptor that are not revealed by crystal struc-
tures (Ulens et al. 2014 ). Site-directed fluorometry of the glycine receptor has been
used to demonstrate that ivermectin, an unusual agonist that binds in the transmem-
brane domain region, induces conformational changes throughout the typical ligand
binding site (Wang and Lynch 2012 ) and in the M2 region, suggesting a mechanism
of cooperative activation between different subunits (Pless and Lynch 2009c). An-
other study used this technique with the novel purpose of measuring conformational
changes driven by phosphorylation of the receptor (Han et al. 2013 ).
Finally, site-directed fluorometry in ligand-gated ion channels is by no means
confined to Cys-loop type receptors. An excellent example is the work done on
ASIC1a, part of the ENaC superfamily. Site-directed fluorometry has demonstrated
that the region just extracellular to M2 in this acid-sensing ion channel undergoes
conformational changes in response to protonation and upon a return to the resting
state following desensitization (Passero et al. 2009 ). Further work has greatly ex-
tended these results, taking advantage of site-specific quenching by an endogenous
tryptophan to demonstrate that upon channel activation one well characterized ex-
tracellular domain known as ‘the finger’ moves away from another known as the
‘β ball’ and showing that the structural pathway taken during gating activation is
likely different from that taken during recovery from desensitization (Bonifacio
et al. 2014 ).
3.5 Additional Applications of Functional Site-Directed
Fluorometry
In some instances when the fluorescence changes are more tractable than the electri-
cal changes, fluorometry alone has been used as a proxy for slow, difficult to record
voltage sensor movement from KCNQ1 (Osteen et al. 2010 , 2012 ; Ruscic et al.
2013 ; Zaydman et al. 2013 ). The reason fluorescence changes are visible while