58 M. Priest and F. Bezanilla
clamp, with no need for protein purification and reconstitution ‒ with those of site-
labeled spectroscopy, such as the ability for the data to provide a higher degree of
information about the conformational changes of the system. The advantage of be-
ing able to study diverse membrane proteins was quickly taken advantage of, with
site-directed fluorometry being applied to a sodium/glucose cotransporter (Loo
et al. 1998 ) and a voltage-gated sodium channel (Cha et al. 1999a) shortly follow-
ing the initial report of the technique. To take advantage of the additional interpre-
tational power of site-directed fluorometry, researchers also began searching for
the particular amino acids responsible for the fluorescence changes they observed
(Blunck et al. 2004 ; Loots and Isacoff 2000 ; Sørensen et al. 2000 ). Over the years,
the strengths of site-directed fluorometry have been extended to a diverse set of
membrane proteins, have expanded in functionality and precision, and have been
used with great success to improve our understanding of how ion channels move in
response to changes in their environment.
2 Methods
In general, functional site-directed fluorometry requires a membrane protein that
contains an amino acid or a group of amino acids that can be accessed by a reactive
fluorescent dye. Typically, the reaction is of a thiol-reactive fluorophore with a cys-
teine located in the region of interest of the protein. However, fluorophores can also
be attached to lysines (Lougheed et al. 2001 ) or to oligohistidine sequences (Guig-
net et al. 2004 ). Following covalent attachment of the fluorescent probe, the con-
formational state of the protein is shifted by application of a ligand or a change in
membrane potential. The optical signal from the dye is measured during this time,
and its changes in fluorescence are then compared to concurrent changes in current
measured from the ion channel or the membrane protein of interest. Useful descrip-
tions of the technique have previously been published by the Bezanilla and Olcese
labs (Cha et al. 1998 ; Gandhi and Olcese 2008 ), and video-supplemented descrip-
tions are also available (Richards and Dempski 2011 ; Rudokas et al. 2014 ). Suc-
cessful site-directed fluorometry experiments require a PCR machine and reagants
for mutagenesis, reagants for RNA synthesis, access to Xenopus laevis oocytes or to
mammalian cells, a microinjector for RNA injection and incubator at 16 °C for in-
cubation, a thiol-reactive fluorophore, an electrophysiological setup for measuring
currents produced by the membrane protein of interest, a light source to excite the
fluorophore, a microscope for focusing on the surface of the oocyte, optical lenses
and dichroic mirrors for passing the appropriate excitation wavelength to the oocyte
from the light source and the emission from the oocyte to the light collector, and a
collector of the emission light such as a photomultiplier tube or photodiode.
The most common technique used to express ion channels in a membrane is to
inject cRNA encoding the protein into Xenopus laevis oocytes, which then express
the channel on their membrane surface (Fig. 1 ). The cRNA is transcribed from DNA
that has been purified from E. coli transformed with a plasmid that contains the