Functional Site-Directed Fluorometry 59
gene of interest. Site-directed mutagenesis of the gene should be done to ensure
that only a single cysteine is accessible for dye labeling; accidental labeling of mul-
tiple residues can produce self-quenching of the dye and fluorescent results that
are inappropriately interpreted as a result. Typically, endogenous cysteines that can
be labeled are mutated to serines due to the molecular similarity between the two
amino acids, and an exogenous cysteine is substituted in place of an amino acid at
the region of interest. Increasingly, cysteine scanning of a region is performed, in
which several constructs are designed with a single cysteine each; in such cases, it
is prudent to demonstrate that more than one construct-dye pair correlates with the
observed ion channel current. The molecular biology required prior to successful
site-directed fluorometry is relatively straightforward (Fig. 1a) and will not be dis-
cussed further here. Similarly, descriptions of oocyte defolliculation and injection
will not be described here.
Following expression of the protein on the surface of the oocyte, various proce-
dures can be attempted to improve accessibility of the fluorescent dye to the thiol
group of the cysteine. One step that is necessary for many sites near the S4 of
voltage-gated ion channels is to place the oocytes in a depolarizing solution during
labeling to expose the residues near the extracellular region of the S4 to regions of
improved accessibility (Mannuzzu et al. 1996 ). Additionally, oocytes can be in-
cubated in (Cha and Bezanilla 1998 ) or pretreated with DTT immediately prior to
labeling (Campos et al. 2007 ) to reduce the thiols and improve labeling. To further
improve signal to noise, the cysteines on endogenously expressed membrane pro-
teins can be treated with a nonpenetrating, nonfluorescent maleimide or other thiol
reactive group prior to surface expression of the ion channel of interest (Chanda
and Bezanilla 2002 ; Mannuzzu et al. 1996 ). Finally, if an endogenous cysteine in
the ion channel of interest is both accessible under typical labeling conditions and
required for protein expression or function, a cysteine can be engineered into the
site of interest with an i + 3 histidine. The cysteine in the Cys-His pair can be easily
blocked by cadmium and background cysteines can then be blocked with a nonfluo-
rescent group. By chelating away the cadmium with EDTA, the engineered cysteine
can then be specifically labeled with any thiol-reactive fluorophore (Puljung and
Zagotta 2011 ).
Numerous fluorophores are used in site-directed fluorometry. The most com-
monly used fluorophores in site-directed fluorometry are tetramethylrhodamine
(TMR), Alexa 488 and Alexa 546. Most frequently, the fluorophore contains a ma-
leimide group for subsequent reaction to the cysteine thiol group (Fig. 1b), but
iodoacetamide and methanethiosulfonate are other common reaction groups. Label-
ing protocols are determined empirically but typically use between 5 and 20 μM
of the dye for 10–30 min. Leaving oocytes in the dye for extended periods of time
can lead to internalization of some dyes, dramatically reducing signal to noise. Fol-
lowing labeling, oocytes are rinsed well in incubation solution and kept in the dark
until experiments are performed. Once oocytes are removed from the labeling solu-
tion, internalization of membrane proteins and gradual loss of fluorophore fluores-
cence can lead to serious difficulties in measuring strong fluorescence signals after
about 2 h.