ANAP Review

CHAPTER THREE
ANAP: A versatile, fluorescent

probe of ion channel gating
and regulation
Michael C. Puljung*
Department of Chemistry and the Neuroscience Program, Trinity College, Hartford, CT, United States
*Corresponding author: e-mail address: michael.puljung@trincoll.edu
Contents
1. Introduction 50
2. Site-specific ANAP incorporation 57
3. ANAP applications in ion channel research 59
3.1 Conformational changes associated with voltage gating 60
3.2 Activation of TRP channels 63
3.3 Binding and activation in ligand-gated channels 64
4. Non-channel applications for ANAP 67
5. Expression of ANAP-tagged channels 67
6. Controls and confounds 69
7. Excitation and detection of ANAP 77
8. Conclusions 79
References 80
Abstract
Fluorescence spectroscopy and microscopy are non-destructive methods that provide
real-time measurements of ion channel structural dynamics. As such, they constitute a
direct path linking the high-resolution structural models from X-ray crystallography and
cryo-electron microscopy with the high-resolution functional data from ionic current
measurements. The utility of fluorescence as a reporter of channel structure is limited
by the palette of available fluorophores. Thiol-reactive fluorophores are small and bright,
but are restricted in terms of the positions on a protein that can be labeled and present
significant issues with background incorporation. Genetically encoded fluorescent
protein tags are specific to a protein of interest, but are very large and usually only used
to label the free N- and C-termini of proteins. L-3-(6-acetylnaphthalen-2-ylamino)-
2-aminopropionic acid (ANAP) is a fluorescent amino acid that can be specifically
incorporated into virtually any site on a protein of interest using amber stop-codon sup-
pression. Due to its environmental sensitivity and potential as a donor in fluorescence
resonance energy transfer experiments, it has been adopted by numerous investigators
to study voltage, ligand, and temperature-dependent activation of a host of ion channels.
Methods in Enzymology, Volume 654 Copyright # 2021 Elsevier Inc. 49

ISSN 0076-6879 All rights reserved.
https://doi.org/10.1016/bs.mie.2021.01.048
50 Michael C. Puljung
Simultaneous measurements of ionic currents and ANAP fluorescence yield exceptional

mechanistic insights into channel function. In this chapter, I will summarize the current
literature regarding ANAP and ion channels and discuss the practical aspects of using
ANAP, including potential pitfalls and confounds.
1. Introduction
Ion channels create and shape all the electrical events of life from fer-
tilization to death (Zheng & Trudeau, 2015). Their coordinated activity
generates nervous impulses, enables sensory perception, induces hormone
secretion, initiates muscle contraction, and keeps the heart beating in time.
Opening an ion channel permits the selective permeation of millions of
ions per second, a flux sufficient to produce measurable electrical currents.
For decades, physiologists, biochemists, and biophysicists have taken advan-
tage of these currents to interrogate ion channel function down to the
single-molecule level and at sub-ms time scales.
Ion channels are membrane proteins, which made them relatively inacces-
sible to the tools of high-resolution structural biology for the first 40 years or
more following their discovery. Instead, channel structures were probed indi-
rectly with thoughtful electrophysiological and mutagenesis experiments. This
changed in the late 1990s with the publication of the first high-resolution chan-
nel structures from X-ray crystallography (Doyle, 1998). More recently, the
trickle of new channel structures transformed into a cascade, thanks to substan-
tial improvements in cryo-electron microscopy (cryo-EM), which can now
produce channel structures at true atomic resolution (Nakane et al., 2020).
The structural models obtained through cryo-EM and crystallography have
broadened our understanding of channel gating and permeability, but usually
only represent one or a handful of channel states. Furthermore, it can be
unclear what channel state is captured in a given structure, or if the structure
of a channel outside of its natural environment even represents a native con-
formation. Thus, there is a need for methods to provide a direct link between
high-resolution structures and the high-resolution functional measurements
available to ion channel physiologists.
Fluorescence spectroscopy provides just such a link. Fluorescence allows
for real-time measurements of protein conformational changes, trafficking,
ligand binding, and protein-protein interactions. Most importantly, fluores-
cence spectroscopy is non-destructive. Thus, fluorescence experiments can
provide dynamic information about “awake, behaving” proteins in their
native environment. For ion channel researchers, this last point is the most
ANAP: A fluorescent probe for ion channels 51
important, as it allows fluorescence measurements to be paired with voltage

clamp, so that structural states of a channel are directly correlated with
functional ones.
Voltage-clamp fluorometry (VCF) and the related patch-clamp fluorom-
etry (PCF) simultaneously monitor currents and fluorescence from tagged
channel subunits (Cha & Bezanilla, 1997; Cowgill & Chanda, 2019;
Mannuzzu, Moronne, & Isacoff, 1996; Zheng & Zagotta, 2000). VCF exper-
iments are typically performed using Xenopus laevis oocytes. Currents are
measured using two-microelectrode voltage clamp (TEVC) or cut-open
oocyte voltage clamp. Typically, fluorophores are excited with a xenon or
mercury lamp and emitted photons are collected using a photodiode or pho-
tomultiplier tube. In PCF, currents from channels expressed in oocytes or cul-
tured mammalian cells are recorded from voltage-clamped cells or excised
membrane patches. Fluorescence is measured with a variety of techniques
including epifluorescence microscopy, confocal microscopy, and total inter-
nal reflection fluorescence (TIRF) microscopy, and through the acquisition
of emission spectra.
Several different fluorophores have been used for VCF and PCF exper-
iments (Cowgill & Chanda, 2019). Thiol-reactive fluorophores exploit the
reactivity and relative scarcity of the amino acid cysteine to label proteins.
This has been particularly useful for labeling cysteines inserted via mutagen-
esis into extracellular channel loops, as very few proteins have free cysteines
on their extracellular side, ensuring low background labeling (Cha &
Bezanilla, 1997; Mannuzzu et al., 1996). On the other hand, the use of
thiol-reactive fluorophores to label intracellular positions on a channel is
complicated by the fact that many channels have multiple free cysteines
on their intracellular side. Cysteine-free mutants of some channels still retain
functionality (Matulef, Flynn, & Zagotta, 1999). However, even if all cys-
teines are removed from a protein of interest, the plasma membrane is full of
other proteins with available cysteines, which would contribute to back-
ground labeling in a membrane environment. Whereas strategies have been
proposed to deal with this problem (Puljung & Zagotta, 2011), it is not a
trivial concern. Finally, the only positions that can be faithfully labeled with
thiol-reactive fluorophores are those that are accessible to bulk solvent,
limiting the number of positions that can be labeled on a target protein.
Fluorescent protein (FP) tags like green fluorescent protein (GFP) are
available in a wide range of colors (Zimmer, 2009). As they are genetically
encoded, they eliminate the problem of background fluorescence from
off-target labeling. However, their general utility is hampered by their large
size (30 kDa), which may disrupt the function of the target protein.
Typically, FP tags can only be used to label the free N- or C-termini of pro-
teins, although they can be inserted into loop regions. Whereas FP tags have
been employed to study changes in ion channel structure in PCF experi-
ments (Miranda et al., 2013; Miranda, Holmgren, & Giraldez, 2018), their
usefulness for providing high-resolution structural information is limited
given their large size relative to most channel protein domains.
Some naturally occurring amino acids (tryptophan, tyrosine, phenylala-
nine) are fluorescent (Lakowicz, 2006). These can be inserted into any site
on a protein of interest via mutagenesis. However, their utility in a cellular or
membrane environment is limited by the fact that many other proteins in a
cell or membrane patch also contain aromatic amino acids. Furthermore,
fluorescent native amino acids must be excited with UV light, requiring
specialized optical components to study them in live cells or membranes.
An ideal fluorophore for capturing ion channel dynamics in living cells or
cell membranes must satisfy several criteria. (1) It must be small, so that it
does not greatly perturb the structure of the labeled channels. (2) It must
be bright enough (high extinction coefficient and/or quantum yield) to pro-
vide ample signal to noise in the presence of background produced by
scattered light and cellular autofluorescence. (3) It must have a short linker,
so that changes in fluorescence faithfully represent the backbone protein
position to which it is attached. (4) It must be excited by visible light so that
it is compatible with the most basic fluorescence microscope setups. (5) Its
incorporation must be specific to the protein and site intended for labeling.
(6) It must have the potential to be attached to any site on a protein, whether
solvent exposed or on the protein’s interior.
Recent decades have seen the development of an expanded genetic code,
capable of incorporating non-canonical amino acids site-specifically into pro-
teins of interest (Braun, Sheikh, Pless, Forsythe, & Wilson, 2020; Pless &
Ahern, 2013). This has allowed investigators to move beyond the limited
chemistry available to the 20 canonical amino acids. Unnatural amino acids
with fluorescent side chains have been developed. L-3-(6-acetylnaphthalen-
2-ylamino)-2-aminopropionic acid (ANAP) is a fluorescent amino acid that
satisfies all of the criteria enumerated above (Chatterjee, Guo, Lee, &
Schultz, 2013; Lee, Guo, Lemke, Dimla, & Schultz, 2009). It is small—
slightly larger than a tryptophan (Fig. 1A)—and reasonably bright. The quan-
tum yield for ANAP has been measured in various solvents and in the
context of labeled protein. Values from 0.22 to 0.5 have been reported
(Gordon, Munari, & Zagotta, 2018; Islam et al., 2019; Lee et al., 2009;
A
(1) (2) nascent (3)
peptide
pANAP
synthetase
O
CH3
+ HN
A
5'
H2 N U
U
L-ANAP A mRNA
C
ANAP-labeled protein
G
O OH 3'
channel
+amber tRNACUA ribosome
amber codon
transfect/inject suppression
B
ANAP salt ANAP ester no ANAP
brightfield
fluorescence
2.0x109
counts
1.0x109
0.0
wt SUR1 no SUR1 T1397TAG T1397TAG H1401TAG H1401TAG A1410TAG A1410TAG
+ANAP +ANAP +ANAP
Fig. 1 Production of ANAP-labeled protein. (A) Steps in the ANAP labeling process.
(1) The plasmid pANAP is introduced into eukaryotic cells by transfection or injection
along with the gene for a protein of interest with the amber stop codon (TAG/UAG)
replacing the codon for the amino acid position intended for labeling. pANAP encodes
an orthogonal, ANAP-specific tRNACUA/synthetase pair. In the presence of ANAP, this
pair results in the production of ANAP-charged tRNACUA. (2) At the ribosome, the
ANAP-charged tRNACUA recognizes the amber stop codon. ANAP is incorporated into
the nascent peptide and translation continues. (3) The final product is ANAP-tagged
protein. (B) Images of HEK-293 cells transfected with a plasmid encoding GFP with
an amber stop codon replacing the codon for amino acid Y40, and pANAP. In the
absence of ANAP, no GFP fluorescence is evident, as only non-fluorescent, truncated
(Continued)
Mitchell, Addy, Chin, & Chatterjee, 2017; Zagotta, Gordon, Senning,

Munari, & Gordon, 2016). ANAP has a short linker, with only one methy-
lene group between the fluorescent 1-[6-(dimethylamino)-2-naphthalenyl]-
1-propanone (Prodan)-based functional group and the alpha carbon. ANAP
is excited by visible light (λmax ¼ 360 nm for ANAP in water, extinction
coefficient 17.5 mM1 cm1) (Chatterjee et al., 2013). Like Prodan,
ANAP is an environmentally sensitive fluorophore, with maximal emission
shifting depending on local polarity (Chatterjee et al., 2013; Lee et al., 2009).
The emission maximum is 490 nm in water and 420 nm in ethyl acetate.
Most importantly, ANAP can be specifically incorporated into any site on
a protein using amber stop-codon suppression (Fig. 1A) (Chatterjee et al.,
2013; Lee et al., 2009).
Following its development in the Schultz Laboratory (Chatterjee et al.,
2013; Lee et al., 2009), the potential for using ANAP as a structural probe
was quickly recognized by the ion channel community. This is, in large
part, the result of a landmark publication by Tanja Kalstrup and Rikard
Blunck, who used ANAP to follow the conformational dynamics of the
Shaker K+ channel (Kalstrup & Blunck, 2013). Since then, the use of
ANAP to study channel dynamics has become widespread. Of the 32 pri-
mary research articles currently published (or in pre-print) using ANAP,
21 of them are ion channel papers (23, if one were to count the Ciona
intestinalis voltage-sensitive phosphatase, CiVSP, as an honorary ion chan-
nel). In this chapter, I will describe the technical aspects of protein labeling
with ANAP, highlighting important controls and potential pitfalls. I will
briefly review the various applications for ANAP in the current literature
that have demonstrated its versatility as a probe of ion channel
structure (Table 1).
Fig. 1—cont’d (at amino acid position 39) GFP is made. 20 μM ANAP (trifluoroacetic salt
or methyl ester) in the cell culture medium allows for the production of full-length GFP as
assayed by GFP fluorescence. (C) Results of a luminescence-based, surface-expression
assay described by Puljung, Vedovato, Usher, and Ashcroft (2019). This experiment tests
for the presence of an extracellular HA epitope on Kir6.2 expressed in HEK-293 cells.
Surface expression requires co-expression of the accessory SUR1 protein. As a positive
control, the surface expression was measured in the presence of wild-type SUR1. The
surface expression in the absence of SUR1 was used as a negative control (median indi-
cated by the dashed line). Various SUR1 constructs with the amber stop codon at differ-
ent positions were assessed for their ability to traffic Kir6.2 to the plasma membrane in
the absence and presence of ANAP. Cells were co-transfected with pANAP and eRF1-
E55D. Boxes represent the 25th and 75th percentile.
Table 1 Ion channel applications using ANAP.

Channel/ Expression
Protein system Application References
spHCN-YFP Xenopus Mapping movement of Dai (2019)
oocytes HCN voltage sensor
spHCN-YFP Xenopus Mapping movement of Dai, Aman, DiMaio, and
oocytes HCN C-linker region Zagotta (2021)
zELK-YFP Xenopus Voltage-dependent Dai, James, and Zagotta
oocytes motion of C-terminal (2018)
intrinsic ligand of
KCNH-family channels
zELK-YFP Xenopus Interaction of the Dai and Zagotta (2017)
oocytes cytoplasmic domains of
KCNH-family channels
during voltage-dependent
potentiation
CNGA1-YFP Xenopus Metal coordination Aman, Gordon, and
oocytes between the cytoplasmic Zagotta (2016)
region of CGNA1 and the
plasma membrane
TRPV1-YFP HEK-293 Conformational changes Zagotta et al. (2016)
in TRPV1 in response to
capsaicin
KATP (SUR) HEK-293 Nucleotide binding to the Puljung et al. (2019)
activating site in KATP ion
channels
KATP HEK-293 Nucleotide binding to the Usher, Ashcroft, and
(Kir6.2-GFP) inhibitory site on KATP Puljung (2020)
channels and correlation of
binding and gating
TRPM8 HEK-293 Cold activation of Yang et al. (2020)
TRPM8 channels
TRPM8 HEK-293 Menthol binding to Xu et al. (2020)
TRPM8 channels
mouse HEK-293 Heat desensitization of Luo et al. (2019)
TRPV1-YFP, TRPV1 channels
platypus
TRPV1-GFP
Continued
Table 1 Ion channel applications using ANAP.—cont’d

Channel/ Expression
Protein system Application References
TRPV1-YFP HEK-293 Conformational changes Yang, (2018)
in TRPV1 in response to
capsaicin
hASIC1a CHO Toxin binding to ASIC Wen et al. (2015)
channels
GlyR Xenopus Mechanism of partial Soh,
oocytes agonism in glycine Estrada-Mondragon,
receptors Durisic, Keramidas, and
Lynch (2017)
ASIC and Xenopus Conformational changes Wulf (2018)
GlyR oocytes in ASIC and GlyR
channels measured with
improved signal/noise
NaV1.5 HEK-293 Conformational changes Shandell, Quejada,
associated with Yazawa, Cornish, and
inactivation of sodium Kass (2019)
channels
Shaker Xenopus Movement of the S4-S5 Kalstrup and Blunck
oocytes linker of Shaker during (2018)
activation and C-type
inactivation
Shaker Xenopus Movement of the internal Kalstrup and Blunck
oocytes pore and voltage sensor (2013)
domain of Shaker in
response to voltage
KCNQ2/3 tSA201 Direct interaction Dai (2020)
and BACE1 between BACE1 and
KCNQ2/3 measured by
FRET
CiVSP Xenopus Movement of the catalytic Kawanabe et al. (2018)
oocytes domain of CiVSP, and Sakata, Jinno,
correlated with movement Kawanabe, and
of the voltage sensor Okamura (2016)
2. Site-specific ANAP incorporation

Non-canonical amino acids can be incorporated at any location in the
primary structure of a protein using amber stop codon suppression (Pless &
Ahern, 2013). The amber stop codon (TAG in DNA, UAG in RNA) is one
of three codons used to terminate translation of a nascent protein in the ribo-
some. In the human genome, this codon is the least frequently used, found at
the end of 20% of genes (Sun, Chen, Xu, & Luo, 2005). To suppress the
amber stop codon, an exogenous, charged tRNA with the appropriate anti-
codon (tRNACUA) is introduced into cells. Rather than terminating the
nascent peptide at the amber stop codon, the exogenous tRNACUA will
bind, its amino acid cargo will be added to the nascent peptide, and trans-
lation will continue. tRNAs can be chemically charged on the bench top
and introduced (usually via injection) into cells (Pless & Ahern, 2013).
Injection limits their use to certain cell types, for example, Xenopus laevis
oocytes.
One of the features that makes ANAP labeling so versatile is the devel-
opment of a transfectable plasmid (pANAP) encoding tRNACUA that is
capable of being charged with ANAP. The plasmid also encodes the tRNA
synthetase enzyme that does the charging (Fig. 1A). The tRNACUA/synthetase
are based on the Escherichia coli amber suppressor tRNACUA and leucyl-tRNA
synthetase (Lee et al., 2009). The tRNACUA/synthetase pair was subjected
to several rounds of positive and negative selection with randomly mutated
leucyl-tRNA synthetase clones in Saccharomyces cerevisiae to produce a
tRNACUA/synthetase pair that is specific for ANAP, recognizes the amber stop
codon, and is completely orthogonal to the eukaryotic translation machinery.
Multiple papers have been published regarding the synthesis of ANAP,
including one in which the synthesis has been optimized to be enantiospecific
(producing only the L enantiomer) and convenient to be performed without
an abundance of specialist equipment (Lee et al., 2009; Wen et al., 2015;
Xiang & Wang, 2011). ANAP is also commercially available in its free acid
(trifluoroacetic salt) form or as a methyl ester, which is a precursor of the free
acid in the multi-step synthesis described by Xiang and Wang (2011).
To produce ANAP-labeled protein, three things must be present in the
cell (Fig. 1A). Naturally, ANAP must be present. This may be introduced via
direct injection (for example, in Xenopus oocytes) or by dissolving ANAP in
the cell culture media. Both the free acid and methyl ester are membrane
permeant and capable of being inserted into proteins. In the case of the
methyl ester, this is presumably following hydrolysis by an endogenous

esterase. Fig. 1B shows that both forms of ANAP can be incorporated into
GFP (as assayed by GFP fluorescence) when included in the bathing
medium. The free-acid and ester forms differ somewhat in their solubility.
The methyl ester is soluble in DMSO or ethanol at 1–3 mM. The free acid is
soluble at 1 mM in aqueous 30–100 mM NaOH (Hsieh et al., 2014; Puljung
et al., 2019). As the acid form is sold as a trifluoroacetic salt with an inde-
terminate number of counter ions per ANAP molecule, it is recommended
to check the concentration of ANAP stocks by the absorbance at 360 nm,
rather than relying on dry weight to determine the concentration.
In addition to ANAP, cells must express a gene for the protein of inter-
est with the amber stop codon, TAG (UAG), replacing the codon
corresponding to the position intended for labeling. It is important that
the gene for the protein of interest does not naturally terminate with
TAG. If it does, the final stop codon must be mutated to either the opal
(TGA) or ochre (TAA) stop codon. The gene with the appropriate
stop-codon substitution is introduced via DNA transfection into cultured
cells or RNA injection into Xenopus oocytes.
In theory, any site on a protein can be labeled with ANAP. In practice,
as for any mutagenesis experiment, some ANAP substitutions do not pro-
duce functional channels. Labeling positions may be chosen based solely on
the site that the investigators wish to study (for example, a ligand-binding
site or voltage sensor). Many investigators have focused on regions in
which a change in the local environment is expected during channel gating
(Kalstrup & Blunck, 2017; Shandell et al., 2019; Soh et al., 2017; Wen
et al., 2015; Wulf, 2018). To maximize the probability of producing func-
tional channels, some groups have emphasized choosing solvent-exposed
sites (Islam et al., 2019; Zagotta et al., 2016) or making conservative sub-
stitutions (for example, aromatic amino acids) (Islam et al., 2019; Usher
et al., 2020). Kalstrup and Blunck (2017) found that scanning every residue
in a protein region of interest is the most productive, an approach adopted
elsewhere. Yang et al. used electrophysiology to screen 142 potential
ANAP-incorporation sites in TRPM8 for cold activation, identifying
15 positive constructs (Yang et al., 2020). Xu et al. used fluorescence emis-
sion spectra to screen 73 ANAP labeling sites on TRPM8 for menthol acti-
vation, identifying 39 positive constructs (Xu et al., 2020). Puljung et al. used
a luminescence-based surface-expression assay developed by Zerangue,
Schwappach, Jan, and Jan (1999) to identify ANAP-incorporated mutants
in one subunit of the KATP channel that support trafficking of the channel
complex to the plasma membrane (Fig. 1C) (Puljung et al., 2019). By exam-
ining surface expression in the absence and presence of ANAP, the authors
could identify and eliminate constructs for which truncated (at the amber
stop codon) protein could reach the plasma membrane. Of 37 sites screened,
only seven constructs exhibited surface expression above background, and
only three demonstrated ANAP-dependent surface expression. Finally,
Ca2+ imaging (when appropriate) offers a potential medium/high through-
put way to screen potential ANAP labeling sites for functional expression
(Zagotta et al., 2016). Like any mutation, incorporation of ANAP into a pro-
tein may alter its local structure and function. Fortunately, electrophysiology
offers direct access to the function of ion channel proteins to test the func-
tionality of ANAP-tagged mutants.
Finally, for tagged protein expression, cells must express the ANAP-
specific tRNACUA/synthetase pair. pANAP (available from AddGene),
which encodes the appropriate tRNACUA/synthetase pair, can be transfected
into cultured cells or injected directly into the nucleus of Xenopus oocytes. In
the presence of ANAP, the orthogonal tRNA synthetase will charge the
orthogonal tRNACUA with ANAP. At the ribosome, rather than terminat-
ing translation at the amber stop codon in the gene of interest, ANAP will be
inserted into the nascent peptide. The ultimate product is full-length protein
with ANAP incorporated at the appropriate site (Fig. 1A).
3. ANAP applications in ion channel research

The environmental sensitivity of ANAP as well as its potential to act
as a F€
orster resonance energy transfer (FRET) donor have contributed to its
versatility as an ion channel probe. ANAP has been used to track ligand
binding, gating conformational changes, and protein-protein interactions
(Table 1). Robust expression of ANAP-tagged channels has been achieved in
Xenopus oocytes, HEK-293 cells, CHO cells, and tSA-201 cells. Non-channel
experiments have also been performed in Xenopus oocytes (Kawanabe et al.,
2018; Sakata & Okamura, 2019), HEK-293 cells (Chatterjee et al., 2013;
Islam et al., 2019; Mitchell et al., 2017), CHO cells (Chatterjee et al., 2013),
HeLa cells (Chatterjee et al., 2013; Park, Ko, Lee, & Shin, 2019; Park, Ko,
Park, Lee, & Shin, 2020), and yeast (Lee et al., 2009). As the tRNACUA/
synthetase pair used for ANAP expression is derived from E. coli, it is not
orthogonal and therefore not suitable for use in prokaryotic expression systems
(Lee et al., 2009). ANAP experiments have been performed on intact cells
and oocytes (Dai, 2020; Dai et al., 2018; Kalstrup & Blunck, 2013, 2015,
2017, 2018; Luo et al., 2019; Shandell et al., 2019; Wen et al., 2015; Xu et al.,
2020; Yang, 2018; Yang et al., 2020), in unroofed plasma membranes
(Puljung et al., 2019; Usher et al., 2020; Zagotta et al., 2016), and in excised
membrane patches under voltage clamp (Aman et al., 2016; Dai, 2019; Dai
et al., 2018; Dai & Zagotta, 2017; Soh et al., 2017; Usher et al., 2020; Wulf,
2018). Below are some examples of ANAP applications drawn from the
channel literature.
3.1 Conformational changes associated with voltage gating

Kalstrup and Blunck used ANAP to follow conformational changes in the
Shaker K+ channel that were correlated with movements of the S4 voltage-
sensor helix (Kalstrup & Blunck, 2013). Previous VCF studies on Shaker had
been limited to positions at the extracellular end of S4 labeled with
thiol-reactive dyes like tetramethylrhodamine (TMR) (Cha & Bezanilla,
1997; Mannuzzu et al., 1996). Using amber stop-codon suppression/
ANAP allowed the authors to label intracellular positions on Shaker as well.
Their experiments were performed in cut-open Xenopus oocytes under VCF.
ANAP was excited with a halogen lamp and changes in fluorescence were
measured using a photodiode, enabling the fast readout times required to fol-
low gating conformational changes. This approach was used in conjunction
with ionic current measurements, gating current measurements (currents
generated primarily by the outward movement of the positively charged
S4 helix across the plasma membrane in response to voltage changes), and
measurements of the conformational change in the outer S4 in double-
labeling experiments with TMR linked to an external cysteine. This enabled
the authors to correlate movements of the outer S4 with conformational
changes at the inner S4, the internal channel gate, and the S4-S5 linker that
couples voltage sensor movement to pore opening (Kalstrup & Blunck,
2013, 2018). The observed fluorescence changes were presumably due to
a change in the polarity of the environment surrounding the ANAP label,
although this cannot be concluded definitively in the absence of emission
spectra. An alternative hypothesis is that ANAP may have been in close
enough proximity to amino acids like tryptophan to allow for quenching
via photoinduced electron transfer, which is known to occur between the
related fluorophore Prodan and tryptophan (Pospı́šil et al., 2014).
A similar approach was employed to study movements associated with
the voltage sensor of Ci-VSP (Kawanabe et al., 2018; Sakata et al., 2016).
Whereas this protein is not an ion channel, its voltage sensing domain is
homologous to that of the voltage-dependent K+ channel superfamily.
Ci-VSP was expressed in Xenopus oocytes. Sensing currents (analogous to

gating currents) were measured alongside changes in the fluorescence of
ANAP incorporated into the cytoplasmic catalytic domain. Movement
of S4 was correlated with conformational changes in the catalytic domain.
However, S4 movement did not bring the catalytic domain closer to the
plasma membrane as there was no change in distance between ANAP in
the catalytic domain and the membrane soluble FRET quencher
dipicrylamine (Sakata et al., 2016).
A different approach was adopted by Dai et al. to monitor the move-
ment of the S4 voltage sensor in hyperpolarization-activated, cyclic
nucleotide-modulated (HCN) channels (Dai, 2019). HCN channels are
homologous to the voltage-activated Shaker K+ channels, but channel
opening is induced by inward S4 movement upon hyperpolarization.
ANAP was inserted at several sites in the S4 of spHCN (from the sea urchin
Strongylocentrotus purpuratus). The fluorescence at each position changed
upon hyperpolarization to 100 mV, presumably due to a change in the
local environment as these residues moved from being embedded in
the plasma membrane to the cytoplasm.
In order to map S4 movements, the authors used a technique called
transition metal ion FRET (tmFRET) (Taraska, Puljung, Olivier,
Flynn, & Zagotta, 2009; Taraska, Puljung, & Zagotta, 2009). This tech-
nique is based on the observation that colored transition metal ions can
quench fluorophores (Richmond, Takahashi, Shimkhada, & Bernsdorf,
2000; Sandtner, Bezanilla, & Correa, 2007). In a typical tmFRET exper-
iment, small fluorophores attached to the protein backbone with short
linkers are used as donors and colored transition metal ions (Ni2+, Co2+,
Cu2+) bound nearby are used as acceptors. Transition metal ions can act
as weak FRET acceptors because their absorbance spectra overlap with
the fluorescence emission spectra of many fluorophores. Because they
are only weak FRET acceptors, transition metals must be very close to
the donor molecules for FRET to occur (half-maximal FRET on the order
of 12–15 Å). Thus, very small distances and changes in distance can be mea-
sured. Metal ions are typically bound to di-histidine motifs introduced by
mutagenesis (Taraska, Puljung, & Zagotta, 2009). Importantly, when used
in conjunction with ANAP, tmFRET allows for high specificity, as both
the donor and acceptor sites are genetically encoded.
Dai et al. used PCF in conjunction with tmFRET and a variation on
tmFRET called ANAP Cyclen-Cu2+ resonance energy transfer (ACCuRET)
in which the acceptor Cu2+ ions are bound to the cysteine reactive chelator
(1-(2-pyridin-2-yldisulfanyl)ethyl)-1,4,7,10-tetraazacyclododecane, TETAC),
to monitor the movement of the S4 voltage sensor of spHCN (Dai, 2019;

Gordon et al., 2018). ANAP at various positions on S4 was quenched by both
Co2+ ions bound to a di-histidine motif in the channel’s N-terminal HCN
domain, and Cu2+ ions bound to TETAC attached to the same domain, in a
voltage-dependent fashion. Using the HCN cryo-EM structure (Lee &
MacKinnon, 2019) and distance constraints generated at positive and negative
voltages, the authors modeled the structure of the down (activated) state of
the voltage sensor. In a recent pre-print, the same authors extend this approach
to examine movements of the C-linker of spHCN, which connects the cyclic
nucleotide-binding domain (CNBD) to the transmembrane domain (Dai
et al., 2021).
FRET between ANAP and bound Co2+ ions was also used to monitor
rearrangements in the intracellular domains of the zebra fish ether-à-go-go
like K+ (zELK) channel expressed in Xenopus oocytes (Dai & Zagotta,
2017). Interactions between the N-terminal eag domain and the
C-terminal cyclic nucleotide-binding homology domain (CNBHD) are
important for proper trafficking and gating of ether-à-go-go-family chan-
nels (Curran et al., 1995; Gustina & Trudeau, 2009, 2013). These inter-
actions were explored using PCF in conjunction with fluorescence
emission spectra. ANAP incorporated into the eag domain was strongly
quenched by Co2+ bound to a pair of histidines in the CNBHD. The
degree of quenching was modified by voltage and the presence of
MgATP. Rearrangement of the intracellular domains of zELK accompa-
nied changes in channel activation.
The CNBHD of zELK is homologous to the CNBD that modulates gat-
ing of both HCN channels and cyclic nucleotide-gated (CNG) channels.
Rather than binding cyclic nucleotides, the CNBHD of zELK is self-ligated
by a C-terminal intrinsic ligand motif (Brelidze, Carlson, Sankaran, &
Zagotta, 2012). VCF was used to monitor the fluorescence of ANAP incor-
porated into the intrinsic ligand of zELK expressed in Xenopus oocytes
(Dai et al., 2018). At two labeling positions, ANAP increased its fluorescence
in response to depolarizing voltage steps. tmFRET between ANAP on the
intrinsic ligand and Co2+ bound to histidines in the CNBHD demonstrated
voltage-dependent movement of the intrinsic ligand relative to the
CNBHD. This conformational change required the N-terminal PAS cap
domain and correlated with voltage-dependent potentiation of zELK.
ANAP was also used to probe inactivation of the cardiac Na+ channel,
Nav1.5 (Shandell et al., 2019). To examine conformational changes in
Nav1.5, the authors developed a K+ depolarization assay. Nav1.5 tagged
with ANAP on the inactivation gate was co-expressed in HEK-293 cells

with a GFP-tagged Kir4.1 inward rectifier K+ channel, so that the cells could
be depolarized by increasing the external K+ concentration. Spectra
acquired during depolarization revealed a shift in the ANAP spectrum to
longer wavelengths, implying that the fluorophore moves to a more polar
environment during inactivation. Deletion of the distal C-terminus of
Nav1.5 eliminated this red shift, implying that the C-terminus was required
to stabilize the inactivation conformational change.
3.2 Activation of TRP channels

Several studies have exploited the spectral properties of ANAP to explore
gating of the temperature-sensitive transient receptor potential (TRP) fam-
ily channels TRPV1 and TRPM8. The N-terminal region of the heat- and
capsaicin-activated TRPV1 channel contains 6 ankyrin repeat domains
(ARDs) that are splayed out horizontally such that they are nearly parallel
with the plasma membrane (Liao, Cao, Julius, & Cheng, 2013). A compar-
ison of the TRPV1 cryo-EM structure with the structure of the related
TRPA1 channel (Paulsen, Armache, Gao, Cheng, & Julius, 2015) led to
the speculation that the ARDs may move relative to the plasma membrane
during the gating process. Zagotta et al. introduced ANAP at three separate
positions in the ARD and measured FRET between ANAP and Co2+
bound to a metal chelating lipid (stearoyl-nitrilotriacetic acid, C18-NTA)
in unroofed plasma membranes (Zagotta et al., 2016). Unroofed membranes
are prepared by sonicating cells on a coverslip, leaving behind adherent frag-
ments of plasma membrane with the intracellular face exposed to bulk solu-
tion (Heuser, 2000). Using this approach, the authors found that FRET
between ANAP and Co2+ bound to C18-NTA in the plasma membrane
was stronger for sites that were predicted to be closer to the plasma mem-
brane based on the cryo-EM structure of TRPV1. Activating TRPV1 with
a saturating concentration of capsaicin did not change the distance between
the ARDs and the plasma membrane.
ANAP was introduced at various positions near the TRPV1 selectivity
filter to study the role of this region in channel activation. By examining
emission spectra acquired from intact HEK-293 cells expressing ANAP-
tagged protein, the authors identified three positions where the peak
ANAP emission shifted to longer wavelengths in the presence of 10μM cap-
saicin, suggesting that amino acids in this region move to an increasingly polar
(presumably aqueous) environment upon channel activation (Yang, 2018).
FRET between ANAP at the N-terminus of mouse TRPV1 and a YFP tag at
the channel’s C-terminus increased during heat-dependent desensitization, as
measured using whole-cell PCF (Luo et al., 2019). No change in FRET was
observed between N-terminal ANAP and C-terminal GFP incorporated into
platypus TRPV1, which does not undergo heat-dependent desensitization.
This observation confirms that desensitization is accompanied by a shortening
of the distance between the N- and C-termini of TRPV1.
A similar approach uncovered three positions in the pore domain of
TRPM8 at which the ANAP fluorescence was red-shifted during cold acti-
vation, suggesting these positions increased their aqueous exposure (Yang
et al., 2020). Emission spectra from whole cells expressing TRPM8 tagged
with ANAP at 73 different sites uncovered widespread conformational
changes during activation by menthol (Xu et al., 2020). Spectral shifts were
observed for ANAP incorporated at sites throughout the pore domain, in
the S2 helix, the S2-S3 linker, and S3-S4 linker.
3.3 Binding and activation in ligand-gated channels

Various researchers have used ANAP to study ligand binding and confor-
mational changes in ligand-gated ion channels. Two studies have used
ANAP to explore activation and inhibition of acid-sensing ion channels
(ASICs). ANAP incorporated into a pore-lining residue of ASIC1a acted
as a reporter of the local confirmation, increasing its fluorescence during
activation by low pH in a PCF experiment (Wulf, 2018). Application of
tetraethylammonium to the intracellular face of the channel modulated
the environment around this residue, even when the channel was closed.
In a separate study, emission spectra were acquired from cells in which
ANAP was incorporated into the acid-binding pocket and first transmem-
brane domain of ASIC1a (Wen et al., 2015). ANAP fluorescence at labeled
positions in both regions was quenched by binding of the toxin mambalgin-1,
an ASIC inhibitor from the black mamba. Quenching in the acid-binding
pocket presumably arose as a direct consequence of toxin binding, as this site
has been proposed to be the binding site. Quenching in the first transmem-
brane domain likely reflected the conformational change induced by toxin
binding.
Ligands that bind to receptors but fail to fully activate them are termed
partial agonists. Soh et al. used the environmental sensitivity of ANAP to study
the nature of partial agonism in the glycine receptor (GlyR), an inhibitory
neurotransmitter receptor (Soh et al., 2017). They expressed channels with

ANAP inserted at several positions at the interface between the transmem-
brane domains and extracellular ligand binding domain of human GlyR
α1. Using VCF, with simultaneous monitoring of receptor currents via
TEVC and ANAP fluorescence using a photomultiplier tube, the authors
tested receptor responses to a panel of GlyR agonists of varying efficacies.
Several positions showed graded fluorescence changes that correlated with
agonist efficacy, demonstrating that the ability to flip the conformation in this
loop region is necessary for a ligand to promote channel opening.
Activity of the retinal cyclic nucleotide-gated CNGA1 channel is poten-
tiated by Ni2+ ions (Gordon & Zagotta, 1995). Potentiation involves a his-
tidine residue (H420) located on an intracellular helix (the A’ helix) adjacent
to the plasma membrane. However, the concentration dependence of Ni2+
potentiation suggests that more than just a single histidine residue must be
involved in binding. Aman et al. (2016) hypothesized that Ni2+ may
be coordinated between H420 and anionic lipids in the plasma membrane.
To explore this hypothesis, the authors used PCF with ANAP at position
417, one helical turn away from H420. Co2+, which also potentiates
CNGA1 quenched ANAP at position 417, presumably by a tmFRET
mechanism. When H420 was mutated to a glutamine, this quenching
was lost. The effect of this mutation, coupled with the short working dis-
tance of tmFRET, provides strong evidence that H420 was directly involved
in transition metal binding. Adding additional metal binding sites to the
membrane with C18-NTA shifted the concentration dependence of Co2+
potentiation to lower concentrations, confirming that channel opening
was promoted by coordinating metal ions between H420 and the plasma
membrane.
Gating of pancreatic ATP sensitive K+ channels (KATP) is affected by
changes in intracellular concentrations of the adenine nucleotides ATP
and ADP (Vedovato, Ashcroft, & Puljung, 2015). Nucleotide modulation
is complicated, with one site on each of four pore-forming Kir6.2 subunits
conferring nucleotide inhibition and two sites on each of four modulatory
sulfonylurea receptor (SUR1) subunits conferring nucleotide-dependent
activation in the presence of Mg2+. The response of KATP to changes in
nucleotide concentrations is shaped by the coordinated activity of all three
classes of site (12 sites in all) working together. Two recent papers have
attempted to isolate individual binding sites on KATP to understand the
details of nucleotide binding to each site and determine the energetic
A 0 TNP-ADP B NH2
100 50 nM O O N N
500 nM 1.0 HO
P
O
P
O N N
fluorescence (a.u.)
O
5 µM OH OH
80
50 µM 0.8 O O
O2N NO2
500 µM TNP-ADP
F/ Fmax
60 1 mM
0.6 O-
N+
O-
40 0.4
20 0.2
0 0.0
450 500 550 600 650 700 10-8 10-7 10-6 10-5 10-4 10-3 10-2
wavelength (nm) [nucleotide] (M)
Fig. 2 Binding of TNP-ADP to ANAP-tagged KATP channels. (A) Emission spectra of ANAP-
labeled KATP channels acquired from unroofed HEK-293 cells. The peak at 470 nm repre-
sents ANAP. TNP-ADP binding quenched the ANAP fluorescence via FRET. A second peak
at 560 nM corresponds to TNP-ADP fluorescence. (B) Normalized ANAP fluorescence (470
nm) from the spectra in (A) plotted as a function of the TNP-ADP concentration. The curve
was fit with a modified Hill equation. The inset shows the structure of TNP-ADP. Panel (A) is
reproduced from Puljung, M., Vedovato, N., Usher, S., & Ashcroft, F. (2019). Activation mech-
anism of ATP-sensitive K+ channels explored with real-time nucleotide binding. eLife, 8,
e41103. doi:10.7554/eLife.41103, published under the Creative commons Attribution license,
http://creativecommons.org/licenses/by/4.0/.
consequences of binding at each site for channel gating (Puljung et al.,

2019; Usher et al., 2020). Both papers used ANAP to label individual bind-
ing sites. Binding was assessed by FRET between ANAP and fluorescent
trinitrophenyl (TNP) nucleotides, which provided the necessary spatial
resolution to separate binding to each site (Fig. 2).
Binding to the stimulatory nucleotide-binding site 2 (NBS2) on SUR
was measured in unroofed membranes (Puljung et al., 2019). Nucleotide
association with NBS2 could be observed by quenching of the donor
(ANAP) peak and an increase in acceptor (TNP-nucleotide) fluorescence
(Fig. 2A and B). The authors monitored the nucleotide dissociation rate
from NBS2 in the presence and absence of Mg2+, concluding that Mg2+
was not necessary for nucleotide binding, but required to promote the con-
formational change in SUR that leads to KATP activation. In a separate
paper, the authors used PCF to simultaneously measure binding to the
inhibitory site on Kir6.2 and nucleotide inhibition of channel currents
(Usher et al., 2020). Fitting the simultaneous binding and gating data
allowed the authors to propose a Monod-Wyman-Changeux-type model
for nucleotide inhibition of KATP, whereby each nucleotide-binding event
was independent and contributed an equal amount of energy to channel
closure.
4. Non-channel applications for ANAP

Outside of the channel field, ANAP has been used for various appli-
cations. Emission spectra of ANAP tagged glutamine binding protein from
E. coli expressed in yeast were acquired at various concentrations of gluta-
mine (Lee et al., 2009). The peak emission wavelength of ANAP blue
shifted by 50 nm in the presence of glutamine, allowing the authors to
detect binding. ANAP was used to follow the sub-cellular localization of
Histone-H3 and the ER resident proteins Grp94 and GalT1 in living cells
(Chatterjee et al., 2013). ANAP has also been used to develop assays for
antibody binding (Islam et al., 2019), in situ protein folding (Hsieh et al.,
2014; Mitchell et al., 2017), and proteolysis in living cells (Mitchell
et al., 2017). Finally, ANAP has been used as a donor fluorophore for
FRET studies monitoring the interaction between the pro-apoptotic pro-
tein Bax and Hsp-70 (Park et al., 2019, 2020) and the interaction between
the Alzheimer’s disease associate protein β-site amyloid precursor protein-
cleaving enzyme 1 (BACE1) and KCNQ2/3 K+ channels (Dai, 2020).
5. Expression of ANAP-tagged channels

Numerous groups have expressed tagged channels (and voltage-
sensitive phosphatases) in Xenopus oocytes. Unlike for a typical oocyte
experiment, in which in vitro transcribed mRNA encoding channel sub-
units is injected into the ooplasm, expression of ANAP-labeled protein
involves two injections (Kalstrup & Blunck, 2017). The plasmid encoding
the tRNACUA/synthetase pair (pANAP) must be injected directly into the
nucleus of oocytes. Injection volumes between 9.2 and 50 nL are reported
for nuclear injections. pANAP concentrations vary from 10 to 200 ng/μL,
with the total amount of DNA varying from 0.2 to 8 ng. In their studies,
Kalstrup and Blunck injected 9.2 nL of 100 ng/μL (a total of 0.92 ng) of
DNA (Kalstrup & Blunck, 2017, 2018).
Injecting the nucleus of an oocyte is more involved than injecting the
ooplasm. The nucleus is located under the animal (darkly pigmented) pole
of the oocyte. Kalstrup and Blunck recommend using a long, thin glass nee-
dle to inject the center of the animal pole at a depth two to three times that
for a typical oocyte injection (Kalstrup & Blunck, 2017). They report a
10%–40% failure rate for their nuclear injections. To verify nuclear injec-
tions in my experiments, I included 5 ng of an additional plasmid,
OH
A HO
P
O OH
B
O
alkaline
phosphatase
+ H2O
1.2 4
N N
O O O O
3
absorbance
0.8
A405/A310
2
0.4
1
0.0 0
300 400 500
nm sham SEAP
Fig. 3 An assay for successful nuclear injections of Xenopus oocytes. (A) Alkaline
phosphatase converts 4-nitrophenyl phosphate (left, peak absorbance 310 nm) to
4-nitrophenol (right, peak absorbance 405 nm). The spectrum of 9 mM 4-nitrophenyl
phosphate before (blue, dashed) or after (red, solid) reaction with 10 U/100 μL calf intes-
tinal phosphatase is shown below. Spectra were acquired using a NanoDrop spectro-
photometer (ThermoFisher). (B) Ratio of the absorbance at 405 nm to absorbance at
310 nm for the bathing media surrounding oocytes injected with CMV-SEAP or sham
injected oocytes. Oocytes were cultured in 96-well plates in the presence of 100 μM
4-nitrophenyl phosphate.
CMV-SEAP (available from AddGene). Nuclear injection of CMV-SEAP

directs the expression of a secreted alkaline phosphatase. Presence of this
enzyme, which indicates a successful nuclear injection, can be assayed
for by the inclusion of a soluble substrate, 4-nitrophenyl phosphate
(100 μM), in the bathing medium and culturing oocytes in individual wells
of a 96-well plate (Fig. 3A). In the presence of the secreted phosphatase,
4-nitrophenyl phosphate (peak absorbance 310 nm) is converted to
4-nitrophenol (peak absorbance 405 nm). The presence of SEAP in a given
well can be inferred from the appearance of the yellow-colored reaction
product. Alternatively, the absorbance at 405 and 310 nm can be measured
from aliquots of the bathing media. Fig. 3B shows a comparison of the A405
nm/A310 nm ratio for the bathing media surrounding sham and SEAP-
injected oocytes. In this particular experiment 12 out of 39 oocytes secreted
phosphatase, a success rate of 30% for nuclear injections. This approach
can be used to optimize nuclear injections or as a selection tool for success-
fully injected oocytes.
Nuclear injection of pANAP is followed by injection of channel mRNA
into the ooplasm. The second injection is performed 1–3 days after the
nuclear injection. Most investigators inject a total of 40–50 nL of RNA
(concentrations vary) mixed with ANAP at a final concentration of

0.5–2 mM. Soh et al. performed separate injections for ANAP and RNA
(Soh et al., 2017). The first injection consisted of 20–40 nL of 2 mM
ANAP. 2–40 nL of channel RNA was injected at the same site after a delay
of 6–8 h. Other investigators have opted not to inject ANAP, but to include
ANAP (methyl ester) in the oocyte storage medium (Aman et al., 2016).
Injection of ANAP represents an economical option, as much smaller
amounts are required for injection than are used when ANAP is added to
the culture media. When possible, ANAP injections should be performed
under red light and oocytes shielded from light (for example, with aluminum
foil) after injection (Kalstrup & Blunck, 2013). These precautions are taken
to minimize photobleaching of ANAP prior to experiments. Recordings are
usually carried out 2–4 days following the last injection.
ANAP-labeled channels have also been produced in cultured HEK-293,
tSA-201, and CHO cells. 0.3–1.2μg of pANAP (per well of a 6-well plate)
were used during transfections. Channel DNA is co-transfected with pANAP.
A HeLa cell line stably expressing pANAP has also been prepared (Park et al.,
2020). ANAP (10–50 μM, typically 20 μM) is introduced to the culture media
at the time of transfection or several hours later. Whereas some groups report
using the methyl ester form of ANAP because it is membrane permeant,
in actuality both the free acid and methyl ester of ANAP are membrane
permeant and result in labeled protein when added to the culture medium
(Fig. 1B). Experiments are typically performed 1–5 days post transfection.
6. Controls and confounds

Depending on the intended application, there are several controls and
pitfalls one might consider when using ANAP to label ion channels. Many of
these considerations are not unique to ANAP, but apply to any experiments
using amber stop-codon suppression.
Truncated protein. Many techniques can be used to verify the expression of
full-length, ANAP-tagged proteins including western blotting (Chatterjee
et al., 2013; Gordon et al., 2018; Hsieh et al., 2014; Islam et al., 2019;
Puljung et al., 2019; Shandell et al., 2019; Usher et al., 2020; Zagotta
et al., 2016), mass spectrometry (Chatterjee et al., 2013; Islam et al.,
2019; Lee et al., 2009; Mitchell et al., 2017), and the addition of
C-terminal FP tags to channel constructs (Aman et al., 2016; Dai, 2019;
Dai et al., 2018; Dai & Zagotta, 2017; Luo et al., 2019; Puljung et al.,
2019; Usher et al., 2020; Yang, 2018; Zagotta et al., 2016). When labeling
D
A
l
ro
55
D
F1 P
nt
55
AP
eR NA
-E
F1 P
AP
co
eR NA
-E
AG
µg pA
R
AP
e
pA
AP
µg A
tiv
pA
SU
FL
p
2 µg
N
si
AN
µg
µg
pA
µg
po
no
no
5
0.
no
5
no
0.
0.
1
260
full-length
140
100 truncated
70
B V13TAG L14TAG T15TAG R16TAG L17TAG A18TAG
50 - + - + - + - + - + - + ANAP
40 full-length
35
25
truncated
Fig. 4 Western blots of ANAP-labeling controls. (A) Western blot for SUR1 protein with a
triple FLAG epitope. Cells were transfected with 1.5 μg of full-length SUR1 in pcDNA4/TO
or SUR with a stop codon at the position corresponding to amino acid 1353 in pcDNA4/
TO. Upper bands are from aggregated protein. The positions of full-length SUR1 and
truncated (after position 1352) protein are indicated. Each lane corresponds to
HEK-293 cells transfected in a single well of a 6-well plate. Cells were co-transfected with
0.5 μg of Kir6.2 in pcDNA4/TO with pANAP and eRF1-E55D transfected as indicated. Cells
were cultured in 20 μM ANAP (trifluoroacetic salt) except where indicated. The positive
control was full-length, FLAG-tagged SUR1. Negative controls were cells transfected
with SUR1 with no FLAG tag or cells that were not transfected with SUR1. (B) Western blot
for HA-tagged Kir6.2 with stop codons at the positions indicated. HEK-293 cells were
co-transfected with 1.5 μg of SUR1 in pcDNA4/TO, 1 μg of pANAP, and 1 μg of pERF1-
E55D per well of a 6-well plate. Cells were cultured in the presence or absence of 20 μM
ANAP (trifluoroacetic salt), as indicated. Truncated protein corresponds to N-terminally
truncated Kir6.2 generated from an internal start site after the initial methionine (Usher
et al., 2020).
proteins via amber stop-codon suppression, invariably some percentage of

the product will be truncated protein prematurely terminated at the amber
stop codon. Fig. 4A shows a western blot performed on SUR1, a subunit of
the pancreatic KATP channel. The SUR1 gene has an amber stop codon
corresponding to amino acid position 1353. The gene also encodes a triple
FLAG epitope upstream from this stop codon, so that both full-length and
truncated proteins could be identified on the blot using the same anti-FLAG
antibody. A positive control (no TAG) marks the position of the full-length
protein (lane 1). When 1.5 μg of SUR1 DNA were transfected with 0.5 μg
of pANAP into a single well of a 6-well plate and cells were cultured in
20 μM ANAP, about 45% of the SUR1 produced was full-length. 55% of
the product was truncated after position 1352 (lower band, lane 5).
Doubling pANAP in the transfection increased the percentage yield of
full-length protein to 80% (lane 4). Similar experiments are reported else-
where involving both the SUR1 and Kir6.2 subunits of KATP (Puljung
et al., 2019; Usher et al., 2020). Western blots performed by Zagotta
et al. show predominantly truncated TRPV1-YFP channels produced when
cells were transfected with 1.2 μg of TRPV1-YFP plasmid and 0.4 μg of
pANAP (Zagotta et al., 2016).
In addition to increasing the amount of pANAP during transfections, pro-
duction of full-length protein can be increased through the expression of
mutated eukaryotic release factors (eRFs). eRFs promote release of a nascent
peptide from the ribosome when a stop codon is reached. Decreasing the rate
of peptide release allows more time for an ANAP-charged tRNACUA to reach
the ribosome. In yeast, the [PSI+] strain, in which part of the native eRF
complex forms aggregates, allowed for greater expression of ANAP-labeled
luciferase (Hsieh et al., 2014). Some groups overexpress a mutated eRF
(eRF1-E55D) to increase the fraction of full-length ANAP-labeled protein
(Gordon et al., 2018; Puljung et al., 2019; Usher et al., 2020). The E55D muta-
tion selectively enhances incorporation of unnatural amino acids at the
amber stop codon with no concomitant increase in read-through for the ochre
or opal stop codons (Schmied, Els€asser, Uttamapinant, & Chin, 2014). Fig. 4A
shows that the additional transfection of 1–2 μg of a plasmid expressing
eRF1-E55D with 0.5μg of pANAP increased the percentage of full-length,
ANAP-tagged SUR1 to 95% and 89%, respectively (Fig. 3A, lanes 2 and 3).
Similar effects have been reported elsewhere (Puljung et al., 2019).
Proteins tagged with unnatural amino acids usually express at lower levels
than their untagged counterparts. For example, the surface expression of
ANAP-labeled KATP channels in Fig. 1C was much lower than for
wild-type channels. Several attempts have been made to bolster expression
of ANAP-labeled channels, although few studies demonstrate the quantita-
tive effects of such interventions. Kalstrup and Blunck cultured their oocytes
in solution containing 5% horse serum to increase protein expression
(Kalstrup & Blunck, 2017). Lowering the incubation temperature of
cultured cells slows cell division and may increase protein produced on a
per-cell basis (Lin et al., 2015; Puljung et al., 2019; Usher et al., 2020).
The most quantitative attempts to maximize production of full-length
ANAP-labeled protein involve titrating the amount of ANAP in the bathing
media, using GFP reporter constructs. These constructs contain an amber
stop codon in their sequence (for example, GFP-Y40TAG) and do not
exhibit any green fluorescence unless pANAP and ANAP are present. As
such, they are a useful positive control for ANAP labeling. ANAP concen-
trations from 100 nM to 500 μM were tested for protein expression (Aman
et al., 2016; Chatterjee et al., 2013; Mitchell et al., 2017). As little as
1–10 μM ANAP was required for robust GFP expression as assayed by fluo-
rescence microscopy (Chatterjee et al., 2013; Mitchell et al., 2017). Zagotta
et al. used fluorescence-detection size-exclusion chromatography (FSEC) to
quantify expression of GFP Y40ANAP in lysates prepared from HEK-293
cells expressing pANAP and cultured in various concentrations of ANAP
methyl ester (Zagotta et al., 2016). They showed increasing expression of
GFP Y40ANAP at concentrations between 100 nM and 30 μM ANAP.
At higher ANAP concentrations, the authors noted fluorescent precipitate
and cellular toxicity. The majority of laboratories use 20 μM ANAP in the
bathing media for protein expression.
Truncated protein produced by premature termination at the amber stop
codon does not contribute to the fluorescence background in an experi-
ment, as ANAP incorporation would lead to amber stop-codon suppression
and the production of full-length constructs. However, prematurely trun-
cated protein may contribute to the ionic current background. Kalstrup
and Blunck provide an illustration of the currents that can be produced
by the isolated voltage-sensor domain of Shaker K+ channels truncated at
position 382 (Kalstrup & Blunck, 2017). To identify the potential for back-
ground currents, control electrophysiology experiments should be con-
ducted for each construct in the absence of pANAP or ANAP. Surface
expression screens like that described by Puljung et al. (Fig. 1C) provide
a medium/high throughput way to identify constructs that only demonstrate
surface expression in the presence of ANAP, that is, constructs for which
truncated protein does not reach the plasma membrane (Puljung et al.,
2019; Zerangue et al., 1999). In the presence of pANAP and ANAP, expres-
sion of truncated protein is suppressed. As such, the background currents in a
given experiment are likely to be much smaller than those identified in no
ANAP or no pANAP controls.
Another potential experimental confound is read-through of the amber

stop codon. This could occur, for example, through recognition of this
codon by the wrong tRNA, or by inappropriate charging of the orthogonal
tRNACUA with an amino acid other than ANAP. The latter possibility is
remote as the orthogonal tRNACUA/synthetase pair used for ANAP label-
ing was subject to negative selection in yeast to ensure specific charging with
ANAP (Lee et al., 2009). However, this does not guarantee that tRNACUA
cannot be charged with native amino acids in other expression systems.
The best control for leak expression from accidental read-through of the
amber codon is to perform experiments in the absence of ANAP. No pANAP
controls do not eliminate the possibility that leak expression could result from
charging of the amber stop codon-recognizing tRNACUA with a different
amino acid. Read-through of the amber stop codon was more prevalent in
[Psi+] yeast (Hsieh et al., 2014). As such, caution should be exercised when
using mutated eRF1-E55D to augment expression of full-length protein
and eRF1-E55D should be included in any controls. Expressing protein with
a C-terminal FP tag in addition to the ANAP labeling site can help control for
leak channel expression, which would be evident from FP fluorescence in the
absence of ANAP. Western blots can also be used to test for read-through. In
Fig. 4A, lanes 6 and 7 shows that no full-length SUR1 protein was produced
in the absence of pANAP or in the absence of ANAP (Puljung et al., 2019).
Similar observations were made by Zagotta et al. using western blots and
FSEC (Zagotta et al., 2016). Surface-expression assays can be performed in
the absence of ANAP and ionic currents can be measured (Kalstrup &
Blunck, 2017; Puljung et al., 2019; Shandell et al., 2019; Soh et al., 2017;
Usher et al., 2020; Wen et al., 2015). Both of these assays will detect leak
expression in addition to expression associated with truncated proteins.
A related issue arises when using amber stop codons to insert ANAP
toward the N-terminus of a protein of interest. If a protein is terminated
via a stop codon within the region encoding the first 70 amino acids, there
is a high probability of re-initiation before the ribosome has a chance to dis-
engage completely from the mRNA (Kalstrup & Blunck, 2015). This will
result in the expression of N-terminally truncated protein produced from an
internal start site. Re-initiation may occur from an internal methionine, but
may use non-canonical start sites. Kalstrup and Blunck demonstrated that
silent mutations in alternative start sites reduced re-initiation, with leak
expression reduced to 1% after simultaneous mutation of six different
alternative start sites (Kalstrup & Blunck, 2015).
Fig. 4B shows a portion of the results from experiments in which the

codons for residues 2 through 24 of Kir6.2, the pore-forming subunit of
KATP, were replaced one at a time with the amber stop codon. Expression
of protein was assessed using western blots by virtue of an HA tag downstream
from the stop codon. Wild-type constructs showed a prominent truncated
band (Usher et al., 2020). This truncation was identified as N-terminal
because addition of a C-terminal GFP tag shifted the truncated band by
the expected amount. The band likely reflects protein translated from an
internal start site, as the truncated band appeared for every construct with stop
codons at positions 2 through 24 in the absence of ANAP. However, muta-
tion of downstream methionines at positions 77 and 88 did not eliminate
expression of this truncated band (data not shown). In addition to the
N-terminal truncation, 17 out of 23 amber stop codon constructs tested
showed some degree of read-through expression of full-length Kir6.2 in
the absence of ANAP. This is particularly evident for the Kir6.2-R16TAG
construct (Fig. 4B, lane 7). Therefore, great caution should be taken when
attempting to incorporate ANAP near the N-terminus of a protein.
Background fluorescence from unincorporated ANAP and off-target
incorporation of ANAP is also a concern. Unincorporated ANAP in the
cytosol and the plasma membrane can both contribute to background
fluorescence. In Xenopus oocytes, injection of ANAP, rather than bathing
oocytes in ANAP (methyl ester) seems to result in lower background in the
plasma membrane (Aman et al., 2016; Dai & Zagotta, 2017). Elimination of
background fluorescence is less critical for VCF experiments where changes
in fluorescence are more important than absolute fluorescence. In VCF,
background fluorescence (due to unincorporated ANAP and oocyte
autofluorescence) can be minimized by imaging the animal pole of the
oocyte, as the pigment blocks much of the background. It has been
reported that fluorescence from unincorporated ANAP undergoes photo-
bleaching at a faster rate than for ANAP incorporated into protein and thus
bleaching can be used to lower background (Aman et al., 2016). For
whole-cell imaging in cultured cells, ANAP can be excluded from the cul-
ture media for 24 h prior to imaging and rinsed away with fresh media (Luo
et al., 2019; Xu et al., 2020; Yang, 2018). Depending on the environment
into which ANAP is incorporated on a protein of interest, background
fluorescence may also be recognized and differentiated from labeled protein
via its peak emission wavelength (Puljung et al., 2019).
Differentiating between the fluorescence signal from labeled protein
and unincorporated ANAP can be difficult. If there are no functional
effects, adding a C-terminal FP tag to channel constructs is recommended.

That way, cells or membranes expressing labeled channels can be identified
by their FP fluorescence. Locating expressing cells by their FP fluorescence
instead of ANAP fluorescence also reduces the photobleaching of ANAP
prior to experiments. To identify the background level from unincorporated
ANAP, full-length, FP-tagged protein (no stop codon) can be expressed in
cells along with pANAP and with the addition of ANAP (Aman et al., 2016;
Dai & Zagotta, 2017; Puljung et al., 2019; Shandell et al., 2019; Usher et al.,
2020). Transfected cells can be identified by virtue of the fluorescent tag,
but no specific ANAP signal is expected to be present. Fig. 5A shows
confocal images acquired from cells expressing KATP channels in the presence
of pANAP and ANAP (Usher et al., 2020). In the top panel, the GFP-
tagged Kir6.2 subunit of KATP has an amber stop codon corresponding to
amino acid position 311. Strong fluorescence for both ANAP and GFP
are found at the membrane (counterstained with a plasma membrane-
incorporated dye). In contrast to this, when the same construct is expressed
with no stop codon, there is still strong GFP fluorescence at the plasma mem-
brane, but ANAP fluorescence is weak and confined to the cytoplasm or
intracellular structures. Thus, the ANAP background in the plasma mem-
brane is quite low. Such experiments represent an upper limit for ANAP
background because overexpression of labeled channels will compete for
ANAP and other resources that contribute to background fluorescence.
Several groups have made plots demonstrating a linear correlation between
ANAP fluorescence and the fluorescence of an FP tag on the same protein in
excised patches or unroofed membranes (Fig. 4B) (Aman et al., 2016; Dai,
2019; Dai et al., 2018; Dai & Zagotta, 2017; Puljung et al., 2019; Shandell
et al., 2019). If a linear fit of such a plot crosses the x-axis near the origin,
then the ANAP background can be considered negligible. Similar plots have
shown a correlation between the ANAP fluorescence and current from
excised patches (Aman et al., 2016; Dai & Zagotta, 2017).
ANAP may be incorporated into other cellular proteins, particularly those
that naturally terminate with the amber stop codon. Gordon et al. used SDS-
PAGE followed by in-gel fluorescence from whole-cell lysates to look for
non-specific ANAP incorporation in other proteins (Gordon et al., 2018).
Hsieh et al. identified incorporation of ANAP into several off-target proteins
in [PSI+] yeast (Hsieh et al., 2014). However, labeling of off target proteins
was greatly reduced by overexpression of the gene for luciferase with an amber
stop codon, again suggesting that competition for resources can reduce
ANAP background when tagged proteins are overexpressed.
A line scan intensity

cell mask GFP ANAP merge
0.4
Kir6.2ANAP-GFP
0.2
F/Fmax
0.0
0.4
Kir6.2-GFP
0.2
0.0
B
10000
1000
FANAP
100
10
1
1 10 100 1000 10000
FGFP
Fig. 5 Controls for background from unincorporated/off-target ANAP. (A) Confocal

images for cells transfected on coverslips in 6-well plates with 0.5 μg of GFP-tagged
Kir6.2 (with or without an amber stop codon corresponding to position 311), 1.5 μg of
SUR1 in pcDNA4/TO, 1 μg of pANAP, and 1 μg of eRF1-E55D. Cells were cultured in the
presence of 20μM ANAP. The plasma membranes were stained with 1 nM CellMask
Deep Red (ThermoFisher). The pale lines in the merge images correspond to the line scans
on the right. (B) Plot showing a correlation between the intensity of ANAP fluorescence
and GFP fluorescence from SUR1 labeled with both fluorophores in unroofed mem-
branes from HEK-293 cells. Panel (A) reproduced from Usher, S. G., Ashcroft, F. M., &
Puljung, M. C. (2020). Nucleotide inhibition of the pancreatic ATP-sensitive K + channel
explored with patch-clamp fluorometry. eLife, 9, e52775. doi:10.7554/eLife.52775, publi-
shed under the Creative commons Attribution license, http://creativecommons.org/
licenses/by/4.0/. Panel (B) reproduced from Puljung, M., Vedovato, N., Usher, S., &
Ashcroft, F. (2019). Activation mechanism of ATP-sensitive K + channels explored with
real-time nucleotide binding. eLife, 8, e41103. doi:10.7554/eLife.41103, published under
the Creative commons Attribution license, http://creativecommons.org/licenses/by/4.0/.
7. Excitation and detection of ANAP

The maximal excitation wavelength for ANAP is 360 nm (Chatterjee
et al., 2013). Excitation has been achieved using halogen lamps, mercury
lamps, LEDs, and laser excitation including 405 nm lasers (Park et al.,
2019; Soh et al., 2017; Usher et al., 2020; Zagotta et al., 2016), argon lasers
(which have a line around 364 nm) (Luo et al., 2019; Xu et al., 2020), and
two-photon excitation with 730 nm lasers (Chatterjee et al., 2013; Wen et al.,
2015). Emitted light has been detected using CCD cameras with epi-
fluorescence microscopy (Aman et al., 2016; Chatterjee et al., 2013; Puljung
et al., 2019; Usher et al., 2020), confocal microscopy (Chatterjee et al.,
2013; Hsieh et al., 2014; Park et al., 2019; Soh et al., 2017; Usher et al.,
2020), and TIRF microscopy (Zagotta et al., 2016). Images are typically
corrected by subtracting a background region near the region of interest. In
VCF experiments, fluorescence is usually collected using a photomultiplier
tube or photodiode, enabling fast readout times (Kalstrup & Blunck, 2013;
Sakata et al., 2016).
Many investigators have used spectrographs coupled to microscopes to
split the emitted ANAP fluorescence into its component wavelengths and
acquire emission spectra of ANAP-labeled protein (Dai, 2019; Dai et al.,
2018; Dai & Zagotta, 2017; Hsieh et al., 2014; Luo et al., 2019; Puljung
et al., 2019; Shandell et al., 2019; Usher et al., 2020; Wen et al., 2015;
Xu et al., 2020; Yang, 2018; Yang et al., 2020; Zagotta et al., 2016).
This involves placing a slit (mask) over a region of interest and reflecting
the collected light off a series of gratings (Puljung et al., 2019). The resulting
image retains spatial information in the y-dimension, but the x-spatial
dimension is replaced with wavelength (Fig. 6A). Spectra can be extracted
from such images using a line-scan or by creating a wavelength-by-
wavelength average of the spectra over a region of interest. Background
is subtracted with an equivalent line scan or average spectrum outside the
region of interest. Alternatively, confocal microscopy with monochromators
can be used to acquire spectra (Wen et al., 2015).
Acquiring emission spectra confers several advantages. First of all, the
peak wavelength provides information about the local polarity around
the ANAP residue. Changes in channel conformation may change the
environment around ANAP and can be detected by a shift in the fluores-
cence. This can also serve as a useful control in FRET experiments to dif-
ferentiate between changes in fluorescence arising from FRET and changes
A BF fluorescence
20 µm
spectrum
40 nm
B
1.0 0 ATP
fluorescence (a.u.)
10 mM ATP
0.8
0.6
0.4
0.2
0.0
450 500 550 600 650 700
nm
C
1.0
fluorescence (a.u.)
0.8
0.6
0.4
0.2
0.0
450 500 550 600 650 700
nm
Fig. 6 Line scan vs averaged spectra. (A) Brightfield, fluorescence, and spectral images
of an unroofed HEK-293 cell membrane expressing ANAP-labeled KATP channels. The
spectral image is produced by passing the emitted fluorescence through a slit (dashed
line in brightfield image) and into a spectrometer. Spatial information is retained in the
y-dimension. The x-spatial dimension is replaced by wavelength. (B) Line scans from a
spectral image of an unroofed membrane expressing ANAP-labeled KATP channels
showing an apparent shift in ANAP fluorescence upon binding of 10 mM ATP.
(C) Averaged spectra from the same unroofed membrane showing no effect of
10 mM ATP. Panel (A) from Puljung, M., Vedovato, N., Usher, S., & Ashcroft, F. (2019).
Activation mechanism of ATP-sensitive K + channels explored with real-time nucleotide
binding. eLife, 8, e41103. doi:10.7554/eLife.41103, published under the Creative commons
Attribution license, http://creativecommons.org/licenses/by/4.0/.
that reflect the local protein environment. Splitting emitted light into
wavelengths also separates ANAP fluorescence from scattered light and
autofluorescence that might otherwise contaminate an image. Use of a
physical mask, like the spectrograph slit, between the sample and the detec-
tor has also been shown to reduce the fluorescence background and
increase signal to noise (Wulf, 2018).
It is recommended that averaged spectra (wavelength-by-wavelength
over a region of interest) be used instead of line scans whenever possible.
Fig. 6B shows an experiment with ANAP-tagged KATP channels in which
a line scan in successive images appeared to show a large shift in polarity
around ANAP subsequent to the addition of 10 mM ATP (Fig. 6B).
However, the averaged spectra for these two images (Fig. 6C) as well as adja-
cent line scans show that this was a misleading result. ATP does not alter the
fluorescence of ANAP incorporated into this site (Puljung et al., 2019).
Therefore, line scans should be interpreted with some caution.
ANAP is prone to photobleaching. Care should be taken when prepar-
ing experiments to minimize light exposure. For oocyte experiments, it has
been suggested that injections and experiments be performed under red light
(Kalstrup & Blunck, 2017). Cells can be shielded with aluminum foil prior to
experimentation. Few groups have explicitly included corrections for pho-
tobleaching in their experiments. Dai et al. and Gordon et al. correct for
photobleaching in their tmFRET experiments implicitly by normalizing
their fluorescence to similar experiments conducted with control constructs
in which there is no metal binding site (Dai et al., 2018; Gordon et al., 2018).
As photobleaching follows an exponential time course, it can be corrected
for by acquiring several exposures prior to or during an experiment, fitting a
plot of the decrement in fluorescence as a function of cumulative exposure
time to a single-exponential decay, and using the fits to correct the other
images (Puljung et al., 2019; Usher et al., 2020; Usher, Ashcroft, &
Puljung, 2021). Photobleaching can be reduced by lowering the intensity
of the excitation light and/or reducing exposure times. The use of triplet
state quenchers like trolox to reduce photobleaching has also been proposed
(Kalstrup & Blunck, 2017), but no examples of its use with ANAP exist in
the literature.
8. Conclusions
Since its introduction, ANAP has proven to be a useful and versatile
probe for understanding the structures of intact, functional ion channels in
their native environment. Studies have taken advantage of its environmental

sensitivity and potential as a FRET donor to follow channel conformational
changes, ligand binding, protein-protein interactions, and temperature sen-
sitivity. As changes in ANAP fluorescence have been shown to track
gating-induced conformational changes in certain channels, it has potential
to be used for high throughput fluorescence screening for drugs and other
ligands that affect ion channel function. Furthermore, as ANAP can confer
fluorescence on a protein while only minimally perturbing its structure, it
may be useful to study protein-protein interactions in situations in which
FP tags may disrupt such associations. The future may provide the ion chan-
nel field with a whole host of fluorescent amino acids of different colors and
properties, or unnatural amino acids with bio-orthogonal reactivity that
allows for conjugation of different fluorophores. Until such a time, ANAP
provides an excellent tool whose potential remains largely untapped.
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CHAPTER THREE

ANAP: A versatile, fluorescent

Methods in Enzymology, Volume 654 Copyright # 2021 Elsevier Inc. 49

Simultaneous measurements of ionic currents and ANAP fluorescence yield exceptional

important, as it allows fluorescence measurements to be paired with voltage

Mitchell, Addy, Chin, & Chatterjee, 2017; Zagotta, Gordon, Senning,

Table 1 Ion channel applications using ANAP.

Table 1 Ion channel applications using ANAP.—cont’d

2. Site-specific ANAP incorporation

methyl ester, this is presumably following hydrolysis by an endogenous

3. ANAP applications in ion channel research

3.1 Conformational changes associated with voltage gating

Ci-VSP was expressed in Xenopus oocytes. Sensing currents (analogous to

to monitor the movement of the S4 voltage sensor of spHCN (Dai, 2019;

with ANAP on the inactivation gate was co-expressed in HEK-293 cells

3.2 Activation of TRP channels

3.3 Binding and activation in ligand-gated channels

neurotransmitter receptor (Soh et al., 2017). They expressed channels with

consequences of binding at each site for channel gating (Puljung et al.,

4. Non-channel applications for ANAP

5. Expression of ANAP-tagged channels

CMV-SEAP (available from AddGene). Nuclear injection of CMV-SEAP

(concentrations vary) mixed with ANAP at a final concentration of

6. Controls and confounds

B V13TAG L14TAG T15TAG R16TAG L17TAG A18TAG

proteins via amber stop-codon suppression, invariably some percentage of

Another potential experimental confound is read-through of the amber

Fig. 4B shows a portion of the results from experiments in which the

effects, adding a C-terminal FP tag to channel constructs is recommended.

A line scan intensity

Fig. 5 Controls for background from unincorporated/off-target ANAP. (A) Confocal

7. Excitation and detection of ANAP

their native environment. Studies have taken advantage of its environmental

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