Restoration of Proper Trafficking to the Cell Surface for
Membrane Proteins Harboring Cysteine Mutations
Angelica Lopez-Rodriguez, Miguel Holmgren*
Neurophysiology Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH),
Bethesda, Maryland, United States of America
A common phenotype for many genetic diseases is that the cell is unable to deliver full-length membrane proteins to the
cell surface. For some forms of autism, hereditary spherocytosis and color blindness, the culprits are single point mutations
to cysteine. We have studied two inheritable cysteine mutants of cyclic nucleotide-gated channels that produce
achromatopsia, a common form of severe color blindness. By taking advantage of the reactivity of cysteine’s sulfhydryl
group, we modified these mutants with chemical reagents that attach moieties with similar chemistries to the wild-type
amino acids’ side chains. We show that these modifications restored proper delivery to the cell membrane. Once there, the
channels exhibited normal functional properties. This strategy might provide a unique opportunity to assess the chemical
nature of membrane protein traffic problems.
Citation: Lopez-Rodriguez A, Holmgren M (2012) Restoration of Proper Trafficking to the Cell Surface for Membrane Proteins Harboring Cysteine Mutations. PLoS
ONE 7(10): e47693. doi:10.1371/journal.pone.0047693
Editor: Ruben Claudio Aguilar, Purdue University, United States of America
Received July 5, 2012; Accepted September 17, 2012; Published October 17, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute Neurological Disorders and Stroke.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com.
Improper targeting of membrane proteins causes many diseases.
Often point mutations to cysteine hinder the delivery of
membrane proteins to the cell surface [1,2,3,4,5,6,7], or to the
correct side of polarized cells [8,9]. Because cysteine is a readily
reactive amino acid, in principle it should be possible to recover
proper trafficking by modifying its chemical structure in order to
mimic the side chain of the wild type amino acid. As a proof of
principle, we have studied two naturally occurring cysteine
mutations in a cyclic nucleotide-gated channel (CNGA3) re-
sponsible for hereditary cone photoreceptor disorders: Y181C
linked to incomplete achromatopsia and R277C linked to
complete and incomplete achromatopsia or cone dystrophy
[10,11]. We have chosen these mutations because proper surface
CNG channel expression can be easily assayed using electrophys-
iological techniques, and because both mutations, which cause
channel retention in the endoplasmic reticulum (ER) [11,12],
change wild type amino acids of drastically different chemistries.
CNG channels open a cationic selective permeation pathway in
response to intracellular cyclic nucleotides [13,14]. In the visual
system, CNG channels are key players in the transduction of light
into electrical signals . In native cells, these channels are
[16,17,18,19,20,21], each containing six transmembrane seg-
ments. Functional homotetramers can be formed by the CNGA1,
A2 or A3 subunits [22,23,24], and these channels are usually
studied as homotetramers in heterologous systems. We have
introduced both achromatopsia-related cysteines in a cysteine-less
CNGA1 channel , and used them as a target for specific
chemical modification with hydroxybenzyl- (MTSHB) and
aminoethyl-methanethiosulfonate (MTSEA). These reagents read-
ily attach to the side chain of cysteines and mimic the chemistry of
tyrosine and arginine, respectively (Fig. 1). Although Y181C and
R277C caused ER retention, after chemical modification both
mutants were targeted to the cell surface, providing a unique
opportunity for their functional characterization.
Materials and Methods
Mutagenesis and Expression
cDNA of a cysteine-less CNGA1 channel was kindly provided
by William Zagotta (University of Washington, Seattle, WA).
Cysteine mutations were introduced in this background using
a QuickChange kit (Stratagene). Amino acid substitutions, as well
as the integrity of the entire coding region of each channel, were
confirmed by DNA sequencing (NINDS sequencing facility). A
CNGA1-Green Fluorescent Protein (CNG-GFP) was created with
standard PCR techniques. cRNAs were synthesized with a T7
promoter-based in vitro transcription protocol (Ambion). Xenopus
oocytes were injected with 50 nl (500 ng/ml) of cRNA and
incubated in ND96 solution (in mM: 96 NaCl, 2 KCl, 1 MgCl2,
1.8 CaCl2, 5 HEPES, pH 7.6) at17uC for two to three days to
allow channel to express. To assess surface expression by
fluorescence, we engineered two cysteine-less background con-
structs: CNGA1-GFP and CNGA1-FLAG. GFP and the FLAG
epitope (DYKDDDDK) were inserted in frame immediately
before the stop codon. In general, CNG channels tolerate these
tags at the carboxy-terminal remarkably well [1,18,19]. This study
was approved by National Institute Neurological Disorders and
Stroke/National Institute on Deafness and Other Communication
PLOS ONE | www.plosone.org1 October 2012 | Volume 7 | Issue 10 | e47693
Disorders Animal Care and Use Committee Protocol Number
R272C mutant channels were rescued using aminoethyl-
methanethiosulfonate (MTSEA), a compound which readily
permeates the membrane of cells, including that of Xenopus
oocytes, in its uncharged form . Treatments were performed
48 hrs after cRNA injection. MTSEA (final concentration 2 mM)
was prepared in ND96 solution and added into wells containing
oocytes. Incubation was performed at 17uC for six hours. Fresh
ND96 solution with MTSEA was replaced every 30 minutes.
These prolonged treatments were not readily tolerated by all
oocytes. We found that after around 4 hours of MTSEA exposure,
about 50% of the oocytes began to show signs of deterioration, as
the animal pole became pale. Those oocytes were not used for any
Y176C mutant channels were treated with hydroxybenzyl-
methanethiosulfonate (MTSHB). Oocytes were injected once with
50 nl of a 40 mM MTSHB stock solution, which was prepared in
an ethanol-DMSO mix (50/50), and incubated at 17uC overnight.
After each treatment, some oocytes were used to analyze the
extent of protein trafficking by fluorescence or immunocytochem-
istry, membrane protein expression assays, and others were used
for electrophysiological characterization.
Biotinylation of Membrane Surface Protein
Six to eight oocytes were incubated for 1 hr at 4uC in ND96,
supplemented with 50 mg/ml gentamicin and 1.0 mg/ml sulfo-
NHS-LC-biotin (Pierce). Then, oocytes were washed several times
with ND96 supplemented with 100 mM glycine and lysed in
200 ml of buffer H (1% Triton X-100, 100 mM NaCl, 20 mM
Tris-HCl, pH 7.4 with protease inhibitors (SIGMA) by trituration.
Lysates were rocked at room temperature for 15 min and then
centrifuged at 13,000 rpm for 3 min. The pellet was discarded and
the supernatant was divided in two equal samples, one containing
total proteins and the other to be used for preparation of cell
membrane proteins. For cell membrane protein isolation, 50 ml of
NeutrAvidinTM Agarose Resin (Thermo Scientific) was added to
the sample and rocked gently at 4uC for at least 30 min. Resin was
washed at least 6 times with buffer H, and finally eluted in buffer
H (supplemented with 10% 2-ME and 50 mM DTT) to the same
volume as the sample containing total proteins, and incubated for
5 min at 95uC.
All samples were deglycosylated using PNGase F (New England
BioLabs) before being loaded into a SDS-PAGE gel and trans-
ferred to a polivinylidene difuoride (PVDF) membrane. Blots were
probed with an anti-GFP mouse monoclonal antibody at a dilution
of 1:5000 (Clontech). Primary antibodies were detected with
a secondary, goat anti-mouse antibody conjugated to horseradish
peroxidase used at a dilution of 1:10000 (Pierce). Membranes were
developed by SuperSignal WestFemto (Thermo Scientific) and
visualized by chemioluminiscence using a FluorChem E Imager
(Cell Biosciences). Analysis was performed using Image Alpha-
View software (Cell Biosciences).
The oocyte’s vitelline layer was removed to reduce background
fluorescence. Oocytes were permeabilized with 0.03% saponin in
ND96 solution for 20 min, washed with ND96 solution, blocked
with 1% BSA in ND96 for 30 min. and incubated overnight with
a 1:250 dilution of a FLAG polyclonal antibody (Santa Cruz
Biotechnology. Inc.) in ND96 at 4uC. Oocytes were washed
several times with ND96 and incubated for 1 hr in a 1:500 dilution
of secondary antibody (Texas Red conjugated donkey anti-goat
IgG; Santa Cruz Biotechnology. Inc.) at 4uC. After several rinses
with ND96, oocytes were imaged using a Zeiss LSM 510 confocal
The recording solution consisted of (in mM): 120 NaCl, 2
EDTA, 10 HEPES (pH=7.4). All reagents were obtained from
SIGMA. Currents from inside-out excised patches  were
acquired using an Axopatch 200B amplifier (Molecular Devices)
and a Digidata 1322 acquisition board (Molecular Devices), and
were sampled between 2.5 to 10 kHz using a low-pass filter at 1 or
2 kHz. Patch electrodes with tip diameters between 8 and 15 mm
were made with borosilicate glass pipettes. Macroscopic data
analysis was performed with pClamp 9 (Molecular Devices) and
Origin 8 (Microcal Software) software. Solutions were changed
using a computer-controlled rapid solution changer (RSC-200;
Biologic Science Instruments).
Recovering Cell Membrane Expression of CNG Channels
Containing the R272C Mutation
To assess whether chemical modification could restore cell
surface expression and functionality, we used the well-character-
ized bovine CNGA1 channel which lacks all native cysteines .
Position R272 in the bovine CNGA1 channel is equivalent to
position R277 in the human CNGA3 channel and is located
within a large domain known as the voltage sensor. From the
crystal structure of a mammalian voltage-activated potassium (KV)
channels , a cousin of CNG channels, this position is part of
Figure 1. Rescuing strategy. A. Via a disulfide bridge, MTSHB
attaches a hydroxyl benzene moiety that mimics tyrosine’s side chain. B.
Via a disulfide bridge, MTSEA introduces a primary amine which mimics
arginine’s side chain.
Restoring Trafficking by Cysteine Modification
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the fourth transmembrane segment (S4) where the critical charges
that sense the transmembrane voltage are spaced every three
amino acids [30,31,32]. Little is known about the role of the
voltage sensor in CNG channels due to technical difficulties, such
as poor surface expression of channels harboring S4 mutations
[4,12]. In all channels within the superfamily of voltage-activated
ion channels, which includes CNG channels, it is known that S4 is
important for maturation [4,12,33,34,35].
To verify that the R272C mutation impedes surface expression
in the cysteine-less CNGA1-GFP background, we injected this
construct into Xenopus oocytes. After two days, we could not
observe channels at the cell surface using fluorescence microscopy.
Further, using functional expression as an indicator, we were
unsuccessful in detecting cGMP-activated ionic currents in more
than 40 excised inside-out membrane patches (not shown).
However, by incubating injected oocytes with a solution contain-
ing 2 mM MTSEA (a membrane permeant reagent  that
leaves a moiety mimicking the side chain of arginine, as shown in
Fig. 1) we restored the proper channel trafficking. Fig. 2A shows
confocal images of an oocyte in which R272C CNGA1-GFP
channels were rescued after ,6 hr of MTSEA treatment. Fig. 2B
shows the time course of fluorescence detection at the cell surface
of six oocytes. A similar MTSEA treatment to cysteine-less
CNGA1-GFP channels has no effect on cell surface expression
Modified channels respond normally to the presence of agonists.
Fig. 3A shows cGMP-activated currents carried by control
MTSEA-treated cysteine-less CNGA1-GFP channels in response
to voltage steps between 280 and +80 mV from a holding
potential of 0 mV in the presence of saturating [cGMP]. Under
similar conditions, ionic currents from MTSEA-modified R272C
mutant channels are comparable (Fig. 3D). Dose-response curves
for cGMP at +60 mV for wild-type channels (Fig. 3B) and
modified R272C channels (Fig. 3E) were also similar. Solid lines
represent Hill equation fits in which KK and n values were
1961 mM and 2.260.2 for wild-type channels and 4360.5 mM
and 1.960.3 for modified 272C channels. Another property of
CNGA1 channels is their sensitivity to saturated concentrations of
the various cyclic nucleotide agonists. In general they barely open
with cAMP, open more with cIMP and open with a high
probability with cGMP. Both, cysteine-less CNGA1-GFP (Fig. 3C)
and modified R272C (Fig. 3F) channels maintained the same
relative efficacy among these agonists. Taken together, these
results demonstrate that attaching a primary amine moiety to
cysteine 272 can successfully mimic the role played by the side
chain of arginine in wild-type channels. Specifically, this modifi-
cation restores targeting to the cell surface and produces channels
that function relatively normal.
How efficiently can a MTSEA-modified R272C CNGA1-GFP
channel be rescued? To approach this question, we assessed total
and cell surface protein expression from pools of six to eight
oocytes (see Methods). For cysteine-less CNGA1-GFP channels,
,45% of the total membrane protein is at the cell surface (Fig. 4;
WT). Untreated R272C channels cannot be detected at the cell
surface (Fig. 4; R272C), consistent with previous observations
[4,12]. However, treatment with MTSEA successfully rescued
R272C CNGA1 channels to the cell surface, at comparable
proportions as cysteine-less CNGA1-GFP channels (Fig. 4;
R272C+MTSEA). These results suggest that the added moiety
to cysteine 272 restored the proper channel conformation allowing
it to pass the various trafficking checkpoints.
Our approach was also successful with other positively charged
residues. Unlike the case for KV channels, the substitution of
cysteine for the positively charged, voltage sensing amino acids
within the S4 transmembrane segment of CNG channels leads to
immature products that get trapped in the ER . We tested the
ability of MTSEA to rescue surface expression of R269C, R275C
and R278C. In each case, mutants were efficiently redirected to
Figure 2. Time course of cell surface expression for R272C CNGA1-GFP channels exposed to MTSEA. A. Representative confocal images
of one oocyte before (T0) and after 2 mM MTSEA exposure. Media with fresh MTSEA was exchanged every 30 min. After ,6 hrs of MTSEA treatment,
GFP fluorescence was detectable at the oocyte’s surface. B. Plot shows the time course of cell surface fluorescence detection in six different oocytes.
Restoring Trafficking by Cysteine Modification
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Figure 3. Functional characterization of CNGA1-GFP channels at the cell surface. A, B & C. Cysteine-less CNGA1-GFP channels. A, Ionic
currents in the presence of saturating [cGMP] (2 mM). cGMP-activated currents shown were acquired in response to 100 ms voltage steps from 280
to +80 mV (every 20 mV) from a holding potential of 0 mV. B. Dose response for cGMP at +60 mV. Solid line represents a normalized Hill equation fit
to the data. The best fit parameter values for KK was 1961 mM, and for the hill coefficient (n) was 2.160.2. Average values for KK and n were
2463 mM and 2.1660.46, respectively (n=4 oocytes). C. Efficacies for different agonists. Wild type CNGA1 channels displayed larger than 95%
maximal probability of opening with saturated concentrations of cGMP, less with cIMP, and much less with cAMP. Nucleotide-activated current
records shown were obtained from the same excised inside out patch with saturating concentrations of cGMP (2 mM; black), cIMP (16 mM; red) and
cAMP (16 mM; blue). D, E & F. Rescued R272C CNGA1-GFP channels. D. Ionic current carried by MTSEA-modified R272C channels. In response to
a comparable experimental protocol as in A, rescued channels were able to conduct ionic current with similar properties as wild type channels. E.
Dose response of modified R272C channels at the cell surface for cGMP. Solid line corresponds to a normalized Hill equation fit to the data. The best
fit parameter values for KK and n were 43.462.5 mM and 1.960.3, respectively. Average values for KK and n were 59610 mM and 1.4860.22,
respectively (n=14 oocytes). F. Agonist efficacies for rescued R272C mutant channels. Nucleotide-activated current records shown were acquired
from the same patch, using the equivalent agonist concentration as in C. Similar observations were made in 3 different patches.
Figure 4. Cell surface expression of CNGA1-GFP channels. A. Representative Western blot of total CNGA1-GFP protein (TP) and biotinylated
CNGA1-GFP cell surface protein (SP). WT, R272C, R272C+MTSEA denote cysteine-less CNGA1-GFP, mutant channels that were not treated with MTSEA
and mutant channels that were modified by MTSEA, respectively. An expected band of ,106 kDa for the deglycosylated wild-type GFP tagged
channel was detected by chemiluminiscence using a GFP antibody. No signal was detected in non injected oocytes. B. Densitometry analysis of the
bands normalized to TP (n=3 different oocytes batches).
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the cell membrane, demonstrating the general applicability of the
technique (Fig. S2).
Restoring Proper Targeting to Y176C Mutant CNG
Position Y176 in CNGA1 channels corresponds to position
Y181 in hCNGA3 channels and is located within the first
transmembrane segment. As in hCNGA3 channels , the
Y176C mutation impeded Y176C CNGA1-GFP channels from
reaching the cell membrane (Fig. 5A). Chemical modification with
MTSHB would attach a moiety to these cysteines that resembles
the original tyrosine side chain (Fig. 1A). This reagent, however, is
very hydrophobic and precipitates in aqueous solutions. We were
unable to find an experimental condition where we could incubate
oocytes in MTSHB to restore function. However, by directly
injecting a 40 mM MTSHB solution into the oocytes, we were
able to recover cell surface expression (Fig. 5B). Likely, the rather
oily environment of an oocyte’s yolk allowed the reagent to stay in
solution at sufficiently high concentrations to modify Y176C. A
direct reagent injection has been successfully used before to probe
proton channel function in a tryptophan mutant . Modified
Y176C CNGA1-GFP channels responded normally to saturating
concentrations of cGMP (Fig. 5C), although current levels were
consistently smaller than cysteine-less and rescued R272C
CNGA1 channels. Likely, a single MTSHB injection (c.f. freshly
applied MTSEA every 30 min for R272C CNGA1 channels) and
the relatively fast hydrolysis of MTS reagents contributed to the
lower levels of rescued Y176C CNGA1 channels. The dose-
response for cGMP at +60 mV for modified Y176C CNGA1-GFP
channels was characteristic of CNGA1 channels (Fig. 5D). The
solid line through the data corresponds to a Hill equation fit with
KK and n values of 1661 mM and 1.360.05. Finally, rescued
channels showed a similar efficacy for saturating concentrations of
different agonists (Fig. 5E) as for the cysteine-less CNGA1-GFP
channels (Fig. 3E). These studies demonstrate that Y176C mutant
channels retained in intracellular compartments can be success-
fully targeted to the cell surface by adding a side-chain to a cysteine
that mimics that of tyrosine. Once at the cell surface, these
channels behave normally.
We describe a method to restore proper maturation and
trafficking of membrane proteins that have been retained within
intracellular organelles due to single point mutations to cysteine.
Because the side chain of cysteine is highly reactive, we reasoned
that modification with reagents that restored the original chemistry
could drive proper maturation. We successfully restored both
trafficking and normal function to CNGA1 mutant channels
R272C and Y176C, both responsible for hereditary cone
photoreceptor disorders [10,11].
Maturation of any protein is a complex, multi-step process
involving a network of intracellular proteins and organelles.
Surely, all genetic mutations leading to defective maturation
cannot be repaired by a single strategy. Thus far, a variety of
experimental approaches have been shown to recover proper
maturation. For example, cell surface expression of mutant HERG
and CNGA3 channels [6,37], as well as deficient lysosomal
Figure 5. Y176C CNGA1-GFP mutant channels. A. Confocal image of an oocyte injected with cRNA encoding for Y176C CNGA1-GFP channels.
We never detected any signs of proper trafficking in .100 oocytes by standard fluorescence microscopy, suggesting that these channels do not
reach the cell surface. B. MTSHB treatment recovers proper trafficking of Y176C mutant channels to the cell surface. Confocal image shows GFP
fluorescent signal at the cell surface of an oocyte expressing Y176C mutant channels. MTSHB was injected , 12 h before acquiring the image (n .25
oocytes). C. Ionic currents in the presence of a saturating cGMP concentration (2 mM), in response to the same voltage protocol as described in Fig.
3A. D. Dose response for cGMP at +60 mV. Solid line represents a normalized Hill equation fit to the data. The best fit parameter values for KK and n
were 1661 mM and 1.3060.05, respectively. From a total of 8 oocytes, the average values for KK and n were 1663 mM and 1.660.1, respectively. E.
Agonist efficacies of rescued Y176C mutant channels. Nucleotide-activated current records shown in the presence of saturating concentrations of
each agonist (same as Fig. 3C) were acquired from the same excised patch. Rescued Y176C channels showed a similar efficacy pattern as cysteine-less
CNGA1-GFP channels. Similar observations were observed in 4 patches.
Restoring Trafficking by Cysteine Modification
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glucocerebrosidase , can be restored simply by lowering the
temperature of incubation. Based on their ability to stabilize
proper folding conformations in the ER, drugs and lipid
chaperones are emerging as new strategies to restore protein
maturation [39,40,41,42,43]. As with other rescue methods, ours
has disadvantages: it is restricted to cysteine mutants and it is not
specific since MTS reagents will modify any accessible cysteine in
a protein. Nevertheless, outside of therapeutics, this method has
applications that could, in principle, offer relevant structural and
functional information about diseases. For example, it could be
used to better understand the chemical nature of the protein
folding failure since many different MTS reagents are available
that attach moieties resembling different amino acid side chains.
Another potential use could be for kinetic studies of folding. For
example, if a cysteine mutation impairs proper folding, modifica-
tion reactions by MTS reagents are sufficiently fast to permit the
temporal resolution of downstream conformational changes,
providing kinetic information on folding steps. A third application
we envision is to use the disease related mutant in the same way
that biophysicists use engineered cysteines as a tool to study state
dependent accessibility. This will provide information of confor-
mational changes at the site of the cysteine mutation.
cysteine-less CNGA1-GFP channels exposed to MTSEA.
A. Representative confocal images of one oocyte before (T0) and
after 2 mM MTSEA exposure. Media with fresh MTSEA was
Cell surface expression time course of
exchanged every 30 min. MTSEA treatment does not affect cell
surface expression of ‘‘wild-type’’ channels.
by chemical modification. All images shown were obtained
after immunocytochemical labeling of oocytes expressing CNGA1
arginine to cysteine mutations in the S4 transmembrane segment.
The absence of fluorescence at the oocytes’ cell membrane (left
column) indicate that these arginine to cysteine mutations in the
S4 segment render immature channels that are unable to reach the
cell surface. After 6 h MTSEA treatment, we were able to restore
proper trafficking to these mutant channels (right column).
Representative of .10 cells in each panel.
Cell surface targeting of S4 cysteine mutants
We thank Dr. William Zagotta for kindly supplying the CNGA1 cysteine-
less construct, Dr. Joshua Rosenthal for helpful discussions, Deepa
Srikumar for technical assistance, Pat Curran and Dan Silverman for
assistance with western blot preparations, Dr. Jeet Kalia for preparing Fig.
1 and the DNA-sequencing facility of National Institute Neurological
Disorders and Stroke where all DNA constructs were sequenced.
Conceived and designed the experiments: AL MH. Performed the
experiments: AL. Analyzed the data: AL MH. Contributed reagents/
materials/analysis tools: AL MH. Wrote the paper: AL MH.
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