Structural studies of ion permeation and Ca2þ
blockage of a bacterial channel mimicking
the cyclic nucleotide-gated channel pore
Mehabaw G. Derebea, Weizhong Zenga,b, Yang Lic, Amer Alamd, and Youxing Jianga,b,1
aDepartment of Physiology and
cShanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China; and
Biology and Biophysics, Eidgenössiche Technische Hochschule Zurich, HPK D11, 8093 Zurich, Switzerland
bHoward Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9040;
dInstitute of Molecular
Edited* by Christopher Miller, Brandeis University, Waltham, MA, and approved November 16, 2010 (received for review September 14, 2010)
Cyclic nucleotide-gated (CNG) channels play an essential role in the
visual and olfactory sensory systems and are ubiquitous in eukar-
yotes. Details of their underlying ion selectivity properties are still
not fully understood and are a matter of debate in the absence of
high-resolution structures. To reveal the structural mechanism of
ion selectivity in CNG channels, particularly their Ca2þblockage
property, we engineered a set of mimics of CNG channel pores
for both structural and functional analysis. The mimics faithfully
represent the CNG channels they are modeled after, permeate
Naþand Kþequally well, and exhibit the same Ca2þblockage
and permeation properties. Their high-resolution structures reveal
a hitherto unseen selectivity filter architecture comprising three
contiguous ion binding sites in which Naþand Kþbind with differ-
ent ion-ligand geometries. Our structural analysis reveals that the
conserved acidic residue in the filter is essential for Ca2þbinding
but not through direct ion chelation as in the currently accepted
view. Furthermore, structural insight from our CNG mimics allows
us to pinpoint equivalent interactions in CNG channels through
structure-based mutagenesis that have previously not been pre-
dicted using NaK or Kþchannel models.
These channels conduct various mono and divalent cations
and, under physiological conditions, are more permeable to Ca2þ
than Naþ(5–9). Ca2þpermeation, mediated through a highly
conserved acidic residue (most commonly Glu) in the selectivity
filter, lowers channel conductance by effectively blocking mono-
valent cation currents (7, 10–18) and plays an essential physiolo-
gical role in visual transduction (1). A Glu-to-Asp mutation
enhances Ca2þbinding whereas a Glu-to-Asn mutation di-
minishes it. In the absence of CNG channel structures, structural
insight into the molecular details underlying ion nonselectivity
has been limited to Kþchannel models (19, 20) and, more
recently, the prokaryotic nonselective cation channel NaK from
Bacillus cereus (21, 22). Although previous studies on NaK have
yielded important insights into Ca2þbinding in cation channels,
they fall short of explaining several key mechanistic differences
between NaK and CNG channels. First, the submillimolar affinity
of external Ca2þbinding observed in NaK is much weaker than
that of most CNG channels (23). Second, an Asp-to-Glu muta-
tion in the NaK filter leads to a higher Ca2þbinding affinity
whereas the opposite holds true for CNG channels (24–26).
Finally, selectivity filter sequences of CNG, Kþ, and NaK chan-
nels differ significantly after the conserved T(V/I)G residues in
both amino acid composition and sequence length, a majority
of CNG channels containing an ETPP motif (Fig. 1A), suggesting
a different filter architecture. To reveal the structural mechanism
of nonselective permeation, and more importantly, Ca2þblock-
agein CNG channels, we finetuned the NaKmodel by generating
a set of chimera channel pores with selectivity filter sequences
matching those of CNG channels.
yclic nucleotide-gated (CNG) channels are central to signal
transduction in the visual and olfactory sensory systems (1–4).
Generating NaK Chimeras That Mimic CNG Channel Pores. A majority
of CNG channels contain an amino acid sequence of ETPP
C-terminal to the T(V/I)G residues that are also conserved in
NaK and Kþselective channel selectivity filters (Fig. 1A). The
NaK channel has an amino acid sequence of DGNFS in the
equivalent region where only the acidic residue (Asp66) is con-
served. Furthermore, the sequence is one residue longer in the
NaK channel. Accordingly, the selectivity filters of commonly
found CNG channels and their mutants (Glu-to-Asp and Glu-to-
its filter sequence of TVGDGNFS to TVGETPP (NaK2CNG-E),
TVGDTPP (NaK2CNG-D), and TVGNTPP (NaK2CNG-N)
(Fig. 1A). Although residue positions equivalent to Val64 in NaK
(position marked by a triangle in Fig. 1A) are occupied by Ile in
packing surrounding the selectivity filter; a NaK mutant contain-
ing a TIGDTPP filter sequence has virtually the same structure as
in this study can accommodate Ile in the filter without structural
change. Therefore all the studies presented here were performed
with Val at position 64.
The CNG-Mimicking NaK Chimeras Are Nonselective. All NaK2CNG
chimeras share biochemical properties similar to wild-type
channel and can be purified as stable tetramers. Giant liposome
patching was employed to assay the functional properties of
the channels and confirm their nonselective nature (Materials
and Methods). In order to observe single channel currents, all
NaK2CNG chimeras used for functional studies carried an
additional Phe92Ala mutation which has been shown to increase
ion flux through the channel pore (23, 27). Fig. 1 B and C show
sample single channel traces for NaK2CNG-E recorded under
bi-ionic conditions (150 mM NaCl and 150 mM KCl in the pipette
and bath solutions, respectively) along with its I-V curve. The
mutant channels exhibit a high single channel conductance in
both directions with a reversal potential of 0 mV, indicating the
channel is indeed nonselective with virtually the same permeabil-
ity for Naþand Kþ. NaK2CNG-D and NaK2CNG-N exhibit
Author contributions: M.G.D. and Y.J. designed research; M.G.D., W.Z., Y.L., and A.A.
performed research; M.G.D., W.Z., Y.L., and Y.J. analyzed data; and M.G.D., A.A., and
Y.J. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID codes 3K0D, 3K03, and 3K06 for Kþcomplexes
of NaK2CNG-E, NaK2CNG-D, and NaK2CNG-N, respectively; and 3K0G, 3K04, and 3K08
for Naþcomplexes of NaK2CNG-E, NaK2CNG-D, and NaK2CNG-N, respectively).
This article contains supporting information online at www.pnas.org/lookup/suppl/
592–597 ∣ PNAS ∣ January 11, 2011 ∣ vol. 108 ∣ no. 2www.pnas.org/cgi/doi/10.1073/pnas.1013643108
similar properties (Fig. S1). All mutant channels also exhibit very
high open probability. Even though we have yet to discover the
channel’s gating mechanism, this high open probability greatly
aides our functional analysis in this study.
Structural Determination of NaK2CNG Chimeras. Structures of all
three NaK2CNG chimeras in complex with various cations were
determined in an open conformation between resolutions of
1.58 and 1.95 Å (Materials and Methods, Fig. S2A, and Table S1).
The Kþcomplex structure of NaK2CNG-D will be used here to
describe the overall structural features, shared by all the mutants,
focusing on the selectivity filter region. The NaK2CNG-D selec-
tivity filter adopts a distinct architecture akin to an intermediate
between NaK and KcsA (Fig. S2 B and C), and contains three
contiguous ion binding sites equivalent to sites 2–4 of a Kþchan-
nel and numbered as such for comparison (Fig. 2A). The external
entryway has a concave funnel shape with the pyrrolidine ring
from the first proline (Pro68) in the filter region, absolutely con-
served in CNG channels, forming the wall of the funnel (Fig. 2A).
Two layers of well-ordered water molecules, four in each, are
observed within the funnel. These water molecules, particularly
the four in the inner layer right above site 2, form a hydration
shell that participates in stabilizing various cations in the funnel
upon entering or exiting the filter.
Protein Packing Around the Selectivity Filter. Although the overall
selectivity filter structure in the three CNG-mimicking mutants
is similar, noticeable differences in protein packing in the region
surrounding it are observed owing to the different amino acid
composition of residue 66 (Fig. 2 B–D and Fig. S3). The side
chain of residue 66 (Glu, Asp, or Asn) in all mutants remains
buried underneath the external surface of the protein and points
away from the ion conduction pathway, but forms different
hydrogen bonding interactions with its neighboring residues in
all three cases. The different hydrogen bonding networks, some
mediated through water molecules, are highlighted in Fig. 2 and
conserved residues are shaded in cyan. Residues in NaK replaced by corresponding CNG channel residues are shaded in orange. Secondary structure assignment
is based on the NaKNΔ19 structures (PDB ID 3E86). Asterisks mark the positions where mutagenesis was performed on bovine CNGA1 based on the NaK2CNG-E
structure and arrows indicate the residues for swap mutations. (B) Single channel traces of NaK2CNG-E at ?80 mV and its I-V curve. Currents were recorded
using giant liposome patch clamping with 150 mM NaCl and 150 mM KCl in the pipette and bath solutions, respectively. Dotted lines mark the zero current
(A) Partial sequence alignment between NaK, Kþchannels (MthK and KcsA) and human, rat, and bovine CNG channel alpha subunits (A1–A3). Semi-
density map (1.62 Å) is contoured at 2.0σ (blue mesh). Red spheres represent the two layers of water molecules within the external funnel. Three ion binding
sites are labeled 2–4 from top to bottom. (B–D) Protein packing around the selectivity filters of (B) NaK2CNG-D, (C) NaK2CNG-E, and (D) NaK2CNG-N. Red
spheres represent water molecules that mediate the hydrogen bonding network. Residue 66 from the neighboring subunit is labeled as D66′, E66′, and N66′,
respectively. All Kþions in the filter are drawn as green spheres.
Selectivity filter structures of CNG-mimicking NaK mutants. (A) Selectivity filter structure of NaK2CNG-D in complex with Kþions. The 2Fo-Fcelectron
Derebe et al.PNAS
January 11, 2011
Fig. S3. The differences in protein packing at residue 66 lead to
subtle changes in the backbone carbonyl position of Gly65 among
the different mutants, which likely accounts for altered ion bind-
ing profiles in the filter, particularly at site 2 (see SI Discussion
and Figs. S4 and S5).
Naþand KþBinding in the Selectivity Filter. Despite similar perme-
ability for Naþand Kþ, we observed clear differences in ion-
ligand geometries between the two ions as illustrated by the
NaK2CNG-D-Naþand −Kþcomplex structures (Fig. 3). The Kþ
complex structure reveals three electron density peaks with
equivalent intensity in the filter, indicating Kþbinding with simi-
lar occupancy at each site (Fig. 3A). A weaker electron density
peak, likely from a Kþion with lower occupancy, is observed
at the center of the external funnel sandwiched between the two
layers of water molecules. In the Naþcomplex, three strong peaks
are observed at sites 3 and 4, two at the upper and lower edges of
site 3, and the other on the lower edge of site 4 (Fig. 3B). In the
cavity just below site 4, a water molecule, not observed in Kþ
complex, participates in the chelation of a Naþion at the lower
edge of site 4. Some weaker electron density is also observed in
the center of site 2 and at the external entrance likely arising from
low occupancy Naþbinding. Although the filter structure remains
unchanged in both complexes (Fig. S6), Kþions tend to bind at
the center of each site, whereas Naþclearly prefers to bind on the
upper or lower edge of each site equatorially with its ligands. The
latter configuration allows for shorter ion-ligand distances and
preferable Naþion binding. Similar patterns of Naþand Kþbind-
ing are also observed in NaK2CNG-E and NaK2CNG-N. Details
of Naþand Kþbinding profiles in all three mutants are discussed
in the SI Text and are summarized in Fig. S5.
Ca2þBinding in the Selectivity Filter. Ca2þcomplexes of the engi-
neered chimeras were obtained by crystal soaking in stabilization
solutions containing 100 mM NaCl∕25 mM CaCl2(Materials
and Methods). These Na/Ca-soaked crystals were compared with
the Naþ-soaked crystals (reference crystals) using FNa∕Ca-soak−
FNa-soakdifference Fourier maps to determine whether and where
Ca2þbinds (Fig. 4A). The FNa∕Ca-soak− FNa-soakdifference map of
NaK2CNG-E indicates that a single Ca2þion binds at site 3
(Fig. 4A, Left). Ca2þbinding in NaK2CNG-D is more complex
and the difference map reveals four major peaks along the ion
conduction pathway: three at sites 2–4 inside the filter and one
within the funnel just above the external entrance (Fig. 4A,
Center). To verify whether these are due to Ca2þbinding and do
not arise from Naþ, which is also present in the soaking condi-
tions, we calculated the anomalous difference Fourier map from
a Ca/Na-soaked NaK2CNG-D crystal collected at longer wave-
length (λ ¼ 1.5 Å) at which Ca2þion is the only element in
the protein that has significant anomalous scattering. As shown
in Fig. 4B, the anomalous difference Fourier map reveals three
peaks at the same positions as the top three observed in the
FNa∕Ca-soak− FNa-soakdifference map, confirming Ca2þbinding
at sites 2, 3, and above the external entrance. In the control
experiment, the anomalous difference Fourier map from a
Naþ-soaked NaK2CNG-D crystal does not show anomalous
signals in the filter. The fourth peak in the FNa∕Ca-soak−
FNa-soakdifference map of NaK2CNG-D is likely due to the
redistribution of Naþat site 4 upon Ca2þbinding.
The FNa∕Ca-soak− FNa-soakdifference map for NaK2CNG-N
reveals a single electron density peak within the funnel at the
same position as the external peak observed in NaK2CNG-D,
(Fig. 4A, Right) indicating that, compared to NaK2CNG-D, Ca2þ
binding inside the selectivity filter is abolished in NaK2CNG-N
but external Ca2þbinding is retained. The position of this exter-
nally bound Ca2þin NaK2CNG-N or NaK2CNG-D is slightly
above the external entrance, at the same position as the hydrated
Naþion observed in the Naþcomplexes (Fig. 4C and Fig. S5).
Refined structures from the Na/Ca-soaked crystals of both
mutants reveal that the external Ca2þis also hydrated by the
inner layer of water molecules similar to Naþand has no direct
interaction with the protein (Fig. 4C). We believe this external
Ca2þbinding observed in both NaK2CNG-N and NaK2CNG-D
is not specific and simply arises from the competition between
hydrated Naþand Ca2þions within the funnel at high Ca2þ
concentration. This conclusion is consistent with our functional
analysis, described below, which shows that in NaK2CNG-N
external Ca2þcauses a reduction in channel current only at high
Ca2þis aPermeable Blockerfor NaK2CNGChimeras.Ca2þblockage in
our CNG-mimicking channels was functionally characterized
using giant liposome patch clamping with various concentrations
of Ca2þadded to the external side of the channel in addition
to 150 mM Kþ. As shown in single channel traces, inward Kþ
currents in NaK2CNG-E and NaK2CNG-D decrease with
increasing [Ca2þ] whereas NaK2CNG-N has almost no sensitivity
to external Ca2þ(Fig. 5A). A plot of unblocked current as a func-
tion of [Ca2þ] gives rise to Kis of 58.3 and 6.3 μM at −80 mV for
NaK2CNG-E and NaK2CNG-D, respectively (Fig. 5B). The
Ca2þbinding affinity in NaK2CNG-E is similar to that observed
in some eukaryotic CNGA2 and A3 channels (2). Higher Ca2þ
affinity in NaK2CNG-D is also consistent with CNG channel
studies which have shown that a Glu-to-Asp mutation in the filter
enhances Ca2þblockage (24–26). We believe the multiple Ca2þ
binding (at sites 2 and 3) in the NaK2CNG-D filter, resulting
from the position of the side-chain carboxylate group of Asp66
and the presence of two water molecules, contributes to this
difference (see SI Discussion). Furthermore, as observed for
CNG channels, Ca2þacts as a permeating blocker in the engi-
neered mimics. The Ca2þpermeation was confirmed using single
channel recording on NaK2CNG-E with 100 mM CaCl2in the
bath solution, which shows a clear Ca2þcurrent, albeit with
low conductance (Fig. 5C, upper trace). Addition of tetrapentyl
ammonium, an open pore blocker for Kþchannels that can also
block NaK from intracellular side, leads to a complete inhibition
of the observed Ca2þcurrent (Fig. 5C, lower trace).
Mutagenesis of CNG Channels Based on the Structure of NaK2CNG
Chimera. We used insights from our chimera models to directly
perform mutagenesis on the bovine retinal CNG channel
(CNGA1) using NaK2CNG-E as a model. In the NaK2CNG-E
structure, the carboxylate of Glu66 forms a hydrogen bond
directly with the hydroxyl group of Thr60 from a neighboring
ion omit map focusing on the selectivity filter of the NaK2CNG-D-Kþcomplex
(1.62 Å) contoured at 4σ. (B) Fo-Fcion omit maps focusing on the selectivity
filter of the NaK2CNG-D-Naþcomplex (1.58 Å) contoured at 4σ. Water
molecules within the external funnel (red spheres) were also omitted in
the map calculation. Kþand Naþions are represented by green and yellow
Kþand Naþbinding in the selectivity filter of NaK2CNG-D (A) Fo-Fc
www.pnas.org/cgi/doi/10.1073/pnas.1013643108 Derebe et al.
subunit (Fig. 2C). To test whether such an interaction exists in
CNG channels, we generated a CNGA1 mutant channel with
the equivalent residues, Glu363 and Thr357, swapped (Fig. 1A).
Whereas the single mutation (T357E) failed to yield functional
channels, the E363T/T357E double mutation gave rise to a func-
tional channel that conducts monovalent cations similar to wild
type, but is no longer blocked by external Ca2þ(Fig. 6 A and B).
An identical residue swap in NaK2CNG-E yielded a channel that
was strikingly similar to the swap mutation of CNGA1: The chan-
nel conducts Naþand Kþjust like NaK2CNG-E but is no longer
sensitive to external Ca2þblockage (Fig. 6C). The data confirm
the direct interaction between Glu363 and Thr357 in CNG chan-
nels and also demonstrate the importance of the exact position of
the glutamate side chain in Ca2þbinding.
Tyr55 in the pore helix appears to be a key player in protein
packing around the selectivity filter of the NaK2CNG chimeras.
In NaK2CNG-E, it forms a short-range hydrogen bond with the
backbone carbonyl oxygen of Glu66 that is expected to be impor-
tant in stabilizing the selectivity filter. This tyrosine is absolutely
conserved in CNG channels and is likely to play a similar role.
Indeed, no obvious current was observed when we replaced the
equivalent Tyr (Tyr352) to Phe in CNGA1 at a voltage range of
dent resulting from voltage-dependent conformational changes
at the filter (28). Furthermore, interaction between Tyr352 and
implicated in the same study, consistent with our structural
Our data on Ca2þblockage yield interesting and unique insights
into the underlying structural mechanisms of specific Ca2þbind-
ing in CNG channels. Contrary to conventional models, the Ca2þ
ion in the filter is exclusively chelated by backbone carbonyl
oxygen atoms and not directly by the acidic side chain which,
particularly in NaK2CNG-E, is oriented tangential to the ion
conduction pathway and buried underneath the external surface
of the protein, seemingly unable to make any direct contact with
main-chain atoms of the selectivity filter (Fig. 2 and Fig. S3).
Ca2þspecificity could arise from the presence of a negative
charge in close proximity that could perturb the charge distribu-
tion along the backbone of the filter residues, making certain
carbonyl oxygen atoms more electronegative and more suited for
Ca2þbinding. Additionally, specificity could also arise from the
side-chain carboxylate of the acidic residue in the NaK2CNG
chimeras being buried in a fairly hydrophobic environment
shielded from the ion conduction pathway by the main-chain
atoms of the selectivity filter residues. This low-dielectric envir-
onment may enhance the electrostatic interaction between the
negative charge and the cation in the filter which, combined with
solutions containing 100 mM NaCl (Na-soak) and in solutions containing both 100 mM NaCl and 25 mM CaCl2(Na/Ca-soak). Maps are contoured at 8σ at a
resolution of 2.0 Å for NaK2CNG-E (Left) and 1.8 Å for NaK2CNG-D (Center) and NaK2CNG-N (Right). (B) Anomalous difference Fourier map (purple mesh, at
1.9 Å and contoured at 5σ) of a Na/Ca-soaked NaK2CNG-D crystal indicates the positions of bound Ca2þions (orange spheres). (C) 2Fo-Fcmaps of Na-soaked
(Upper, 1.62 Å) and Na/Ca-soaked (Upper, 1.63 Å) NaK2CNG-N crystals reveal the hydration of an externally bound Naþ(yellow sphere) or Ca2þion (orange
sphere) by the inner layer of water molecules within the funnel. External Ca2þin NaK2CNG-D is bound in the same hydrated form.
Ca2þbinding in the selectivity filter of NaK mutants. (A) FNa∕Ca-soak-FNa-soakdifference Fourier maps between mutant crystals soaked in stabilization
(Right traces) recorded at −80 mV in the presence of various concentrations of external Ca2þ. Both pipette and bath solutions contain symmetrical 150 mM KCl.
Additionally, 30 μM tetrapentyl ammonium (TPeA) was added to the bath solution to ensure that recorded currents are from mutant channels oriented
with their external side facing the bath solution. (B) Plot of the fraction of unblocked single channel currents recorded at −80 mV as a function of external
Ca2þconcentrations. Data points are mean ? SEM from five measurements and are fitted to the Hill equation with Kiof 58.3 ? 6.3 μM and Hill coefficient
n ¼ 0.98 ? 0.1 for NaK2CNG-E, and Kiof 6.3 ? 0.62 μM and n ¼ 1.22 ? 0.1 for NaK2CNG-D. (C) Single channel traces of NaK2CNG-E recorded at −80 mV
with 150 mM KCl in the pipette and 100 mM CaCl2in the bath solution. The single channel in the recordings had its intracellular side facing the bath solution
and was blocked by addition of 30 μM TPeA in bath solution (bottom trace).
Ca2þblockage and permeation in NaK mutants. (A) Single channel traces of NaK2CNG-E (Left traces), NaK2CNG-D (Center traces), and NaK2CNG-N
Derebe et al. PNAS
January 11, 2011
the presence of four such negative charges in a channel tetramer,
may help stabilize the doubly charged Ca2þion.
The structural study presented here provides an accurate
picture of how Ca2þbinds in the selectivity filter as well as the
position of conserved acidic residues mediating it. In light of
other striking similarities between the two groups, we feel these
NaK2CNG chimera structures represent accurate structural
models for CNG channel pores, complementing long-standing
electrophysiological studies and providing unique insights into
the structural mechanism of CNG channel function. The efficacy
of these models is further verified by our data on chimera struc-
ture-based mutagenesis studies of the bovine retinal CNG chan-
Although further study is needed to work out exact details of
Ca2þbinding mechanisms, we believe our observations very likely
hold true for CNG channels. A similar mechanism may also apply
to voltage-gated Ca2þchannels whose Ca2þbinding mechanism
has been suggested to be similar to CNG channels (2, 4), utilizing
the four conserved glutamate resides in the filter, one from each
homologous domain, to confer Ca2þspecificity (29–31). Finally,
our high-resolution structures also open the door for computa-
tional simulations aimed at providing a more theoretical explana-
tion for Ca2þbinding in tetrameric cation channels.
Materials and Methods
Protein purification, crystallization, and structure determination of all
NaK2CNG chimeras followed our recent study of NaKNΔ19, a truncated
NaK channel lacking the N-terminal M0 helix (27). Similarly, these mutant
structures were determined in an open conformation between resolutions
of 1.58 and 1.95 Å (Table S1 and SI Materials and Methods). All chimeras used
for functional studies contain an extra Phe to Ala mutation at a position
equivalent to Phe92 of NaK in order to enhance the single channel conduc-
tance, and the proteins were reconstituted in lipid vesicles composed of
3∶1 ratio of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine and 1-palmitoyl-
2-oleoyl-phosphatidylglycerol (Avanti Polar Lipids) at a protein/lipid ratio
of 0.1 μg∕mg as described (21, 32). Giant liposomes were obtained by air
drying followed by rehydration. Giant liposome patches were performed as
described in SI Text. The gene encoding bovine retinal cyclic GMP-gated
channel (CNGA1) was cloned into the pcDNA3.1(+) plasmid and the channel
was expressed in HEK 293 cells. The channel currents were recorded 2–4 d
after transfection using patch clamp in a whole-cell configuration. Detailed
descriptions of materials and methods are provided in SI Text.
ACKNOWLEDGMENTS. Use of the Advanced Photon Source and the Advanced
Light Source was supported by the US Department of Energy, Office of
Energy Research. We thank the beamline staff for assistance in data
collection. This work was supported in part by The Howard Hughes Medical
Institute and by grants from the National Institute of Health (GM079179 to
Y.J.) and The David and Lucile Packard Foundation. M.G.D. was supported by
Ruth L. Kirschstein National Research Service Award Predoctoral Fellowship
(5 F31 GM07703) from the National Institutes of Health.
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its E363T/T357E swapped mutation (Lower) recorded at −80 mV in the presence (red traces) and absence (black traces) of 100 μM extracellular Ca2þ. Channels
were activated by 1 mM cGMP in the pipette solution. (B) Plot of the fraction of unblocked inward currents (I/Io) at −80 mV as a function of external Ca2þ
concentrations. Data points are mean ? SEM from five measurements and are fitted to Langmuir functions with Kiof 7.1 μM for wild-type CNGA1. (C) Single
channel traces of NaK2CNG-E (E66T/T60E) swapped mutation recorded at ?80 mV with 150 mM NaCl in the pipette and 150 mM KCl in the bath solution (top
two traces). Addition of 1 mM Ca2þat extracellular side (bath solution) has no effect on single channel conductance at −80 mV (bottom trace).
Mutagenesis of bovine CNGA1 channel based on the structure of NaK2CNG-E. (A) Macroscopic currents of wild-type bovine retinal CNGA1 (Upper) and
www.pnas.org/cgi/doi/10.1073/pnas.1013643108Derebe et al.