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
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Derebe et al. PNAS
January 11, 2011