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CYCLIC NUCLEOTIDE-GATED
CHANNELS: SHEDDING LIGHT ON
THE OPENING OF A CHANNEL PORE
Galen E. Flynn, J. P. Johnson Jr and William N. Zagotta
Few proteins have been described functionally in such detail as ion channels. All ion channels
open and close their ion-conducting pores, a process referred to as gating. The recent
crystallization of the P-loop-containing channel KcsA has cast channel function in a new light.
Results relating to a variety of P-loop-containing channels are converging on a common
mechanism in which separation of the inner helices that line the pore results in channel opening.
At the same time, differences some subtle and some perhaps more profound have
emerged between channel types. Here we highlight the evidence for a specific conformational
change during the gating of cyclic nucleotide-gated channels, and compare and contrast this
evidence to that obtained for other channels.
ALLOSTERISM (ALLOSTERY) Ion channels are allosteric proteins that regulate ion the inactivation ball of voltage-dependent K+ channels.
The property of a fluxes across membranes. Ion channels can transport This gate, which is tethered to the intracellular amino
macromolecule by which its ions across the membrane at rates of about 108 s1. At terminus, physically plugs the pore after the channel has
function is modified by the the same time, channels select specific ions, often with opened, a mechanism referred to as the ball-and-chain
binding of an effector to a site
other than the binding site of
an accuracy greater than 99.9%. In addition to selecting inactivation 25. Other mechanisms of gating have also
the principal reactant, inducing permeant ions, channels undergo conformational been proposed that involve a hinged lid over the pore, or
a conformational shift in the changes that open and close their ion-permeable pores, a a pinching off of the intracellular or extracellular pore6.
macromolecule. process referred to as gating. Unlike most other allosteric The elucidation of the gating mechanism and identifica-
proteins, conformational changes associated with chan- tion of the gate have been and remain central questions
P LOOP
A conserved structural motif nel gating can be directly observed on a sub-millisecond in the study of ion channels.
found in many different timescale in a single ion channel protein using patch- Years of electrophysiological recording and molecular
ion channels, which constitutes clamp recording techniques1. It is because of the com- cloning have revealed that the hundreds of ion channel
part of the channel pore. plexity and functional precision of these allosteric pro- proteins can be classified into a relatively small number
teins, and the availability of an exquisitely sensitive assay, of discrete gene families on the basis of their primary
that ion channels provide such a powerful system for structure. The biggest family comprises the P-LOOP-
studying ALLOSTERY in proteins. containing channels. This family includes the voltage-
Gating the allosteric transition that opens and dependent Na+, Ca2+ and K+ channels, large-conductance
closes the pore is tightly controlled by allosteric mod- Ca2+-activated K+ (BK) channels, inward-rectifier K+
Department of Physiology
and Biophysics, ulators, such as ligand binding, membrane voltage and (IRK) channels, G-protein-coupled inward-rectifier
Howard Hughes Medical mechanical force. Whereas gating refers to this whole K+ (GIRK) channels, ATP-sensitive K+ (KATP) channels,
Institute, University of process, the gate generally refers only to the structure in and last, but certainly not least, cyclic nucleotide-gated
Washington, Seattle, the pore that prevents ion flow. The gate could be elec- (CNG) channels (FIG. 1). All members of this family share
Washington 98195, USA.
Correspondence to W.N.Z.
trostatic in nature, but most channel physiologists think a common pore domain. The pore domain contains two
e-mail: of it as a physical obstruction of the pore. Perhaps the transmembrane (TM) segments referred to as M1 and
zagotta@u.washington.edu most studied and best-understood example of a gate is M2, or S5 and S6 with an intervening P loop. All
a b
Selectivity filter
2P CHANNELS
Channel proteins that contain
two pore-forming domains in Outer Pore
each subunit. They constitute helix helix
the so-called KCNK channel
family, and function largely as f
regulated K+-selective leak
channels.
Inner
SITE-DIRECTED MUTAGENESIS
Helix
The generation of a
mutation at a predetermined
position in a DNA sequence.
The most common method Figure 2 | Structure of the KcsA channel. a | Top view of the KcsA channel showing four subunits, each in a different colour. A K+
involves the use of a chemically ion is shown in purple. b | Side view of two diagonally opposed subunits. Inner helices, shown in yellow, line the permeation
synthesized mutant DNA pathway. Three K+ ions in the permeation pathway are shown in purple. Oxygens involved in coordinating K+ ions are shown for the
strand that can hybridize with amino acids Thr-Val-Gly-Tyr-Gly in the selectivity filter. Adapted with permission from REF. 7 1998 American Association for the
the target molecule. Advancement of Science.
photoreceptors (CNG3)19. Further subunits have also binding domains (CNBDs) of cyclic nucleotide-depen-
been identified (CNG46)2024. These subunits do not dent protein kinases and transcription factors, and can
form functional channels by themselves, but when coex- accommodate one cyclic GMP or cyclic AMP molecule
pressed with CNG13, they produce functional hetero- per subunit. The binding of cyclic nucleotide induces a
meric channels. In addition to the retina and olfactory conformational change in the carboxy-terminal region
epithelium, CNG channels have been identified in sev- that is allosterically coupled to the opening conforma-
eral other vertebrate tissues, including heart, kidney and tional change in the pore (see BOX 1). This allosteric cou-
brain, where their function remains unknown2531. CNG pling generates the cyclic nucleotide-dependent opening
channels are not activated by changes in membrane of CNG channels.
voltage, but are activated by the direct binding of cyclic Members of the P-loop-containing family of chan-
nucleotide to an intracellular domain of the channel nels share considerable sequence similarity throughout
located in the carboxy-terminal region. This domain the P loop and inner-helix regions (over 50%; FIG. 3).
shows sequence similarity to the cyclic nucleotide- CNG channels are no exception, resembling KcsA in the
sequences that define the pore helix, selectivity filter and
Pore helix Inner helix inner helix. This sequence similarity, along with several
studies using SITE-DIRECTED MUTAGENESIS, supports the
hypothesis that CNG channels also share structural fea-
tures with KcsA3236. In addition, these studies have led to
KcsA TYPRALWWSVETATTVGYGDLYPVTLW--GRLVAVVVMVAGITSFGLVTAALATWFVGRE
a model for the structural rearrangements of the pore
CNG1 KYVYSLYWSTLTLTTIG-ETPPPVRDS--EYFFVVADFLIGVLIFATIVGNIGSMISNMN
associated with the opening of CNG channels that might
CNG2 EYIYCLYWSTLTLTTIG-ETPPPVKDE--EYLFVIFDFLIGVLIFATIVGNVGSMISNMN
also apply to other members of this family36,37. In this
review, we examine each of the structural elements of
IRK1 SFTAAFLFSIETQTTIGYGFRCVTDECPIAVFMVVFQSIVGCIIDAFIIGAVMAKMAKPK
the pore, and discuss their possible roles in the gating of
GIRK2 GFVSAFLFSIETETTIGYGYRVITDKCPEGIILLLIQSVLGSIVNAFMVGCMFVKISQPK
CNG and related channels.
KATP SFSSAFLFSIEVQVTIGFGGRMVTEECPLAILILIVQNIVGLMINAIMLGCIFMKTAQAH
BK TYWECVYLLMVTMSTVGYGDVYAKTTL--GRLFMVFFILGGLAMFASYVPEIIELIGNRK
Pore helix
Shaker SIPDAFWWAVVTMTTVGYGDMTPVGFW--GKIVGSLCVIAGVLTIALPVPVIVSNFNYFY
A key element in the structure of the pore of KcsA is the
Figure 3 | Sequence alignment of the pore helix, selectivity filter and inner helix for pore helix. There is also evidence for a pore helix in
several channels of the P-loop-containing family. Amino acids in blue are conserved among CNG channels. Mutant CNG1 channels that contain
several channels; amino acids in red are identical for all channels. The selectivity filter is site-directed cysteine substitutions at positions in the
highlighted in yellow; residues lining the pore are highlighted in green. KcsA, Streptomyces pore helix have been probed with the cysteine-modify-
lividans, accession number S60172; cyclic nucleotide-gated channel 1 (CNG1), Bos taurus,
Q00194; CNG2, Rattus norvegicus, Q00195; inward-rectifier K+ channel 1 (IRK1), Cavia
ing reagents (2-aminoethyl)methanethiosulphonate
porcellus, P52185; G-protein-coupled inward-rectifier K+ channel 2 (GIRK2), Mus musculus, (MTSEA) and (2-(trimethylammonium)ethyl)methane-
AAA91457; ATP-sensitive K+ channel (KATP), M. musculus, Q61743; large-conductance Ca2+- thiosulphonate (MTSET) to determine the accessibility,
activated K+ channel (BK), M. musculus, A48206; Shaker, Drosophila melanogaster, S00479. and therefore exposure, of these amino acids32,33,35. The
In KcsA, the inner helix lines the inner vestibule. Recent 0.6
l/Imax
work has shown that the analogous structure in CNG
0.4
channels, the S6 segment, serves a very similar role.
Mutant CNG1 channels that contained site-directed 0.2
change during gating. How could ion channels so closely related N482C
differ so much in the location of the gate? One possibility is a notable Y483C
difference in the S6 amino-acid sequence. Shaker K+ channels have
F484C
two prolines in the middle of their S6 segment. Prolines are predicted
to break -helices and, as a consequence, might impose a kink in the Y485C
Post-TM segments
In CNG channels, the region between the S6 TM seg-
a b
ment and the CNBD (residues 400500 in bovine
109 CNG1) is known as the C-linker. In addition to its
Current (A)
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URLs
BK Shaker
http://www.ncbi.nlm.nih.gov/LocusLink/list.cgi?Q=kcnma1[sym]%20 http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=32780
or%20kcnmb1&ORG=Hs SK
IRK http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=3780
http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=3759 MscL
GIRK http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pro
http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=3760 tein&list_uids=547924&dopt=GenPept
KATP Herg
http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=3767 http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=3757
CNG Kv
http://www.ncbi.nlm.nih.gov/LocusLink/list.cgi?Q=cnga1%20or%20cn http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=3736
gb1&ORG=Hs The ion channel web page
X-ray crystal structure of KcsA http://phy025.lubb.ttuhsc.edu/Neely/ionchann.htm
http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form=6& The ligand-gated ion channel database
db=t&Dopt=s&uid=12521 http://www.pasteur.fr/recherche/banques/LGIC/LGIC.html
KcsA Families of transport proteins
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=Pro http://www.biology.ucsd.edu/%7Emsaier/transport/toc.html
tein&list_uids=3402029&dopt=GenPept
Biographies
In 1995, Galen E. Flynn received her Ph.D. in neurobiology from the University of Washington. Before joining William N. Zagottas laboratory, she
studied the molecular mechanisms of the block of sodium channels by inactivation-gate peptides with William A. Catterall. At present, her research
is focused on the molecular mechanisms involved in the gating of cyclic-nucleotide-gated channels.
J. P. Johnson Jr earned his Ph.D. in pharmacology from Vanderbilt University (1999) while studying the function of voltage-gated K+ channels with
Paul B. Bennett. At present, he is a senior fellow of the Howard Hughes Medical Institute at the University of Washington Department of Physiology
and Biophysics. There, his postdoctoral work with William N. Zagotta continues to centre on ion-channel function. His research is focused on the
coupling of ligand-binding and gating in cyclic-nucleotide-gated channels.
William N. Zagotta received his Ph.D. degree in neuroscience from Stanford University, where he worked with Richard W. Aldrich on the activation
and inactivation of Shaker K+ channels. He continued his studies as a postdoctoral fellow at Stanford, working with Richard W. Aldrich and Denis
Baylor, where he developed an interest in the ion channels involved in phototransduction. William Zagotta is now Professor of Physiology and
Biophysics at the University of Washington School of Medicine, and an associate investigator of the Howard Hughes Medical Institute.
At a glance
The term gating refers to the allosteric transition that opens and closes the pore of an ion channel. Channel gating has been the focus of intense
investigation, but its structural basis remains elusive. The crystal structure of KcsA, a bacterial potassium channel, has provided a framework for
new studies of gating in many channel proteins, including cyclic nucleotide-gated (CNG) channels, the focus of this review.
The sequence of CNG channels is similar to that of KcsA in the region around the pore domain, having a pore helix, a selectivity filter and an inner
helix. Site-directed cysteine substitutions at the presumptive pore helix of CNG1 have provided evidence for the rotation of this helix during gating.
Similarly, kinetic analysis and studies with channel blockers have provided indirect evidence for movement of the selectivity filter during gating.
In the case of the inner helix, a conformational change in this region also seems to occur during channel gating, as illustrated by the spontaneous
formation of disulphide bridges between the inner helices of different CNG subunits when the channel is closed, but not when it is open.
The linker between the inner helix and the intracellular cyclic nucleotide-binding domain is crucial for the allosteric coupling between ligand binding
and channel opening. It has been found that histidine residues that are present in part of the linker region are capable of coordinating Ni2+ ions
between subunits, indicating their spatial proximity. Histidine-substitution experiments show that this region of the linker rotates during gating.
On the basis of these and other observations, a new structural model for CNG channel gating is emerging. Opening of the channel involves a
clockwise rotation of the distal portion of the linker segment. This rigid body movement unwinds the helical bundle at the bottom of the inner
helix, leading to a significant increase in the diameter of the pore. The movement of the inner helix then initiates rearrangements in a gate that is
presumably located in the selectivity filter.