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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

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a P loop The missing link for studies of allostery in ion chan-


Extracellular nels has been structural data. In 1998, the structure of the
first ion channel pore was revealed after the elucidation
M1 M2 of the X-ray crystal structure of KcsA, a K+ channel from
Streptomyces lividans7 (FIG. 2). KcsA is a tetramer of iden-
Intracellular tical subunits arranged with fourfold symmetry around
N a centrally located pore. A single KcsA subunit has two
membrane-spanning helices, the outer and inner helices,
C and a re-entrant P loop. The P loop of KcsA enters the
b
P loop membrane as an -helix (pore helix), then exits back
Extracellular extracellularly as an uncoiled strand. It is in this strand
+ that permeant ions are coordinated by the backbone car-
+
S1 S2 S3 S4 S5 S6 bonyl oxygens of the amino acids Thr-Val-Gly-Tyr-Gly,
+ which have been recognized as the signature sequence of
+
Intracellular K+-selective channels8. Intracellular to the selectivity filter
is a large (10 in diameter) water-filled vestibule, which
N
also holds a permeant ion7,9. The partial negative charge
C
of the oriented pore helices electrostatically stabilizes the
ion in the inner vestibule10. The inner membrane-span-
Figure 1 | Membrane topology for subunits of the two
main groups of the P-loop-containing family of ion
ning helices line the vestibule of the channel and cross
channels. a | Predicted membrane topology of inward-rectifier the membrane at an angle to form a helix bundle on the
K+ (IRK), G-protein-coupled inward-rectifier K+ (GIRK), ATP- intracellular side in what has been referred to as an
sensitive K+ (KATP) and KcsA channels. b | Predicted membrane inverted teepee structure7. The helix bundle defines the
topology of cyclic nucleotide-gated (CNG), voltage-dependent intracellular entrance to the pore, which is sometimes
K+ (KV), Shaker, large-conductance Ca2+-activated K+ (BK) and referred to as the smokehole11. The KcsA structure has
small-conductance Ca2+-activated K+ (SK) channels. The S4
transmembrane segment is highly charged.
provided a framework for new studies of permeation
and gating in all members of the P-loop-containing
family of ion channels.
members of this family are tetrameric or pseudo- This review will highlight one member of the P-loop-
tetrameric. In the case of some channels, such as the containing channel family the CNG channels and
+ 2+
2P CHANNELS or voltage-dependent Na and Ca channels, compare recent evidence for conformational changes in
two or four subunits are concatenated in a single poly- the pore to that of other members of the P-loop channel
peptide chain, creating a pseudo fourfold symmetry6. family. CNG channels were first identified in photo-
Voltage-dependent and CNG channels have four addi- receptors12 and olfactory receptors13, where they pro-
tional TM segments per subunit: S1S4. The S4 segment duce the primary electrical signal in response to sensory
is highly charged, and in voltage-dependent channels, stimuli (for review, see REF. 14). These channels were
this segment is the sensor for the allosteric modulation first cloned from rod photoreceptors15 (CNG1), and
of gating by membrane voltage. later from olfactory epithelium (CNG2)1618 and cone

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.

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Box 1 | Cyclic nucleotide-binding domain


Cyclic nucleotide-gated (CNG) channels are activated by the
direct binding of cyclic GMP or cyclic AMP to a cyclic
nucleotide-binding domain (CNBD) in the intracellular
carboxy-terminal region14. This domain has significant
sequence similarity to the CNBD of several other cyclic
nucleotide-binding proteins, including the Escherichia coli
catabolite gene activator protein (CAP). The structure of CAP
bound to cAMP has been determined by X-ray
crystallography93. The CNBD comprises eight -strands in a
-roll conformation (blue), followed by two -helices: the B-
and C-helices (red). Recently, a chimaera of the CNBD of
CNG1 and the DNA-binding domain of CAP has also been
crystallized and shown, at low resolution, to have a structure
similar to CAP88. Functional studies of CNG channels using
mutagenesis and cysteine modification have identified several
sites of interaction between the CNBD and the cyclic
nucleotide (yellow)94100. There is a particularly interesting
interaction between D604 in the C-helix and the purine ring Channel closed Channel open
of cGMP. Interactions between the ligand and D604
determine the agonist efficacy and cyclic nucleotide specificity
of the channel96,101,102. Moreover, this interaction occurs during the opening of the channel. These results, combined with cysteine-accessibility studies100,
indicate that the opening of the channel is associated with a rearrangement in the CNBD, moving the C-helix closer to the -roll. This conformational
change in the CNBD is allosterically coupled to the opening conformational change in the CNG channel pore by a mechanism that is so far unknown.

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

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a between inactivation in voltage-gated channels and the


P366C closing of CNG channels.
T364C
Selectivity filter
Wt:E363C The amino acids that follow the pore helix in KcsA form
its selectivity filter. Several lines of evidence indicate that
Wt:L358C
the selectivity filter of CNG channels is also formed by the
Wt:T355C amino acids following its pore helix. Most notably, replac-
L351C
ing the pore sequence of bovine CNG1 with the sequence
of the fish CNG2 confers the larger pore diameter and sin-
V348C gle-channel conductance of CNG2 onto CNG1 (REF. 40).
Interestingly, replacing the selectivity-filter sequence of
1 10 100 1,000 Shaker K+ channels with the CNG channel sequence
Modification rate (M1 s1) altered the selectivity profile of Shaker, making it a non-
SHAKER
A voltage-gated channel, the selective cation channel like the CNG channel41.Although
activation of which leads to the b CNG channels lack the signature sequence for K+ selectivi-
appearance of a transient K+ ty8, they contain an acidic residue (E363; FIG. 4b) that acts
current. It takes its name from
as an external binding site for monovalent cations, divalent
Drosophila with mutations in the
gene that encodes this protein. cations and protons, as in other Ca2+- and Na+-selective
These flies display a violent channels4249. These results show the importance of this
shaking phenotype when under region in permeation as well as in selectivity.
anaesthesia. New evidence indicates that the selectivity filter of
Figure 4 | State-dependence of modification of amino
C-TYPE INACTIVATION
CNG channels undergoes a conformational change in
acids in the pore helix of CNG channels. a | Rates of
Two distinct molecular modification of the pore-helix residues of cyclic nucleotide-
association with gating. CNG channels that are partially
mechanisms for K+ channel gated channel 1 (CNG1) by (2-aminoethyl)methanethio- activated have permeation properties that differ from
inactivation have been sulphonate (MTSEA) for both open (open circles) and closed those of fully activated channels5052. In addition, intra-
described: N-type, which
(filled circles) states. Wt, wild type. Adapted with permission cellular tetracaine a local anaesthetic gives a state-
involves occlusion of the pore by
from REF. 35 2000 Elsevier Science. b | Homology model of dependent block of CNG channels5355. In blocking these
an intracellular domain of the
the pore helix and selectivity filter of CNG1, based on the channels, the affinity of tetracaine is about three orders
channel, and C-type, which
structure of KcsA. The homology model was generated using
involves a conformational of magnitude higher in the closed state than in the open
change in the outer pore. the sequence alignment of CNG1 to KcsA, shown in FIG. 3,
and the SWISS-MODEL server. Limited energy minimizations state55. Surprisingly, this state-dependent block by tetra-
ELECTRON PARAMAGNETIC were also done105. Red residues are modified preferentially in caine is abolished by the mutation E363G56. In addition
RESONANCE SPECTROSCOPY the closed state by MTSEA; green residues are modified to effects on permeation and block, mutations at E363
When an atom with an unpaired preferentially in the open state. E363, shown in blue, is the also affect the allosteric equilibrium constant for channel
electron is placed in a magnetic external divalent-binding site36. gating, lowering the opening probability in E363 mutant
field, the spin of the unpaired
channels relative to wild-type CNG1 channels by about
electron can align, either in the
same direction as the field, or in 100-fold33,46,55,57. Although indirect, these studies point to
the opposite direction. EPR is presence of a pore helix in CNG1 was shown by the the possibility that the selectivity filter moves during
used to measure the absorption pattern of reactivity to modification by extracellular channel opening, and lead to the speculation that it
of microwave radiation that MTSEA, in which every third to fourth position is modi- might act as a gate in CNG channels.
accompanies the transition
between those two states.
fied, consistent with the presence of an -helical structure Movement of the selectivity filter is also associated
at the beginning of the P loop35. In addition, this putative with gating in K+ channels. It has long been known that
PROBE MOBILITY CNG1 pore helix has been suggested to undergo a con- certain permeant ions, such as Rb+, exert a foot-in-the-
In an EPR experiment, changes formational change during gating33,35.V348C, L351C and door effect, slowing the rate of channel closure5860. A
in the mobility of spin-labelled
T355C are modified only in the closed state from the movement of the selectivity filter has been directly
residues are indicative of
rearrangements in tertiary or extracellular side, whereas W353C, on the opposite side of observed in KcsA using ELECTRON PARAMAGNETIC RESONANCE
quaternary contacts; positive the helix, is modified only in the open state35 (FIG. 4). State- SPECTROSCOPY (EPR). Residues on the intracellular end of
values indicate increased steric dependent modification of these residues indicates a pos- the selectivity filter show changes in PROBE MOBILITY
contacts (reduced mobility) and sible rotation of the pore helix during gating. (Ho) and intersubunit PROBE-TO-PROBE DISTANCE (),
negative values point to
increased motional freedom.
Studies on voltage-dependent K+ channels have also indicating that the intracellular side of the selectivity fil-
reported movement of the pore helix in association with ter undergoes a rearrangement in association with open-
PROBE-TO-PROBE DISTANCE gating. Perhaps most notably, the W434F mutation in the ing61. By contrast, residues on the extracellular side of
In an EPR experiment, this pore helix causes SHAKER K+ channels to undergo perma- the selectivity filter show no change in either parameter,
parameter provides an
nent C-TYPE INACTIVATION, but leaves the activation-gating indicating that this region is immobile61.
indication of changes in inter-
subunit proximity. In the case of mechanism intact38,39.W434, one of a pair of neighbouring Certain mutations in the selectivity filter of K+ chan-
P-loop-containing channels, aromatic residues in the pore helix, interacts with amino nels have particularly intriguing effects on gating. These
values lower than 1 indicate that acids in the selectivity filter7.It has been suggested that these mutations have been shown to cause the appearance of
the spin-labelled residues move interactions hold the selectivity filter open, and that inacti- pronounced subconductance states in single-channel
closer to the axis of symmetry of
the channel, whereas larger
vation involves a collapsing of the selectivity filter. records 6265. In voltage-dependent channels, these
values point to motion away Aromatic amino acids are conserved at some of these subconductance states are associated with partial activa-
from the symmetry axis. positions in the CNG channel, indicating a possible link tion of the channels. Furthermore, the subconductance

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levels in Shaker mutants have been shown to have dif- a


S399C
ferent ionic selectivities from that of the fully open Sat. cGMP
state62. These results provide evidence that the selectivity 0 cGMP
filter might participate in K+-channel gating.
1.0

Inner helix 0.8

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

cysteine substitutions at positions in the S6 were studied 0.0


to determine the effects of positively charged MTSET 0 4,000 8,000 12,000
on permeation. The modification of residues that were Time (s)
predicted to point towards the pore were shown to have b
large effects on permeation, whereas the modification of
residues that were predicted to point away from the pore
had little or no effect36. These results are consistent with
a structural homology between CNG and KcsA chan-
nels in this region. This structural homology might also
extend to other channels, such as KATP channels66, IRK
channels67 and GIRK channels68,69.
A conformational change occurs in the CNG1
helix bundle during channel opening. Spontaneous Closed Open
disulphide bonds between different CNG1 subunits
Figure 5 | Conformational changes in the helix bundle
form at S399C residues, which are located at the cyto-
reported by disulphide bond formation at S399C in
plasmic end of S6 (REF. 36). But to form disulphide CNG1. a | State-dependence of spontaneous disulphide bond
bonds, the intracellular ends of the S6 helices of formation at S399C of cyclic nucleotide-gated channel 1
CNG1 must be reasonably close together. So, whereas (CNG1). Following excision, this patch was initially exposed to
disulphide bonds form spontaneously between S399C saturating (Sat., 2.5 mM) cyclic GMP for about 1 hr. The patch
residues in the closed state, they do not form when the was then exposed repeatedly to 0 cGMP for 500 s followed by
channels are open (FIG. 5a). This indicates that the 2.5 mM cGMP for 10 s. The time-dependent decrease in
cGMP-activated current is shown. b | A possible mechanism
smokehole enlarges during channel opening by a con- for conformational changes in S6 associated with the opening
formational change in S6 (FIG. 5b). Further support for and closing of CNG1 channels. Shown is a view of four S6
this conformational change comes from the state- helices from the extracellular side of a CNG1 channel in the
dependence of modification of inner-vestibule closed (left) and open (right) states. Yellow amino acids are
residues. V391C is modified more quickly by MTSET S399; red amino acids are V391. A silver atom is shown in blue
when the channel is open than when it is closed36. on the same CPK SCALE (CoreyPaulingKoltun scale) as the
side chains. The tops of the S6 helices are in the same position
Enlarging the smokehole could explain how large
for both closed and open states. Adapted with permission from
quaternary ammonium compounds, such as tetra- REF. 36 2001 Elsevier Science.
butylammonium, could enter the inner vestibule and
block CNG channels in a voltage-dependent manner70.
Enlarging the smokehole also explains how the Shaker for these channels. Interestingly, similar experiments in
ball peptide, a 20-amino-acid peptide, blocks open voltage-dependent K+ channels support a rather different
CNG channels in a voltage-dependent manner and conclusion (BOX 2).
prevents channel closure71. To account for all these Movement of the pore-lining M2 helices in associa-
results, a conformational change in the helix bundle tion with gating was also shown in KcsA using EPR
of CNG channels must occur. techniques. Residues throughout the M2 of KcsA show
Is the helix bundle a gate for CNG channels? As the changes in Ho with gating. Interestingly, there is a
S399C residues of CNG1 are close enough to form a gradual increase in Ho near the intracellular side of
disulphide bond spontaneously in the closed state, it is the membrane61. In addition, measurements indicate
conceivable that channel closure would prevent ions that most of the residues of M2 move away from the
from entering the inner vestibule, and that the smoke- central axis of the pore. As a consequence of this move-
hole might act as the activation gate for CNG channels. ment, it has been proposed that the intracellular ends of
However, Ag+ enters the inner vestibule of CNG1 chan- the M2 helices move by a translation and rotation that
nels in both the closed and open states, unencumbered enlarges the entrance of the inner vestibule of KcsA61.
by the conformation of the S6 helices, and at a rate that A similar conclusion was reached for GIRK channels.
CPK MODEL is limited only by diffusion36. As Ag+ is nearly the same Mutations in the M2 of GIRK channels that result in
A space-filling atomic model in size as the permeant ions, these results indicate that, channels that are constitutively open have been identi-
which the atoms are represented
as spheres, the radii of which are
although the S6 helices of CNG channels undergo a fied68,69. These results indicate that many P-loop chan-
proportional to the van der conformational change associated with channel nels undergo similar movements of the inner helices
Waals radius of the atom. opening, the helix bundle cannot be the activation gate during gating.

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Box 2 | Gated access in Shaker channels


The location of the gate in voltage-dependent channels might differ
T469C
from that of cyclic nucleotide-gated (CNG) channels. Two lines of
I470C EA
evidence support a difference in the S6 segment of Shaker K+
channels. Cysteine-accessibility methods were used to determine the A471C

rates of intracellular (2-(trimethylammonium)ethyl)methane- L472C


thiosulphonate (MTSET) modification of S6 residues in both the P473C
open and closed conformations. The plot (adapted with permission
V474C
from REF. 103 1997 Elsevier Science) shows that sites in the inner
P475C
vestibule were modified significantly faster in the open state (open
circles) than in the closed state (filled squares). These results point to V476C

gated access to the inner vestibule of Shaker K+ channels, and I477C


indicate that the inner half of the Shaker S6 segment acts as a gate V478C
that regulates access to the pore. Furthermore, coordination of Cd2+
S479C
between cysteine residues at position 474 of multiple subunits has no
N480C
effect on channel opening103. These results indicate that the outer half
of the S6 segment is static and does not undergo a conformational F481C

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

S6 of Shaker channels104. Other voltage-dependent channels, such as H486C

BK and Herg channels, have at least one helix-breaking proline in


100 101 102 103 104 105 106
their S6 segments. A kink in the Shaker S6 segment could explain
some of the differences between Shaker and CNG channels. Modification rate (M1 s1)

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)

obvious role in tethering the ligand-binding domain


1010 (residues 500693) to the rest of the channel protein, the
+Ni2+ significance of the C-linker is just beginning to be under-
1011 stood. Throughout this region, residues have been shown
K416H
to exhibit state-dependent modification, and mutations
have been shown to have large effects on gating7279.
107 106 105 104 103 Recently, a fluorophore attached to C481 in the distal
[cGMP] (M) C-linker has been shown to exhibit state-dependent fluo-
rescence quenching80. These results have pointed to the
Inhibit C-linker as a crucial component in the allosteric coupling
108
between ligand binding and channel opening.
The coordination of Ni2+ between histidine residues of
Potentiate
Current (A)

multiple subunits reveals a close association between the


109
first 20 amino acids of the C-linker (post-TM segments)
in the functional tetrameric channel37,72,73,81. The H420
+Ni2+ residue from neighbouring subunits in bovine CNG1
1010 Q409H channels creates a Ni2+-binding site at the interface
107 106 105 104 103 between subunits73. Furthermore, a histidine in rat CNG2
at a position equivalent to 417 supports Ni2+ binding72.
[cGMP] (M)
Recently, a histidine scan of the post-TM segment has
Figure 6 | Ni2+ effects on histidine-substituted channels indicate a rotation of the revealed a number of positions that produce high-affinity
post-TM segment during gating. a | Shown are doseresponse curves for cyclic GMP in the Ni2+-binding sites37. As the Ni2+ interactions seem to
absence (filled squares) and presence (open squares) of 1 M Ni2+. The arrow indicates the occur through coordination by individual histidines from
change in current at subsaturating cGMP concentrations. The upper panel shows potentiation multiple subunits, the post-TM segments of neighbour-
by Ni2+ binding to K416H residues; the lower panel shows inhibition by Ni2+ binding to Q409H ing subunits must be in close proximity. The pattern of
residues. b | The post-transmembrane segment shown as an -helix. Histidine substitution at the
positions that produce Ni2+-binding sites, together with a
red positions produced Ni2+-inhibited channels; histidine substitution at the green positions
produced Ni2+-potentiated channels. Substitution at blue residues produced Ni2+-insensitive predicted -helical structure82, indicates that the post-
channels. Mutating grey residues yielded non-functional channels. Adapted with permission from TM segments form a right-handed helical bundle just
REF. 37 2001 Macmillan Magazines Ltd. cytoplasmic to the S6 segments.

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Other ion channels have -helical structures after


the last TM segment. On the basis of EPR data, the
post-TM regions of KcsA have also been suggested to
be -helical, and predicted to form a right-handed heli-
cal bundle83 similar to that proposed for the CNG
channels37. The calmodulin-binding domain of the
small-conductance Ca2+-activated K+ (SK) channel,
which corresponds to the post-TM segment, was recently
crystallized and shown to have an -helical conforma-
tion84. Also, the mechanosensitive channel MscL is a Closed Open
homopentamer that is not a member of the family of
P-loop-containing channels; however, the segment fol-
lowing M2 in MscL is -helical and forms a left-handed
helical bundle cytoplasmic to the pore85.
Although a fourfold symmetrical structure for the
post-TM segments seems likely in view of the fourfold
symmetry of the helix bundle of KcsA, the data on CNG
channels do not rule out the possibility that the helices
might actually associate as a dimer-of-dimers86. For
instance, Ni2+ coordination in homomeric CNG1 chan-
nels and heteromeric CNG1/CNG4 channels involves
identical histidines from two neighbouring subunits,
which form a single Ni2+-binding site81,87. Moreover, the
gating of heteromeric channels, in which some subunits
contain non-functional CNBDs, was found to be consis-
tent with a coupled-dimer model86. The CNBD of CNG
channels is positioned on the carboxy-terminal side of the
post-TM segments, but a chimaera of the CNBD of Figure 7 | Model of the conformational changes in the
helix bundle and post-TM segment of CNG1 channels
CNG1 and the DNA-binding domain of the Escherichia during gating. Bottom and side views of the model of cyclic
coli catabolite gene activator protein (CAP) crystallizes as nucleotide-gated channel 1 (CNG1) are shown in the closed
a dimer88. As for other P-loop channels, the calmodulin and open states. Histidine substitution at the red positions
binding domains in the post-TM segment of SK chan- produced Ni2+-inhibited channels. Histidine substitution at the
nels crystallize as dimers in the presence of calmodulin84, green positions produced Ni2+-potentiated channels. Position
as do the domains that regulate the conductance of K+ 399 is shown in yellow. Adapted with permission from REF. 37
2001 Macmillan Magazines Ltd. See movie online.
(RCK domains) in the carboxy-terminal region of an
E. coli K+ channel89. Whether the post-TM segments
associate with a twofold or a fourfold symmetry in closed state (red residues in FIG. 6b). The offsetting distri-
tetrameric channels is still open to question. butions of potentiating and inhibiting residues indicates
that there is a clockwise rotation of the post-TM seg-
Rearrangements of post-TM segments ment relative to the central axis of the pore on channel
Ni2+ binding to the post-TM segments of CNG channels opening. To support Ni2+ coordination in the closed
provides more structural information than that relating state, the inhibitory residues from different subunits
to their proximity. Ni2+ binding has one of two effects on must be close together and aligned when the channel is
channel gating: potentiation or inhibition37,72,73,90,91. closed. When the channel opens, the post-TM segments
At some positions, Ni2+ binds preferentially to the open rotate clockwise, and the potentiating residues move
state of the channels, and thereby potentiates the into alignment to coordinate Ni2+ in the open state.
response of the channel to partial agonists and low con- These simple movements could produce the observed
centrations of full agonists (FIG. 6a). At other positions, patterns of Ni2+ reactivity and state-dependence.
Ni2+ binds preferentially to the closed state of the chan- On the basis of the new information on pore move-
nels and produces inhibition (FIG. 6a). The state-depen- ments in CNG1 channels, and considering what is
dence of Ni2+ binding in the post-TM segments indicates known about the structure of KcsA, a new structural
that this region is involved in the allosteric coupling model for CNG channel gating is emerging (FIG. 7). In
between ligand binding and channel opening. this model, opening of the channel involves a clockwise
Histidine substitution experiments in the post-TM rotation of the distal portion of the post-TM segment.
segments reveal a striking pattern of Ni2+ modulation of This rigid body movement unwinds the helical bundle
channel function. Histidine substitution of amino acids at the bottom of S6, leading to a significant increase in
at positions 416 and 420, separated by four amino acids, the diameter of the smokehole. The movement of the
permits Ni2+ binding to the open state (green residues in S6 segments initiates rearrangements in a gate that is
FIG. 6b). By contrast, histidine substitution of the amino presumably located in the selectivity filter. This sce-
acids at positions 409, 413 and 417 of CNG1, again sep- nario allows the C-linker to act as a lever arm, twisting
arated by four amino acids, permits Ni2+ binding to the open the channel and transferring the conformational

NATURE REVIEWS | NEUROSCIENCE VOLUME 2 | SEPTEMBER 2001 | 6 4 9


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REVIEWS

change of the ligand-binding domain to the pore of the Conclusion


channel protein. Although the precise manner in which channels move
One difficulty in defining the movement of such a during gating remains elusive, some common themes are
model is the establishment of a fixed pivot point the beginning to emerge on the structure and allosteric con-
point around which the helices move away from, but do formational changes that are associated with channel
not rotate relative to, the central axis of the pore during opening. The last TM segment seems to line the pore of
gating. The location of the pivot point defines the direc- all P-loop-containing channels, and rotational and trans-
tion in which residues move relative to the central axis of lational movements of this segment are associated with
the pore. In models like the one shown in FIG. 7, the the opening of many of these channels. Furthermore, it
residues above the pivot point will rotate anticlockwise, seems that there is frequently an association of the post-
and those below the pivot point will rotate clockwise, rela- TM region of multiple subunits, and that this region
tive to the central axis of the pore. In CNG1 channels, we might act as a lever arm for transmitting changes in an
assumed that this pivot point is located at the top of the S6 intracellular domain to open a gate located in the pore.
helices. If the pivot point is at the top of the S6, then all the The CNG channels seem to be sitting at a fortunate con-
residues below it will rotate clockwise relative to the central vergence point, as features of channel structure and gating
axis of the pore, as we proposed for the post-TM seg- previously seen in many different channels come together
ment37. An analysis of gating movements in KcsA using in the framework of a single model channel: CNG1.
EPR-derived distance restraints indicates that the pivot
point might be lower, near the smokehole92. This could
explain the apparent discrepancy between the clockwise Links
rotation of the post-TM region in CNG channels, and the DATABASE LINKS BK | IRK | GIRK | KATP | CNG | X-ray
apparent anticlockwise rotation of the inner helix in KcsA. crystal structure of KcsA | KcsA | Shaker | SK | MscL | Herg | KV
More experiments to localize the pivot point accurately are FURTHER INFORMATION SWISS-MODEL server | The ion
needed to determine the direction of rotation that occurs channel web page | The ligand-gated ion channel database |
in the inner helices and post-TM segments during gating. Families of transport proteins

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Electron paramagnetic resonance spectroscopy 84. Schumacher, M. A., Rivard, A. F., Bachinger, H. P. & structural implications. Nature 403, 321325 (2000).
experiments indicate that the opening of KcsA Adelman, J. P. Structure of the gating domain of a Ca2+- 105. Guex, N. & Peitsch, M. C. SWISS-MODEL and the Swiss-
involves a translation and rotation of the TM1 and activated K+ channel complexed with Ca2+/calmodulin. PdbViewer: an environment for comparative protein
TM2 segments. Nature 410, 11201124 (2001). modeling. Electrophoresis 18, 27142723 (1997).

NATURE REVIEWS | NEUROSCIENCE VOLUME 2 | SEPTEMBER 2001 | 6 5 1


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ONLINE

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.

2001 Macmillan Magazines Ltd

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