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Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating
Journal of Genetics and Genomics (Formerly Acta Genetica Sinica) June 2007, 34(6): 477-485
Review
Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating Zhengchao Wang1, Yongqing Jiang2, Lizhi Lu2, Ruihua Huang1, Qingchao Hou1, Fangxiong Shi1,
①
1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China; 2. Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Abstract: Cyclic nucleotide-gated ion channels (CNGs) are distributed most widely in the neuronal cell. Great progress has been made in molecular mechanisms of CNG channel gating in the recent years. Results of many experiments have indicated that the stoichiometry and assembly of CNG channels affect their property and gating. Experiments of CNG mutants and analyses of cysteine accessibilities show that cyclic nucleotide-binding domains (CNBD) bind cyclic nucleotides and subsequently conformational changes occurred followed by the concerted or cooperative conformational change of all four subunits during CNG gating. In order to provide theoretical assistances for further investigation on CNG channels, especially regarding the disease pathogenesis of ion channels, this paper reviews the latest progress on mechanisms of CNG channels, functions of subunits, processes of subunit assembly, and conformational changes of subunit regions during gating. Keywords: cyclic nucleotide-gated ion channel (CNG); conformational change; gating
Cyclic nucleotide-gated ion channels (CNGs) are nonselective cation channels, which were initially discovered in the plasma membrane of the outer segment of the vertebrate rod photoreceptors and observed in cone photoreceptors, olfactory sensory neurons (OSNs), and other neuronal and nonneuronal cell [1−6]
types
logical ligand for certain CNGs is cGMP, whereas for others it is cAMP. To a certain extent, all CNGs can be activated by both cGMP and cAMP; however, the efficiencies can vary by several orders of magnitude in different CNG channels because of their different affinities to these two ligands. These differences may
. CNGs play critical roles in phototransduc-
be due to the stoichiometry and assembly of CNG
tion and olfactory transduction. In visual and olfactory sensory cells, CNGs are gated by the second
channels. Recently, two reviews were done to discuss
messengers, guanosine 3′ , 5′ -cyclic monophosphate
roles, and regulatory mechanisms of CNG chan-
(cGMP), and adenosine 3′ , 5′ -cyclic monophosphate
nels[6,7]. However, reviews about gating and working
(cAMP), respectively, and convert stimuli into electrical signals that are processed as visual or olfactory
mechanisms on this particular channel are still un-
information[3−14]. It is well known that the physio-
paper reviews the latest progress on mechanisms of
molecular structures, physiological and pathological
available in Chinese published reports. Therefore, this
Received: 2006-11-07; Accepted: 2007-01-08 This work was supported by the Provincial Key Projects for Scientifical and Technological Research of Zhejiang Province (No. 2006C12058), and National Natural Science Foundation of China (No. 30571335) and a Grant-in-Aid for Innovative Training of Doctoral Students in Jiangsu Province, China. ① Corresponding author. E-mail: [email protected]; Tel/Fax: +86-25-8439 9112 www.jgenetgenomics.org
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CNG channels, functions of subunits, processes of subunit assembly, and conformational changes of subunit regions during gating in detail. In vertebrates, there are at least six CNG subunit genes, CNGA1, CNGA2, CNGA3, CNGA4, CNGB1, and CNGB3, with homology between families ranging from 30% to 70%[5, 6]. These subunits are generically designated as principle subunits (or A subunits) and modulatory subunits (or B subunits). CNGAs are defined as functional subunits of channels because they are functional when four different CNGAs are assembled, whereas CNGBs are only defined as regulatory subunits of channels because CNGBs are nonfunctional if four different CNGBs are assembled together. This functional definition, however, is not always consistent with the phylogenetic assignment of subunit type, for example, CNGA4. Although certain types of CNG subunits can form functional homomultimeric channels when expressed in a heterologous system, in vivo they appear to be constructed as heterotetramers with one or more mediated subunit composed of 2−3 different types of subunits per [1−14]
. Native ion channels can prechannel complex cisely tune to their physiological roles in neuronal signaling. This tuning frequently involves the controlled assembly of heteromeric channels comprising of multiple types of subunits. Many fundamental and specialized properties of CNGs, such as open probability, ligand sensitivity, ion permeability, and odorant adaptation vary according to the subunits expressed (A and B subunit types) and their stoichiometry[5−16]. Findings in recent studies have suggested that the identity of the β-subunit (CNGB1 or CNGB3) is an important determinant for heteromeric channel assembly and arrangement and that the order of subunits affects the conductance of the channel and also affects channel gating[5−19].
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age independent, their transmembrane topology, sequence similarity, and tetrameric assembly place them within the superfamily of voltage-gated ion channels[5, 6]. Therefore, CNGs, like the voltage-dependent K+ channel family, form as tetramers with each subunit containing six putative transmembrane segments (S1-S6), a charged S4 region, a P region between S5 and S6, and cytoplasmic amino and carboxyl termini[5, 6]. However, certain subunit regions are important for gating: one is the intracellular cyclic nucleotide-binding domain (CNBD); another one is an iris-like structure formed by a loop which connects the S5 and S6 segments; and the other one is the helix bundle[20−26]. The S6 region of the bovine rod CNG channel (CNGA1) shares a sequence similarity with both bacterial potassium channel from Streptomyces lividans (KcsA) and a voltage-gated potassium channel (Shaker B). On the basis of sequence alignment between CNGA1 and KcsA, a homology model was constructed to represent the putative pore structure of CNG channels. Notably, the CNG pore model is different from KcsA in the selectivity filter, as CNG channels lack the GYG signature sequence of potassium-selective channels. Outside the selectivity filter, the S6 α-helices enter the membrane at an angle to form a helix bundle on the intracellular side[25−27]. As in KcsA, this inverted teepee structure forms the inner vestibule of the channel. Understanding the nature of subunit interactions and their contribution to channel gating requires knowledge of subunit stoichiometry and arrangement as heteromultimers. The cell type-specific expression of CNG genes determines the make up of the sensory transduction channels. Functional and biochemical and fluorescently tagged subunits and fluorescence resonance energy transfer (FRET) and other approaches help in assembling CNGs[8−14]. The stoichiometry of native heteromeric CNG channels in olfac-
1
Overview of CNG Subunit Structures
tory, rod, and cone has been worked out. For example,
Although activated by a ligand and nearly volt-
in olfactory receptor neurons, CNGs require three www.jgenetgenomics.org
Zhengchao Wang et al.: Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating
479
types of subunits, CNGA2, CNGA4, and CNGB1b, to
partial agonist cAMP and inosine 3′ , 5′ -cyclic mo-
exhibit properties necessary for olfactory transduc-
nophosphate (cIMP), can bind to the CNBDs of bo-
[5, 6,12]
. In a photoreceptor, Rod CNGs are made up
vine CNGA1 channels. However, the bound cGMP
of CNGA1 and CNGB1, while cone CNG channels
promotes the opening allosteric transition approxi-
tion
[5−13]
are comprised of CNGA3 and CNGB3 subunits
.
mately one order of magnitude better than bound
However, information in other tissues is very limited.
cIMP and three orders of magnitude better than bound
Many studies have suggested that the spatial ar-
cAMP[5, 6,
rangement of subunits with the heterotetramer has a
that occur in CNBD during channel gating, the sub-
[5−19]
15]
. To probe the conformational changes
. Therefore,
stituted cysteine accessibility method (SCAM) is used.
many scientists are working on the assembly of CNG
It is found that the residue in the β-roll, C505, is more
subunits in different tissues.
accessible in unligand channels than in ligand chan-
profound effect on channel functions
nels, whereas the residue in the C helix, G597C, is
2
Molecular Mechanisms of CNG Gating It is realized that channel activation is involved
in the initial binding of ligand, followed by the opening of allosteric transition, and that conformational changes of all four subunits are involved in channel opening[17,
32, 33]
. When cyclic nucleotides bind to
CNBDs, CNBDs undergo conformational changes, followed by the conformational changes of C-linker, S6 region, and P-pore in each subunit. In this part, roles of each region of subunits during gating are taken into consideration. 2.1
CNBD The cyclic nucleotide-binding domain (CNBD)
is an intracellular domain in C-terminal region, and shares sequence similarity with other cyclic nucleotide-binding proteins, including cGMP- and cAMPdependent protein kinases (PKG and PKA, respectively) and the Escherichia coli catabolite gene activator protein (CAP)[20, 21]. This domain is thought to contain a β-roll and two α-helices, designated as the B and C helices. It is not well known how the binding of the ligand to an intracellular domain leads to the opening of the pore. The binding of the ligand to the CNBD is followed by an allosteric conformational change coupled to the opening of the pore[17, 32, 33]. Various cyclic nucleotides, including the full agonist cGMP and the www.jgenetgenomics.org
more accessible in closed channels than in open channels[20]. These led to a molecular mechanism for channel activation that the ligand initially binds to the β-roll, followed by an opening allosteric transition involving the relative movement of the C helix toward the β-roll. The movement of the C helix relative to the βroll leading to the opening of the pore still needs to be studied further. By introducing the single cysteine into the C helices of cysteineless CNGA1 (CNGA1Cysfree) subunits and using both protein chemistry and electrophysiology to examine the intersubunit disulfide bond formation, many disulfide bonds can be formed at consecutive positions, especially I600C and L601C, indicating that C helix is flexible in closed channels[21]. The formation rate of intersubunit disulfide bonds similar to that in channels containing four cysteines suggests that C helices of CNGA1 subunits are in close proximity to form intersubunit disulfide bonds. Although all cysteine accessibility, orientation, and chemical reactivity contribute to the rate of disulfide bond formation, this rate has been shown to depend mostly on the distance between the two cysteines. Together, these suggest that during channel opening the distances among I600C, L601C, and L607C residues on multiple subunits increase[21, 33]. That is, when ligand initially binds to the β-roll, the bound ligand will stabilize a relative movement of the
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Journal of Genetics and Genomics
C helix toward the β-roll in each subunit, which pulls the C helices away from each other. When disulfide bonds are formed between C helices primarily in closed channels, they will inhibit the relative movement of the C helices toward the β-rolls, and the channel closes (Fig. 1). All of these make a better understanding of the quaternary structure of the CNG channels and the conformational changes during channel gating.
Fig. 1 Intersubunit disulfide bond formation inhibits the opening allosteric transition [5, 6, 19, 20, 31] The model describes the closed-stated inhibition. For simplification, only two subunits are shown, each containing a CNBD. The illustration shows the CNBDs of two diagonal channel subunits during channel activation. The B and C helices are shown as cyclinders, with multimerization of channel subunits occurring along the C helices in the closed state.
2.2
C-linker In CNG channels, binding of cGMP or cAMP
drives a conformational change that leads to the opening of an ion-conducting pore. The region implicated in the coupling of ligand binding to opening of the pore is the C linker. Crosslinking of endogenous cysteines is used to investigate interregion proximity, and an individual
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Vol.34 No.6 2007
close proximity in the tertiary structure. The characteristics of C481 in the CNGA1 C-linker region allow it to be a reporter of disulfide bond formation. Since it is modified preferentially when the channels are in the open state, disulfide bonds that are involved decrease the free energy of the open state relative to the closed state[19]. Therefore, opening of the channels in a C481 disulfide bond is more favorable. Although rare conformations of a protein can certainly be trapped by disulfide bond formation, the propensity of C linker regions to participate in the C481-C481 disulfide bond suggests that the proximity between C linker regions may be prevalent in functional channels[19, 33]. Formation of a disulfide bond between cysteines in neighboring subunits indicates that the regions containing these cysteines lie in close proximity in functional channels. C481 is positioned just a few amino acids away from the CNBD. In the closed state, C481 is shown as being either inaccessible or nonreactive. Opening of the channel involves a conformational change of C481 so that it becomes accessible or reactive. Since C481 residues of neighboring subunits are in close proximity, a disulfide bond forms between them under oxidizing conditions. In addition, the region of the N-terminus containing C35 is also close to this part of the C linker region. Since the C481 residues of neighboring subunits are essentially in the same place, C35 in the N-terminal region can reach these two C481 residues[17]. Formation of the C35-C481 disulfide bond is favored compared with the formation of the C481-C481 disulfide bond (Fig. 2)[19].
amino acid (C481) in each C linker region of two neighboring subunits can form a disulfide bond. Fur-
2.3
S6 region
thermore, a disulfide bond between a cysteine residue
To probe the architecture of the CNG channel
at position 35 of the N-terminal region (C35) and a
pore, Simoes and his colleagues took the advantage of
cysteine residue at position 481 of the C linker region
the unique biochemistry of cysteine and substituted
(C481) can be formed either within a subunit or be-
the amino acid into S6 sites of a cysteine-free CNG1
. Forma-
channel[26]. In contrast, for those positions that are
tion of this disulfide bond alters channel functions,
further away from the pore, modification is expected
suggesting that N-terminal and C-linker regions lie in
to have little effects on permeation, since the positive
[31, 34]
tween subunits using tandem dimmers
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Zhengchao Wang et al.: Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating
481
Fig. 2 Integrative model for interaction among C-linker regions and between the N-terminus and the C-linker regions[5, 18, 26] The closed state of the channel is represented on the left part of the figure, with C481 inaccessible/nonreactive. The right and bot tom panels represent the open state of the channel (after addition of cAMP/cGMP) with C481 accessible/reactive.
charge is likely to be buried behind the helix away from the permeation pathway. Formation of a spontaneous disulfide bond at S399C in the absence of cyclic nucleotide suggests that these residues are in close proximity in the closed state[25]. To confer it in the open state, the disulfide bond formation was investigated by maintaining S399C channels in saturating cGMP[25]. Thus, state-dependent formation of a spontaneous disulfide bond at S399C indicates a conformational change in the helix bundle that is associated with channel gating, which suggests that the smoke hole may widen as the channel opens. Since a conformational change occurs in the helix bundle that is associated with gating, it is necessary to know whether the helix bundle acts as an activation gate for CNG channels[26]. If so, cysteine residues lining the inner vestibule should be protected from internal methanethiosulfonate (MTS) ethyltrimethylammonium (MTSET) modification in the closed state. To investigate it, Flynn and his colleagues determined the state-dependent accessibility of several cysteine mutants that line the pore and span the helix bundle by measuring the rates of modification for each site in both open and closed states. To determine whether small cations pass through the helix bundle in the closed state, Flynn et al.[25] measured closed- and open-state rates of V391C modification www.jgenetgenomics.org
by each of the three cationic modifiers: MTSET, MTSEA, and Ag+. The smallest of these modifiers is Ag+, which is similar in size to a potassium ion. These experiments suggested that the modification of S399C decreases the rate of modification of V391C in the closed state but not in the open state, resulting in an increase in the apparent state dependence of modification of V391C. This indicates that MTSEA enters the inner vestibule through the helix bundle, not via an alternate pathway[25]. Since MTSEA and Ag+ can pass into the inner vestibule, even when CNG channels are closed, the cytoplasmic ends of the S6 helices are in close proximity to each other. There is an opening sufficient to permit the passage of small cations. These imply that the gate is beyond the helix bundle and movement of the S6 helices is coupled with conformational changes in the selectivity filter as previously proposed[24]. A view of four S6 helices from the extracellular side of a CNG channel in the closed (left) and open (right) states is shown at overlook (Fig. 3). The side chain groups of amino acids S399 (white) and V391C (spot) are highlighted. A sliver atom is shown (black) on the same CPK scale as the side chains. The top of the S6 helices is the same position as closed and open states.
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Fig. 3
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A possible mechanism for conformational changes
associated with opening and closing of CNG channels[12, 24, 25] View of four S6 helices from the extracellular side of a CNG channel in the closed (left) and open (right) states is shown. Highlighted are side chain groups of amino acids S399 (white) and V391C (spot). A sliver atom is shown (black) on the same CPK scale as the side chains. The top of the S6 helices is the same position for both closed and open states.
Fig. 4 P region and rearrangement of CNG channel subunits during gating[12-16, 21, 32-33] A: P region of CNG channel forms a loop that extends parallel
2.4
units contributes a single P region loop form the selectivity
P region
to the plane of the membrane illustration of P region consistent with the accessibility results. Each of the four identical sub-
P region connects the S5 and S6 segments, forming a loop that extends toward the central axis of the channel[22, 23]. Residues in the loop between the S5 and S6 transmembrane segments are the major determinants of their ion permeation properties. This loop was proposed to form the blades of an iris-like structure, which constricts the large diameter pore. Al-
filter in the open state (shown at horizontal cross sections). In
though residues in the S4–S5 loop and in the S5 and
extending parallel to the plane of the membrane. B: Rear-
S6 transmembrane segments also affect ion conduction, the P region is the major determinant of ion selectivity in CNG channels[17, 23]. Moreover, these regions determine the pore diameter of CNG channels. To investigate the structure and the role of the P region in gating, the accessibility of 11 cysteine- sub stituted P region residues to small, charged sulfhydryl reagents, methanethiosulfonate (MTS) derivatives, MTS-ethylammonium (MTSEA) and MTS-ethylsul fonate (MTSES) and Ag+, applied to the inside-out configuration of the membrane patches in the open and closed states of the bovine retinal CNG channel should be determined[22]. The center of the iris, possi bly involving residues T16-E19, forms a short, narrow region of the pore that serves as the selectivity filter. Since the channel must contain a gate to prevent ion permeation in the closed state, the P region itself, in addition to forming the ion selectivity filter, must
rangement of CNG channel subunits during gating. A plausible
the closed state, the pore may be occluded by subtle conformational changes in this region. The illustration is meant to emphasize the fact that the P region is likely to form a thin iris-like structure, providing access to certain substituted cysteine side chains from either the external or internal side of the membrane without passage through the selectivity filter at the center of the iris. For simplicity, the P region has been drawn
extension to the general allosteric model can account for the number of closed and opened states in the empirical connected state diagram.
function as the channel gate, the structure of which changes when the channel opens (Fig. 4A). 2.5
Gating rearrangement Recordings of site-specific fluorescence have
shown great promise in understanding conformational changes in signaling proteins. These recordings and patch-clamp current recording from inside-out patches allow direct monitoring of the gating movements of CNGs[32, 33]. As a result, fluorescence signals are reliably observed when fluorophore is covalently attached to a site between CNBD and the pore. Iodide, an anionic quencher, has a higher quenching efficiency in the channel, s closed state. Thallium ion, a www.jgenetgenomics.org
Zhengchao Wang et al.: Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating
cationic quencher, has a higher quenching efficiency
References
in the open state. The state and charge dependence of
1
quenching suggests movements of charged or dipolar
concerted or cooperative conformational change of all
and cells. Neuropharmacology, 1994, 33(11): 1275−1282. 2
Friedmann NK. Cyclic nucleotide-gated channels in non-
3
Kaupp UB. The cyclic nucleotide-gated channels of vertebrate
sensory organs. Cell Calcium, 2000, 27(3): 127−138.
four subunits, and the stability of the opening transi-
photoreceptors and olfactory epithelium. Trends Neurosc,
tion depends largely on the number and type of cyclic [5, 17]
nucleotides bound to CNG channels (Fig. 4B)
3
.
Distler M, Biel M, Flockerzi V, Hofmann F. Expression of cyclic nucleotide-gated cation channels in non-sensory tissues
residues near the fluorophore during CNG channel activation[32]. Therefore, CNG gating is involved in a
483
1991, 14(4): 150−157. 4
Menin A. Cyclic nucleotide-gated channels in visual and olfactory transduction. Biophys Chem, 1995, 55(3): 185−196.
Conclusions and Prospects
5
CNG proteins form ion-permeable pores across
6
Kaupp UB, Seifert R. Cyclic nucleotide-gated ion channels. Physiol Rev, 2002, 82(3): 769−824. Wang ZC, Huang RH, Pan LM, Li XB, Shi FX. Molecular
plasma membranes and have at least two well-defined
structures, physiological roles and regulatory mechanism of
conformational states: opened and closed. In the open
cyclic nucleotide-gated ion channels. Chin J Biochem Mol Biol, 2006, 22(4): 282−288 (in Chinese with an English ab-
state, CNGs function as a conduit through which select ions move passively into or out of the cells; while
stract). 7
Wang ZC, Luo JB, Pang US, Shi FX. Involvement of cyclic
in the closed state, the pore is occluded, thus prevent-
nucleotide-gated ion channel in disease pathogenesis. Prog
ing ion conduction. Conformational changes in and
Vet Medic, 2006, 27(8): 1−4 (in Chinese with an English ab-
between the subunits are often allosterically coupled with the molecular rearrangements in the pore. Thus,
stract). 8
subunits in rod cyclic nucleotide-gated channels. Neuron,
allosteric coupling alters the free energy of CNG channel gating, which is modulated by cyclic nucleotide binding. Although the mechanistic details of this coupling between binding and gating are yet to be understood, there is an attempt to gain a complete picture of CNG channel gating. CNGs are nonselective cation channels, which are very important in phototransduction and olfactory transduction and in neuronal pathfinding or synaptic
Shammat IM, Gordon SE. Stoichiometry and arrangement of 1999, 23(4): 809−819.
9
Weitz D, Ficek N, Kremmer E, Bauer PJ, Kaupp UB. Subunit Stoichiometry of the CNG Channel of Rod Photoreceptors. Neuron, 2002, 36(5): 881−889.
10 Zheng J, Trudeau MC, Zagotta WN. Rod cyclic nucleotide-gated channels have a stoichiometry of three CNGA1 subunits and one CNGB1 subunit. Neuron, 2002, 36(5): 891−896. 11 Zhong H, Lai J, Yau KW. Selective heteromeric assembly of cyclic nucleotide-gated channels. Proc Natl Acad Sci USA, 2003, 100(9): 5509−5513.
plasticity. The level and existence of ion and cation in
12 Zheng J, Zagotta WN. Stoichiometry and assembly of olfac-
the animal tissue and blood have attracted the atten-
tory cyclic nucletide-gated channels. Neuron, 2004, 42(3):
tion of scientists[35, 36]. Mutations in CNG genes give rise to retinal degeneration, color blindness, and many other diseases[6, 7, 37−45]. In particular, mutations in the
411−421. 13 Peng CH, Rich ED, Vamum MD. Subunit configuration of heteromeric cone cyclic nucleotide-gated channels. Neuron, 2004, 42(3): 401−410.
A and B subunits of the CNG channel expressed in
14 Liu DT, Tibbs GR, Siegelbaum SA. Subunit stoichiometry of
human cones cause various forms of complete and
cyclic nucleotide-gated channels and effects of subunit order
incomplete achromatopsia. Therefore, further studies on the molecular mechanisms of CNG gating would be helpful in the prevention and cure of the channelopathies on CNG channels. www.jgenetgenomics.org
on channel function. Neuron, 1996, 16(5): 983−990. 15 Varnum MD, Black KD, Zagotta WN. Molecular mechanism for ligand discrimination of cyclic nucleotide-gated channels. Neuron, 1995, 15(3): 619−625. 16 Bradley J, Reisert J, Frings S. Regulation of cyclic nucleo-
484
Journal of Genetics and Genomics
遗传学报
Vol.34 No.6 2007
tide-gated channels. Curr Opin Neurobiol, 2005, 15(3):
ity and pore properties of heteromeric channels. J Biol Chem,
343−349.
2003, 278(36): 34533−34540.
17 Giorgetti A, Nair AV, Codega P, Torre V, Carloni P. Structural basis of gating of CNG channels. FEBS Lett, 2005, 579(9): 1968−1972. 18 Trudeau MC, Zagotta WN. An Intersubunit interaction regulates yrafficking of rod cyclic nucleotide-gated channels and is disrupted in an inherited form of blindness. Neuron, 2002, 34(2): 197−207. 19 Rosenbaum T, Gordon SE. Dissecting intersubunit contacts in cyclic nucleotide-gated ion channels. Neuron, 2002, 33(5): 703−713. 20 Matulef K, Flynn GE, Zagotta WN. Molecular rearrangements in the ligand-binding domain of cyclic nucleotide-gated channels. Neuron, 1999, 24(4): 443−452. 21 Matulef K, Zagotta W. Multimerization of the ligand binding domains of cyclic nucleotide-cated channels. Neuron, 2002, 36(1): 93−103. 22 Sun ZP, Akabas MH, Goulding EH, Karlin A, Siegelbaum SA. Exposure of redidues in the cyclic nucleotide-gated channel pore P region structure and function in gating. Neuron, 1996, 16(1): 141−149. 23 Becchetti A, Gamel K, Torre V. Cyclic nucleotide-gated channels. Pore topology studied through the accessibility of reporter cysteines. J Gen Physiol, 1999, 114(3): 377−392. 24 Liu J, Siegelbaum SA. Change of pore helix conformational state upon opening of cyclic nucleotide-gated channels. Neuron, 2000, 28(3): 899−909. 25 Flynn GE, Zagotta WN. Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels. Neuron, 2001, 30(3): 689−698. 26 Simoes M, Garneau L, Klein H, Banderali U, Hobeila F, Roux B, Parent L, Sauve R. Cysteine mutagenesis and computer modeling of the S6 region of an intermediate conductance IKCa channels. J Gen Physiol, 2002, 120(1): 99−116. 27 Giorgetti A, Carloni P. Molecular modelling of ion chanels: structural predictions. Curr Opin Chem Biol, 2003, 7(1): 150−156. 28 Gordon SE, Varnum MD, Zagotta WN. Direct interaction between amino- and carboxyl-terminal domains of cyclic nucleotide-gated channels. Neuron, 1997, 19(2): 431−441. 29 Peng C, Rich ED, Thor CA, Varnum MD. Functionally important calmodulin binding sites in both N- and C-terminal regions of the cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit. J Biol Chem, 2003, 278(27): 24617−24623. 30 Peng C, Rich ED, Varnum MD. Achromatopsia-associated mutation in the human cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit alters the ligand sensitiv-
31 Kwon RJ, Ha TS, Kim W, Park CS. Binding symmetry of extracellular divalent cations to conduction pore studied using tandem dimers of a CNG channel. Biochem Biophys Res Commun, 2002, 29(4): 478−485. 32 Zheng J, Zagotta WN. Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry. Neuron, 2000, 28(2): 369−374. 33 Johnson J, Zagotta WN. Rotation movement during cyclic nucleotide-gated channel opening. Nature, 2001, 412(6850): 917−921. 34 Ruiz M, Karpen JW. Opening mechanism of a cyclic nucleotide-gated channel based on analysis of single channels locked in each liganded state. J Gen Physiol, 1999, 113(6): 873−895. 35 He XL, Wang HD, Yuan Y, Wang ZY. Detection of Pb, Cd, Cu and Cr in chicken tissues by graphite furnace atomic absorption spectrometry. Animal Husbandry and Veterinary Medicine, 2005, 37 (1): 15−17 (in Chinese with an English abstract). 36 Tao SH, Han ZK, Wang GJ. Effect of ipriflavone on growth performance and serum biochemical parameters in broilers. Animal Husbandry and Veterinary Medicine, 2007, 39 (2): 18−21 (in Chinese with an English abstract). 37 Wang ZC, Shi FX. Phosphodiesterase 4 and compartmentalization of cyclic AMP signaling. Chin Sci Bull, 2007, 52(1): 34−46. 38 Kruse LS, Sandholdt N T, Gammeltoft S, Olesen J, Kruuse C. Phosphodiesterase 3 and 5 and cyclic nucleotide-gated ion channel expression in rat trigeminovascular system. Neurosci Lett, 2006, 404(1-2): 202−207. 39 Brown RL, Strassmaier T, Brady J D, Karpen JW. The pharmacology of cyclic nucleotide-gated channels: emerging from the darkness. Curr Pharm Des, 2006, 12(28): 3597−3613. 40 Michalakis S, Reisert J, Geiger H, Wetzel C, Zong X, Bradley J, Spehr M, Huttl S, Gerstner A, Pfeifer A, Hatt H, Yau KW, Biel M. Loss of CNGB1 protein leads to olfactory dysfunction and subciliary cyclic nucleotide-gated channel trapping. J Biol Chem, 2006, 281(46): 35156−35166. 41 Qu W, Moorhouse A J, Chandra M, Pierce KD, Lewis, TM, Barry PH. A single P-loop glutamate point mutation to either lysine or arginine switches the cation-anion selectivity of the CNGA2 channel. J Gen Physiol, 2006, 127(4): 375−389. 42 Liu C, Varnum MD. Functional consequences of progressive cone dystrophy-associated mutations in the human cone photoreceptor cyclic nucleotide-gated channel CNGA3 subunit. Am J Physiol Cell Physiol, 2005, 289(1): C187−198. 43 Michalakis S, Geiger H, Haverkamp S, Hofmann F, Gerstner A, Biel M. Impaired opsin targeting and cone photoreceptor
www.jgenetgenomics.org
Zhengchao Wang et al.: Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating
migration in the retina of mice lacking the cyclic nucleotide-gated channel CNGA3. Invest Ophthalmol Vis Sci, 2005, 46(4): 1516−1524. 44 Nishiguchi KM, Sandberg MA, Gorji N, Berson EL, Dryja TP. Cone cGMP-gated channel mutations and clinical findings in patients with achromatopsia, macular degeneration, and other
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hereditary cone diseases. Hum Mutat, 2005, 25(3): 248−258. 45 Patel KA, Bartoli KM, Fandino RA, Ngatchou AN, Woch G, Carey J, Tanaka JC. Transmembrane S1 mutations in CNGA3 from achromatopsia 2 patients cause loss of function and impaired cellular trafficking of the cone CNG channel. Invest Ophthalmol Vis Sci, 2005, 46(7): 2282−2290.
环核苷酸门控离子通道门控的分子机理 王正朝1, 蒋永清2, 卢立志2, 黄瑞华1, 侯清超1, 石放雄1 1. 南京农业大学动物科技学院, 南京 210095； 2. 浙江省农业科学院畜牧兽医研究所, 杭州 310021 摘 要: 环核苷酸门控离子通道(CNG)最广泛地分布于神经细胞。近年来关于 CNG 通道门控的分子机制的研究取得了很大 的进步。研究表明, CNG 通道的组成及组装影响通道的特性及门控。近年来有关 CNG 突变体的研究及半胱氨酸残基亲和性 的分析表明, 环核苷酸首先结合到 CNG 通道 C 端的环核苷酸结合域(CNBD)上引起 CNBD 空间构像改变, 然后 4 个亚单元 发生空间构像的协调改变, CNG 通道开放。本文详细讨论了 CNG 通道的门控机制、各亚单元之间的相互作用、组装的过程 及其空间构想的变化, 为 CNG 通道的进一步研究, 尤其是离子通道疾病方面提供理论指导。 关键词: 环核苷酸门控离子通道；构像改变；门控 作者简介: 王正朝(1978−), 男, 安徽人, 博士研究生, 主要研究方向: 分子与细胞生物学。E-mail: [email protected]
www.jgenetgenomics.org
Review
Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating Zhengchao Wang1, Yongqing Jiang2, Lizhi Lu2, Ruihua Huang1, Qingchao Hou1, Fangxiong Shi1,
①
1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China; 2. Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Abstract: Cyclic nucleotide-gated ion channels (CNGs) are distributed most widely in the neuronal cell. Great progress has been made in molecular mechanisms of CNG channel gating in the recent years. Results of many experiments have indicated that the stoichiometry and assembly of CNG channels affect their property and gating. Experiments of CNG mutants and analyses of cysteine accessibilities show that cyclic nucleotide-binding domains (CNBD) bind cyclic nucleotides and subsequently conformational changes occurred followed by the concerted or cooperative conformational change of all four subunits during CNG gating. In order to provide theoretical assistances for further investigation on CNG channels, especially regarding the disease pathogenesis of ion channels, this paper reviews the latest progress on mechanisms of CNG channels, functions of subunits, processes of subunit assembly, and conformational changes of subunit regions during gating. Keywords: cyclic nucleotide-gated ion channel (CNG); conformational change; gating
Cyclic nucleotide-gated ion channels (CNGs) are nonselective cation channels, which were initially discovered in the plasma membrane of the outer segment of the vertebrate rod photoreceptors and observed in cone photoreceptors, olfactory sensory neurons (OSNs), and other neuronal and nonneuronal cell [1−6]
types
logical ligand for certain CNGs is cGMP, whereas for others it is cAMP. To a certain extent, all CNGs can be activated by both cGMP and cAMP; however, the efficiencies can vary by several orders of magnitude in different CNG channels because of their different affinities to these two ligands. These differences may
. CNGs play critical roles in phototransduc-
be due to the stoichiometry and assembly of CNG
tion and olfactory transduction. In visual and olfactory sensory cells, CNGs are gated by the second
channels. Recently, two reviews were done to discuss
messengers, guanosine 3′ , 5′ -cyclic monophosphate
roles, and regulatory mechanisms of CNG chan-
(cGMP), and adenosine 3′ , 5′ -cyclic monophosphate
nels[6,7]. However, reviews about gating and working
(cAMP), respectively, and convert stimuli into electrical signals that are processed as visual or olfactory
mechanisms on this particular channel are still un-
information[3−14]. It is well known that the physio-
paper reviews the latest progress on mechanisms of
molecular structures, physiological and pathological
available in Chinese published reports. Therefore, this
Received: 2006-11-07; Accepted: 2007-01-08 This work was supported by the Provincial Key Projects for Scientifical and Technological Research of Zhejiang Province (No. 2006C12058), and National Natural Science Foundation of China (No. 30571335) and a Grant-in-Aid for Innovative Training of Doctoral Students in Jiangsu Province, China. ① Corresponding author. E-mail: [email protected]; Tel/Fax: +86-25-8439 9112 www.jgenetgenomics.org
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Journal of Genetics and Genomics
CNG channels, functions of subunits, processes of subunit assembly, and conformational changes of subunit regions during gating in detail. In vertebrates, there are at least six CNG subunit genes, CNGA1, CNGA2, CNGA3, CNGA4, CNGB1, and CNGB3, with homology between families ranging from 30% to 70%[5, 6]. These subunits are generically designated as principle subunits (or A subunits) and modulatory subunits (or B subunits). CNGAs are defined as functional subunits of channels because they are functional when four different CNGAs are assembled, whereas CNGBs are only defined as regulatory subunits of channels because CNGBs are nonfunctional if four different CNGBs are assembled together. This functional definition, however, is not always consistent with the phylogenetic assignment of subunit type, for example, CNGA4. Although certain types of CNG subunits can form functional homomultimeric channels when expressed in a heterologous system, in vivo they appear to be constructed as heterotetramers with one or more mediated subunit composed of 2−3 different types of subunits per [1−14]
. Native ion channels can prechannel complex cisely tune to their physiological roles in neuronal signaling. This tuning frequently involves the controlled assembly of heteromeric channels comprising of multiple types of subunits. Many fundamental and specialized properties of CNGs, such as open probability, ligand sensitivity, ion permeability, and odorant adaptation vary according to the subunits expressed (A and B subunit types) and their stoichiometry[5−16]. Findings in recent studies have suggested that the identity of the β-subunit (CNGB1 or CNGB3) is an important determinant for heteromeric channel assembly and arrangement and that the order of subunits affects the conductance of the channel and also affects channel gating[5−19].
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Vol.34 No.6 2007
age independent, their transmembrane topology, sequence similarity, and tetrameric assembly place them within the superfamily of voltage-gated ion channels[5, 6]. Therefore, CNGs, like the voltage-dependent K+ channel family, form as tetramers with each subunit containing six putative transmembrane segments (S1-S6), a charged S4 region, a P region between S5 and S6, and cytoplasmic amino and carboxyl termini[5, 6]. However, certain subunit regions are important for gating: one is the intracellular cyclic nucleotide-binding domain (CNBD); another one is an iris-like structure formed by a loop which connects the S5 and S6 segments; and the other one is the helix bundle[20−26]. The S6 region of the bovine rod CNG channel (CNGA1) shares a sequence similarity with both bacterial potassium channel from Streptomyces lividans (KcsA) and a voltage-gated potassium channel (Shaker B). On the basis of sequence alignment between CNGA1 and KcsA, a homology model was constructed to represent the putative pore structure of CNG channels. Notably, the CNG pore model is different from KcsA in the selectivity filter, as CNG channels lack the GYG signature sequence of potassium-selective channels. Outside the selectivity filter, the S6 α-helices enter the membrane at an angle to form a helix bundle on the intracellular side[25−27]. As in KcsA, this inverted teepee structure forms the inner vestibule of the channel. Understanding the nature of subunit interactions and their contribution to channel gating requires knowledge of subunit stoichiometry and arrangement as heteromultimers. The cell type-specific expression of CNG genes determines the make up of the sensory transduction channels. Functional and biochemical and fluorescently tagged subunits and fluorescence resonance energy transfer (FRET) and other approaches help in assembling CNGs[8−14]. The stoichiometry of native heteromeric CNG channels in olfac-
1
Overview of CNG Subunit Structures
tory, rod, and cone has been worked out. For example,
Although activated by a ligand and nearly volt-
in olfactory receptor neurons, CNGs require three www.jgenetgenomics.org
Zhengchao Wang et al.: Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating
479
types of subunits, CNGA2, CNGA4, and CNGB1b, to
partial agonist cAMP and inosine 3′ , 5′ -cyclic mo-
exhibit properties necessary for olfactory transduc-
nophosphate (cIMP), can bind to the CNBDs of bo-
[5, 6,12]
. In a photoreceptor, Rod CNGs are made up
vine CNGA1 channels. However, the bound cGMP
of CNGA1 and CNGB1, while cone CNG channels
promotes the opening allosteric transition approxi-
tion
[5−13]
are comprised of CNGA3 and CNGB3 subunits
.
mately one order of magnitude better than bound
However, information in other tissues is very limited.
cIMP and three orders of magnitude better than bound
Many studies have suggested that the spatial ar-
cAMP[5, 6,
rangement of subunits with the heterotetramer has a
that occur in CNBD during channel gating, the sub-
[5−19]
15]
. To probe the conformational changes
. Therefore,
stituted cysteine accessibility method (SCAM) is used.
many scientists are working on the assembly of CNG
It is found that the residue in the β-roll, C505, is more
subunits in different tissues.
accessible in unligand channels than in ligand chan-
profound effect on channel functions
nels, whereas the residue in the C helix, G597C, is
2
Molecular Mechanisms of CNG Gating It is realized that channel activation is involved
in the initial binding of ligand, followed by the opening of allosteric transition, and that conformational changes of all four subunits are involved in channel opening[17,
32, 33]
. When cyclic nucleotides bind to
CNBDs, CNBDs undergo conformational changes, followed by the conformational changes of C-linker, S6 region, and P-pore in each subunit. In this part, roles of each region of subunits during gating are taken into consideration. 2.1
CNBD The cyclic nucleotide-binding domain (CNBD)
is an intracellular domain in C-terminal region, and shares sequence similarity with other cyclic nucleotide-binding proteins, including cGMP- and cAMPdependent protein kinases (PKG and PKA, respectively) and the Escherichia coli catabolite gene activator protein (CAP)[20, 21]. This domain is thought to contain a β-roll and two α-helices, designated as the B and C helices. It is not well known how the binding of the ligand to an intracellular domain leads to the opening of the pore. The binding of the ligand to the CNBD is followed by an allosteric conformational change coupled to the opening of the pore[17, 32, 33]. Various cyclic nucleotides, including the full agonist cGMP and the www.jgenetgenomics.org
more accessible in closed channels than in open channels[20]. These led to a molecular mechanism for channel activation that the ligand initially binds to the β-roll, followed by an opening allosteric transition involving the relative movement of the C helix toward the β-roll. The movement of the C helix relative to the βroll leading to the opening of the pore still needs to be studied further. By introducing the single cysteine into the C helices of cysteineless CNGA1 (CNGA1Cysfree) subunits and using both protein chemistry and electrophysiology to examine the intersubunit disulfide bond formation, many disulfide bonds can be formed at consecutive positions, especially I600C and L601C, indicating that C helix is flexible in closed channels[21]. The formation rate of intersubunit disulfide bonds similar to that in channels containing four cysteines suggests that C helices of CNGA1 subunits are in close proximity to form intersubunit disulfide bonds. Although all cysteine accessibility, orientation, and chemical reactivity contribute to the rate of disulfide bond formation, this rate has been shown to depend mostly on the distance between the two cysteines. Together, these suggest that during channel opening the distances among I600C, L601C, and L607C residues on multiple subunits increase[21, 33]. That is, when ligand initially binds to the β-roll, the bound ligand will stabilize a relative movement of the
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Journal of Genetics and Genomics
C helix toward the β-roll in each subunit, which pulls the C helices away from each other. When disulfide bonds are formed between C helices primarily in closed channels, they will inhibit the relative movement of the C helices toward the β-rolls, and the channel closes (Fig. 1). All of these make a better understanding of the quaternary structure of the CNG channels and the conformational changes during channel gating.
Fig. 1 Intersubunit disulfide bond formation inhibits the opening allosteric transition [5, 6, 19, 20, 31] The model describes the closed-stated inhibition. For simplification, only two subunits are shown, each containing a CNBD. The illustration shows the CNBDs of two diagonal channel subunits during channel activation. The B and C helices are shown as cyclinders, with multimerization of channel subunits occurring along the C helices in the closed state.
2.2
C-linker In CNG channels, binding of cGMP or cAMP
drives a conformational change that leads to the opening of an ion-conducting pore. The region implicated in the coupling of ligand binding to opening of the pore is the C linker. Crosslinking of endogenous cysteines is used to investigate interregion proximity, and an individual
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Vol.34 No.6 2007
close proximity in the tertiary structure. The characteristics of C481 in the CNGA1 C-linker region allow it to be a reporter of disulfide bond formation. Since it is modified preferentially when the channels are in the open state, disulfide bonds that are involved decrease the free energy of the open state relative to the closed state[19]. Therefore, opening of the channels in a C481 disulfide bond is more favorable. Although rare conformations of a protein can certainly be trapped by disulfide bond formation, the propensity of C linker regions to participate in the C481-C481 disulfide bond suggests that the proximity between C linker regions may be prevalent in functional channels[19, 33]. Formation of a disulfide bond between cysteines in neighboring subunits indicates that the regions containing these cysteines lie in close proximity in functional channels. C481 is positioned just a few amino acids away from the CNBD. In the closed state, C481 is shown as being either inaccessible or nonreactive. Opening of the channel involves a conformational change of C481 so that it becomes accessible or reactive. Since C481 residues of neighboring subunits are in close proximity, a disulfide bond forms between them under oxidizing conditions. In addition, the region of the N-terminus containing C35 is also close to this part of the C linker region. Since the C481 residues of neighboring subunits are essentially in the same place, C35 in the N-terminal region can reach these two C481 residues[17]. Formation of the C35-C481 disulfide bond is favored compared with the formation of the C481-C481 disulfide bond (Fig. 2)[19].
amino acid (C481) in each C linker region of two neighboring subunits can form a disulfide bond. Fur-
2.3
S6 region
thermore, a disulfide bond between a cysteine residue
To probe the architecture of the CNG channel
at position 35 of the N-terminal region (C35) and a
pore, Simoes and his colleagues took the advantage of
cysteine residue at position 481 of the C linker region
the unique biochemistry of cysteine and substituted
(C481) can be formed either within a subunit or be-
the amino acid into S6 sites of a cysteine-free CNG1
. Forma-
channel[26]. In contrast, for those positions that are
tion of this disulfide bond alters channel functions,
further away from the pore, modification is expected
suggesting that N-terminal and C-linker regions lie in
to have little effects on permeation, since the positive
[31, 34]
tween subunits using tandem dimmers
www.jgenetgenomics.org
Zhengchao Wang et al.: Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating
481
Fig. 2 Integrative model for interaction among C-linker regions and between the N-terminus and the C-linker regions[5, 18, 26] The closed state of the channel is represented on the left part of the figure, with C481 inaccessible/nonreactive. The right and bot tom panels represent the open state of the channel (after addition of cAMP/cGMP) with C481 accessible/reactive.
charge is likely to be buried behind the helix away from the permeation pathway. Formation of a spontaneous disulfide bond at S399C in the absence of cyclic nucleotide suggests that these residues are in close proximity in the closed state[25]. To confer it in the open state, the disulfide bond formation was investigated by maintaining S399C channels in saturating cGMP[25]. Thus, state-dependent formation of a spontaneous disulfide bond at S399C indicates a conformational change in the helix bundle that is associated with channel gating, which suggests that the smoke hole may widen as the channel opens. Since a conformational change occurs in the helix bundle that is associated with gating, it is necessary to know whether the helix bundle acts as an activation gate for CNG channels[26]. If so, cysteine residues lining the inner vestibule should be protected from internal methanethiosulfonate (MTS) ethyltrimethylammonium (MTSET) modification in the closed state. To investigate it, Flynn and his colleagues determined the state-dependent accessibility of several cysteine mutants that line the pore and span the helix bundle by measuring the rates of modification for each site in both open and closed states. To determine whether small cations pass through the helix bundle in the closed state, Flynn et al.[25] measured closed- and open-state rates of V391C modification www.jgenetgenomics.org
by each of the three cationic modifiers: MTSET, MTSEA, and Ag+. The smallest of these modifiers is Ag+, which is similar in size to a potassium ion. These experiments suggested that the modification of S399C decreases the rate of modification of V391C in the closed state but not in the open state, resulting in an increase in the apparent state dependence of modification of V391C. This indicates that MTSEA enters the inner vestibule through the helix bundle, not via an alternate pathway[25]. Since MTSEA and Ag+ can pass into the inner vestibule, even when CNG channels are closed, the cytoplasmic ends of the S6 helices are in close proximity to each other. There is an opening sufficient to permit the passage of small cations. These imply that the gate is beyond the helix bundle and movement of the S6 helices is coupled with conformational changes in the selectivity filter as previously proposed[24]. A view of four S6 helices from the extracellular side of a CNG channel in the closed (left) and open (right) states is shown at overlook (Fig. 3). The side chain groups of amino acids S399 (white) and V391C (spot) are highlighted. A sliver atom is shown (black) on the same CPK scale as the side chains. The top of the S6 helices is the same position as closed and open states.
482
Fig. 3
Journal of Genetics and Genomics
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A possible mechanism for conformational changes
associated with opening and closing of CNG channels[12, 24, 25] View of four S6 helices from the extracellular side of a CNG channel in the closed (left) and open (right) states is shown. Highlighted are side chain groups of amino acids S399 (white) and V391C (spot). A sliver atom is shown (black) on the same CPK scale as the side chains. The top of the S6 helices is the same position for both closed and open states.
Fig. 4 P region and rearrangement of CNG channel subunits during gating[12-16, 21, 32-33] A: P region of CNG channel forms a loop that extends parallel
2.4
units contributes a single P region loop form the selectivity
P region
to the plane of the membrane illustration of P region consistent with the accessibility results. Each of the four identical sub-
P region connects the S5 and S6 segments, forming a loop that extends toward the central axis of the channel[22, 23]. Residues in the loop between the S5 and S6 transmembrane segments are the major determinants of their ion permeation properties. This loop was proposed to form the blades of an iris-like structure, which constricts the large diameter pore. Al-
filter in the open state (shown at horizontal cross sections). In
though residues in the S4–S5 loop and in the S5 and
extending parallel to the plane of the membrane. B: Rear-
S6 transmembrane segments also affect ion conduction, the P region is the major determinant of ion selectivity in CNG channels[17, 23]. Moreover, these regions determine the pore diameter of CNG channels. To investigate the structure and the role of the P region in gating, the accessibility of 11 cysteine- sub stituted P region residues to small, charged sulfhydryl reagents, methanethiosulfonate (MTS) derivatives, MTS-ethylammonium (MTSEA) and MTS-ethylsul fonate (MTSES) and Ag+, applied to the inside-out configuration of the membrane patches in the open and closed states of the bovine retinal CNG channel should be determined[22]. The center of the iris, possi bly involving residues T16-E19, forms a short, narrow region of the pore that serves as the selectivity filter. Since the channel must contain a gate to prevent ion permeation in the closed state, the P region itself, in addition to forming the ion selectivity filter, must
rangement of CNG channel subunits during gating. A plausible
the closed state, the pore may be occluded by subtle conformational changes in this region. The illustration is meant to emphasize the fact that the P region is likely to form a thin iris-like structure, providing access to certain substituted cysteine side chains from either the external or internal side of the membrane without passage through the selectivity filter at the center of the iris. For simplicity, the P region has been drawn
extension to the general allosteric model can account for the number of closed and opened states in the empirical connected state diagram.
function as the channel gate, the structure of which changes when the channel opens (Fig. 4A). 2.5
Gating rearrangement Recordings of site-specific fluorescence have
shown great promise in understanding conformational changes in signaling proteins. These recordings and patch-clamp current recording from inside-out patches allow direct monitoring of the gating movements of CNGs[32, 33]. As a result, fluorescence signals are reliably observed when fluorophore is covalently attached to a site between CNBD and the pore. Iodide, an anionic quencher, has a higher quenching efficiency in the channel, s closed state. Thallium ion, a www.jgenetgenomics.org
Zhengchao Wang et al.: Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating
cationic quencher, has a higher quenching efficiency
References
in the open state. The state and charge dependence of
1
quenching suggests movements of charged or dipolar
concerted or cooperative conformational change of all
and cells. Neuropharmacology, 1994, 33(11): 1275−1282. 2
Friedmann NK. Cyclic nucleotide-gated channels in non-
3
Kaupp UB. The cyclic nucleotide-gated channels of vertebrate
sensory organs. Cell Calcium, 2000, 27(3): 127−138.
four subunits, and the stability of the opening transi-
photoreceptors and olfactory epithelium. Trends Neurosc,
tion depends largely on the number and type of cyclic [5, 17]
nucleotides bound to CNG channels (Fig. 4B)
3
.
Distler M, Biel M, Flockerzi V, Hofmann F. Expression of cyclic nucleotide-gated cation channels in non-sensory tissues
residues near the fluorophore during CNG channel activation[32]. Therefore, CNG gating is involved in a
483
1991, 14(4): 150−157. 4
Menin A. Cyclic nucleotide-gated channels in visual and olfactory transduction. Biophys Chem, 1995, 55(3): 185−196.
Conclusions and Prospects
5
CNG proteins form ion-permeable pores across
6
Kaupp UB, Seifert R. Cyclic nucleotide-gated ion channels. Physiol Rev, 2002, 82(3): 769−824. Wang ZC, Huang RH, Pan LM, Li XB, Shi FX. Molecular
plasma membranes and have at least two well-defined
structures, physiological roles and regulatory mechanism of
conformational states: opened and closed. In the open
cyclic nucleotide-gated ion channels. Chin J Biochem Mol Biol, 2006, 22(4): 282−288 (in Chinese with an English ab-
state, CNGs function as a conduit through which select ions move passively into or out of the cells; while
stract). 7
Wang ZC, Luo JB, Pang US, Shi FX. Involvement of cyclic
in the closed state, the pore is occluded, thus prevent-
nucleotide-gated ion channel in disease pathogenesis. Prog
ing ion conduction. Conformational changes in and
Vet Medic, 2006, 27(8): 1−4 (in Chinese with an English ab-
between the subunits are often allosterically coupled with the molecular rearrangements in the pore. Thus,
stract). 8
subunits in rod cyclic nucleotide-gated channels. Neuron,
allosteric coupling alters the free energy of CNG channel gating, which is modulated by cyclic nucleotide binding. Although the mechanistic details of this coupling between binding and gating are yet to be understood, there is an attempt to gain a complete picture of CNG channel gating. CNGs are nonselective cation channels, which are very important in phototransduction and olfactory transduction and in neuronal pathfinding or synaptic
Shammat IM, Gordon SE. Stoichiometry and arrangement of 1999, 23(4): 809−819.
9
Weitz D, Ficek N, Kremmer E, Bauer PJ, Kaupp UB. Subunit Stoichiometry of the CNG Channel of Rod Photoreceptors. Neuron, 2002, 36(5): 881−889.
10 Zheng J, Trudeau MC, Zagotta WN. Rod cyclic nucleotide-gated channels have a stoichiometry of three CNGA1 subunits and one CNGB1 subunit. Neuron, 2002, 36(5): 891−896. 11 Zhong H, Lai J, Yau KW. Selective heteromeric assembly of cyclic nucleotide-gated channels. Proc Natl Acad Sci USA, 2003, 100(9): 5509−5513.
plasticity. The level and existence of ion and cation in
12 Zheng J, Zagotta WN. Stoichiometry and assembly of olfac-
the animal tissue and blood have attracted the atten-
tory cyclic nucletide-gated channels. Neuron, 2004, 42(3):
tion of scientists[35, 36]. Mutations in CNG genes give rise to retinal degeneration, color blindness, and many other diseases[6, 7, 37−45]. In particular, mutations in the
411−421. 13 Peng CH, Rich ED, Vamum MD. Subunit configuration of heteromeric cone cyclic nucleotide-gated channels. Neuron, 2004, 42(3): 401−410.
A and B subunits of the CNG channel expressed in
14 Liu DT, Tibbs GR, Siegelbaum SA. Subunit stoichiometry of
human cones cause various forms of complete and
cyclic nucleotide-gated channels and effects of subunit order
incomplete achromatopsia. Therefore, further studies on the molecular mechanisms of CNG gating would be helpful in the prevention and cure of the channelopathies on CNG channels. www.jgenetgenomics.org
on channel function. Neuron, 1996, 16(5): 983−990. 15 Varnum MD, Black KD, Zagotta WN. Molecular mechanism for ligand discrimination of cyclic nucleotide-gated channels. Neuron, 1995, 15(3): 619−625. 16 Bradley J, Reisert J, Frings S. Regulation of cyclic nucleo-
484
Journal of Genetics and Genomics
遗传学报
Vol.34 No.6 2007
tide-gated channels. Curr Opin Neurobiol, 2005, 15(3):
ity and pore properties of heteromeric channels. J Biol Chem,
343−349.
2003, 278(36): 34533−34540.
17 Giorgetti A, Nair AV, Codega P, Torre V, Carloni P. Structural basis of gating of CNG channels. FEBS Lett, 2005, 579(9): 1968−1972. 18 Trudeau MC, Zagotta WN. An Intersubunit interaction regulates yrafficking of rod cyclic nucleotide-gated channels and is disrupted in an inherited form of blindness. Neuron, 2002, 34(2): 197−207. 19 Rosenbaum T, Gordon SE. Dissecting intersubunit contacts in cyclic nucleotide-gated ion channels. Neuron, 2002, 33(5): 703−713. 20 Matulef K, Flynn GE, Zagotta WN. Molecular rearrangements in the ligand-binding domain of cyclic nucleotide-gated channels. Neuron, 1999, 24(4): 443−452. 21 Matulef K, Zagotta W. Multimerization of the ligand binding domains of cyclic nucleotide-cated channels. Neuron, 2002, 36(1): 93−103. 22 Sun ZP, Akabas MH, Goulding EH, Karlin A, Siegelbaum SA. Exposure of redidues in the cyclic nucleotide-gated channel pore P region structure and function in gating. Neuron, 1996, 16(1): 141−149. 23 Becchetti A, Gamel K, Torre V. Cyclic nucleotide-gated channels. Pore topology studied through the accessibility of reporter cysteines. J Gen Physiol, 1999, 114(3): 377−392. 24 Liu J, Siegelbaum SA. Change of pore helix conformational state upon opening of cyclic nucleotide-gated channels. Neuron, 2000, 28(3): 899−909. 25 Flynn GE, Zagotta WN. Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels. Neuron, 2001, 30(3): 689−698. 26 Simoes M, Garneau L, Klein H, Banderali U, Hobeila F, Roux B, Parent L, Sauve R. Cysteine mutagenesis and computer modeling of the S6 region of an intermediate conductance IKCa channels. J Gen Physiol, 2002, 120(1): 99−116. 27 Giorgetti A, Carloni P. Molecular modelling of ion chanels: structural predictions. Curr Opin Chem Biol, 2003, 7(1): 150−156. 28 Gordon SE, Varnum MD, Zagotta WN. Direct interaction between amino- and carboxyl-terminal domains of cyclic nucleotide-gated channels. Neuron, 1997, 19(2): 431−441. 29 Peng C, Rich ED, Thor CA, Varnum MD. Functionally important calmodulin binding sites in both N- and C-terminal regions of the cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit. J Biol Chem, 2003, 278(27): 24617−24623. 30 Peng C, Rich ED, Varnum MD. Achromatopsia-associated mutation in the human cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit alters the ligand sensitiv-
31 Kwon RJ, Ha TS, Kim W, Park CS. Binding symmetry of extracellular divalent cations to conduction pore studied using tandem dimers of a CNG channel. Biochem Biophys Res Commun, 2002, 29(4): 478−485. 32 Zheng J, Zagotta WN. Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry. Neuron, 2000, 28(2): 369−374. 33 Johnson J, Zagotta WN. Rotation movement during cyclic nucleotide-gated channel opening. Nature, 2001, 412(6850): 917−921. 34 Ruiz M, Karpen JW. Opening mechanism of a cyclic nucleotide-gated channel based on analysis of single channels locked in each liganded state. J Gen Physiol, 1999, 113(6): 873−895. 35 He XL, Wang HD, Yuan Y, Wang ZY. Detection of Pb, Cd, Cu and Cr in chicken tissues by graphite furnace atomic absorption spectrometry. Animal Husbandry and Veterinary Medicine, 2005, 37 (1): 15−17 (in Chinese with an English abstract). 36 Tao SH, Han ZK, Wang GJ. Effect of ipriflavone on growth performance and serum biochemical parameters in broilers. Animal Husbandry and Veterinary Medicine, 2007, 39 (2): 18−21 (in Chinese with an English abstract). 37 Wang ZC, Shi FX. Phosphodiesterase 4 and compartmentalization of cyclic AMP signaling. Chin Sci Bull, 2007, 52(1): 34−46. 38 Kruse LS, Sandholdt N T, Gammeltoft S, Olesen J, Kruuse C. Phosphodiesterase 3 and 5 and cyclic nucleotide-gated ion channel expression in rat trigeminovascular system. Neurosci Lett, 2006, 404(1-2): 202−207. 39 Brown RL, Strassmaier T, Brady J D, Karpen JW. The pharmacology of cyclic nucleotide-gated channels: emerging from the darkness. Curr Pharm Des, 2006, 12(28): 3597−3613. 40 Michalakis S, Reisert J, Geiger H, Wetzel C, Zong X, Bradley J, Spehr M, Huttl S, Gerstner A, Pfeifer A, Hatt H, Yau KW, Biel M. Loss of CNGB1 protein leads to olfactory dysfunction and subciliary cyclic nucleotide-gated channel trapping. J Biol Chem, 2006, 281(46): 35156−35166. 41 Qu W, Moorhouse A J, Chandra M, Pierce KD, Lewis, TM, Barry PH. A single P-loop glutamate point mutation to either lysine or arginine switches the cation-anion selectivity of the CNGA2 channel. J Gen Physiol, 2006, 127(4): 375−389. 42 Liu C, Varnum MD. Functional consequences of progressive cone dystrophy-associated mutations in the human cone photoreceptor cyclic nucleotide-gated channel CNGA3 subunit. Am J Physiol Cell Physiol, 2005, 289(1): C187−198. 43 Michalakis S, Geiger H, Haverkamp S, Hofmann F, Gerstner A, Biel M. Impaired opsin targeting and cone photoreceptor
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Zhengchao Wang et al.: Molecular Mechanisms of Cyclic Nucleotide-Gated Ion Channel Gating
migration in the retina of mice lacking the cyclic nucleotide-gated channel CNGA3. Invest Ophthalmol Vis Sci, 2005, 46(4): 1516−1524. 44 Nishiguchi KM, Sandberg MA, Gorji N, Berson EL, Dryja TP. Cone cGMP-gated channel mutations and clinical findings in patients with achromatopsia, macular degeneration, and other
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hereditary cone diseases. Hum Mutat, 2005, 25(3): 248−258. 45 Patel KA, Bartoli KM, Fandino RA, Ngatchou AN, Woch G, Carey J, Tanaka JC. Transmembrane S1 mutations in CNGA3 from achromatopsia 2 patients cause loss of function and impaired cellular trafficking of the cone CNG channel. Invest Ophthalmol Vis Sci, 2005, 46(7): 2282−2290.
环核苷酸门控离子通道门控的分子机理 王正朝1, 蒋永清2, 卢立志2, 黄瑞华1, 侯清超1, 石放雄1 1. 南京农业大学动物科技学院, 南京 210095； 2. 浙江省农业科学院畜牧兽医研究所, 杭州 310021 摘 要: 环核苷酸门控离子通道(CNG)最广泛地分布于神经细胞。近年来关于 CNG 通道门控的分子机制的研究取得了很大 的进步。研究表明, CNG 通道的组成及组装影响通道的特性及门控。近年来有关 CNG 突变体的研究及半胱氨酸残基亲和性 的分析表明, 环核苷酸首先结合到 CNG 通道 C 端的环核苷酸结合域(CNBD)上引起 CNBD 空间构像改变, 然后 4 个亚单元 发生空间构像的协调改变, CNG 通道开放。本文详细讨论了 CNG 通道的门控机制、各亚单元之间的相互作用、组装的过程 及其空间构想的变化, 为 CNG 通道的进一步研究, 尤其是离子通道疾病方面提供理论指导。 关键词: 环核苷酸门控离子通道；构像改变；门控 作者简介: 王正朝(1978−), 男, 安徽人, 博士研究生, 主要研究方向: 分子与细胞生物学。E-mail: [email protected]
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