Cyclic nucleotide–gated ion channels or CNG channels are ion channels that function in response to the binding of cyclic nucleotides. CNG channels are nonselective cation channels that are found in the membranes of various tissue and cell types, and are significant in sensory transduction as well as cellular development. Their function can be the result of a combination of the binding of cyclic nucleotides (cGMP and cAMP) and either a depolarization or a hyperpolarization event. Initially discovered in the cells that make up the retina of the eye, CNG channels have been found in many different cell types across both the animal and the plant kingdoms. CNG channels have a very complex structure with various subunits and domains that play a critical role in their function. CNG channels are significant in the function of various sensory pathways including vision and olfaction, as well as in other key cellular functions such as hormone release and chemotaxis. CNG channels have also been found to exist in prokaryotes, including many spirochaeta, though their precise role in bacterial physiology remains unknown.
Discovery The discovery of CNG channels is related to the discovery of intracellular messengers responsible for the mediation of responses in retinal photoreceptors. Before their discovery, it was thought that cyclic nucleotides played a role in phosphorylation. In 1985, it was discovered that cGMP was able to directly activate the light-dependent response of rod ion channels by studying light-adapted retina of frogs. CNG channels were also found in cone photoreceptors, chemo sensitive cilia of olfactory sensory neurons, and the pineal gland. After the identification of amino acids from purified proteins, cloning and functional expression of CNG channels were performed. Molecular cloning allowed for the discovery of similar channels in many other tissues. In 2000, scientists performed studies using mouse retina and molecular cloning to find a new subunit of the channel, CNG6.
Function CNG channels have important functions in signal transduction in retinal photoreceptors and olfactory receptor neurons. They are directly activated by cyclic nucleotides, and approximately 4 cyclic nucleotides are needed to activate each channel. CNG channels are nonselective and allow many alkali ions to flow into or out of a cell expressing CNG channels on its membrane. This flow of ions can result in either depolarization or hyperpolarization. CNG channels can be activated by cAMP or cGMP exclusively, or sometimes by a combination of both cNMPs, and some channels are more selective than others. Even though the activity of these channels show little voltage dependence, they are still considered voltage-dependent channels. Calcium, calmodulin, and phosphorylation modulate the opening of CNG channels.
The main role of CNG channels is sensory transduction in various tissues. Many studies have shown CNG channels in rod and cone photoreceptors, and they have also been found in the brain, the heart, the kidneys, and gonads.
CNG channel homologs in Caenorhabditis elegans, Drosophila melanogaster, and Limulus polyphemus have unknown functions. Studies have shown homologs in C. elegans might have functions in chemosensation.
Color blindness and retinal degeneration result when CNG channels have mutations. Mutations specifically in the subunits A and B result in complete and incomplete achromatopsia.
Structure A CNG channel consists of four subunits around a central pore. Each protein subunit consists of 6 transmembrane segments (S1-S6), a P-loop, intracellular amino terminal region, and carboxy terminal region. The P-loop and S6 segments around the pore, which plays a role in ion conduction. There is a cyclic nucleotide binding domain (CNBD) and connection region to the S6 segment in the carboxy terminal. There is a post-CNDB region in the amino terminal.
Alpha subunits Cyclic nucleotide gated channel alpha-subunits include Cyclic nucleotide-gated channel alpha 1 Cyclic nucleotide-gated channel alpha 2 Cyclic nucleotide-gated channel alpha 3 Cyclic nucleotide-gated channel alpha 4
Beta subunits Cyclic nucleotide gated channel beta-subunits include: Cyclic nucleotide-gated channel beta 1 Cyclic nucleotide-gated channel beta 3
Pore The structure of the pore is similar to other ion channels that contain P-loops. The P-loop enters the membrane of the pore from the extracellular side and exits to the intracellular side. The P loop enters as an alpha helix and exists as an uncoiled strand. Helices that cover the inner membrane line the channel. These also form a 6 helix bundle that signifies the entrance. In order to open the pore, a conformational change must occur in the inner 6 helix bundle.
Cyclic nucleotide binding domain A Cyclic nucleotide-binding domain is an intracellular domain located in the C-terminus region and has a similar sequence to other cyclic nucleotide-binding proteins. The domain is believed to be made up of a β-pleated sheet and two α-helices. The β-pleated sheet is made up of eight antiparallel strands. The α helices are named the B and C helices. A ligand initially binds to the β-pleated sheet, and through allosteric regulation causes the movement to an α-helix toward the β-pleated sheet. The α-helix is flexible in closed channels. When an α-helix of a CNGA1 subunit is close to another α-helix, they form intersubunit disulfide bonds. This occurs mainly in closed channels, inhibiting movement of the α-helix towards the β-pleated sheet. When a ligand binds to the β-pleated sheet, this bound cyclic nucleotide stabilizes the movement of the α-helix toward the β-pleated sheet in each subunit, pulling the α-helices away from each other.
C-linker The C-linker is a region that connects the CNBD to the S6 segment. The C-linker region contributes to the contact between channel subunits as well as promotes tetramerization, the forming of tetramers. There are many residues that play a role in modulation of CNG channels. This process uses metals such as nickel, zinc, copper, and magnesium. The C-linker region is involved in the coupling of ligand binding to the opening of the pore. The C linker region forms disulfide bonds with N-terminal regions. Disulfide bonds alter the channel function therefore they most likely lie close to the tertiary structure. Disulfide bonds decrease the free energy of the open state compared to the closed state. The specific cysteine residue C481 on the C-linker region is located only a few amino acids away from the binding domain. In the closed state C481 is nonreactive; C481 must undergo a conformational change so that it is accessible for the opening of the channel. Disulfide bonds form between neighboring subunits and C481. Simultaneously there is a C35 cysteine residue at the N-terminal of the C-linker region that can reach two C481 residues, making a favorable disulfide bond compared to a C481-C481 bond.
S6 region Spontaneous disulfide bond formation is state-dependent, implying that the conformational change in the helix bundle is affiliated with channel gating. When the CNG ion channels are closed, the cytoplasmic ends of the S6 helices are close to each other. Small cations are able to move through an opening, which implies that the gate is beyond the helix bundle and that S6 helices are in conjunction with conformational changes in the selectivity filter.
P region The P region forms a loop, the pore loop, connecting the S5 and S6 regions, which extend to the central axis of the channel. Ionic properties are determined by the residues in the loop between S5 and S6 transmembrane segments. The P region dictates the ion selectivity of the cyclic-nucleotide gated ion channel, which also determine the pore diameter of CNG channels. The P region functions as a channel gate since it prevents ion permeation in the closed state. The pore may be hindered by small conformational changes in this region. The P region acts as an ion selectivity filter that changes structure in the open conformation. In the open state, four identical subunits contribute a single P-loop region, which forms a selectivity filter.
CNG channel family In vertebrates, the CNG channel gene family consists of six members and belong to a larger group of voltage-gated ion channels. These genes are divided based on sequence similarity into two subtypes CNGA and CNGB. The CNG channels consist of and alpha subunit, known as CNGA1 and a beta subunit CNGB1. An aspect of the alpha subunit is that it is able to form a functional channel on its own, called a homotetrameric channel. This homotetrameric channel can be of scientific interest because it can be used to further understand the nature of ligand binding and selectivity of a particular channel of interest. Additional genes that code for CNG channels have been cloned from Caenorhabditis elegans and Drosophila melanogaster. A subunit of a CNG channel CNGA1, previously called the rod α subunit, was expressed in rod photoreceptors and produced functional channels that were gated by cGMP when expressed externally in either Xenopus oocytes or in a human embryonic kidney cell line (HEK293). In humans, mutated CNGA1 genes result in an autosomal recessive form of retinitis pigmentosa, a degenerative form of blindness. CNGB1, previously called the rod β subunit, is a second subunit of the rod channel. Unlike CNGA1, CNGB1 subunits expressed alone do not produce functional CNG channels, but coexpression of CNGA1 and CNGB1 subunits produces heteromeric channels with modulation, permeation, pharmacology, and cyclic-nucleotide specificity comparable to that of native channels.
CNG channels form tetramers, and recent studies indicate that native rod channels consist of three CNGA1 subunits and one CNGB1 subunit. CNGA3 subunits, previously called the cone α subunits, form functional channels when its expression occurs exogenously. On the other hand, CNGB3, previously called the cone β subunit, does not. Mutations in human CNGA3 and CNGB3 are involved in complete achromatopsia, which is a rare, autosomal recessive inherited and congenital disorder characterized by the complete failure in color distinction.
CNGA2, previously called the olfactory α subunit, CNGA4, previously called the olfactory β subunit, and CnGB1b are involved in transduction of odorant signals in olfactory sensory neurons for which the subunit stoichiometry and arrangement are unknown.
In invertebrates, a CNG channel subunit called CNG-P1 has been cloned from D. melanogaster and is expressed in antennae and the visual system, an indication that CNG channels may be linked to the transduction of light in invertebrates. A second putative CNG-like subunit called CNGL, cloned from D. melanogaster, is found to be expressed in the brain. Two CNG channel subunits, Tax-2 and Tax-4, have been cloned in C. elegans and are responsible for chemosensation, thermosensation, and normal axon outgrowth of some sensory neurons in C. elegans.
The binding event The ligand might be placed at the bottom of the cavity due to interactions with the phosphate binding cassette (PBC). This cavity refers to a region in the CNBD formed by the β roll, a two-looped β helix. Changes induced by ligand binding occur in α helices (αA, αB, and αC and PBC helix). The β roll only undergoes small changes during binding. After the ligand is seated, αB and αC helices arrange themselves so that they form a cap over the cavity. How binding affects the αA helix is still unclear.
Cooperative and non-cooperative activation The steep concentration between CNG channels and ligand concentration shows that at least two or three cyclic nucleotides are needed. It is believed that the second ligand is required for the channel to transition from closed to open. When the third and fourth ligands bind, the open state of the channel becomes stabilized.