User:Songforsunshine5/Cyclic nucleotide gated ion channel (Rough Draft)

Project Proposal for Cyclic nucleotide gated ion channel

Proposed by: Angi Guo, Junwon Park, Meg Lewis, and Peter Clarner.

Discovery
Cyclic nucleotide-gated ion channels were first discovered in the plasma membrane of rod photoreceptors, in which they were responsible for the primary electrical signals in response to light.

General Function

 * respond to cGMP and cAMP
 * neuronal pathway finding
 * plasticity

Physiological Significance

 * In Photoreceptors
 * In Olfactory neurons
 * In the Brain

In Spermatozoa
cAMP and cGMP mediate several cellular responses for instance swimming behavior, acrosomal exocytosis, and chemoattraction. In the sea urchin species, Strongylocentrotus purpuratus, speract, a short peptide, was studied. Speract activates a receptor-type GC and stimulates a rise of intracellular cGMP concentrations. Speract also increases the concentration of calcium. Although there has yet to be any establishment of a direct causal relationship, the previously mentioned observations suggest that cGMAP activates calcium conductance. CNG channels are prime candidates for the calcium-entry pathway, due to their high calcium permeability. CNG channels have yet to be detected by homology screening. In mammals, testicular CNG channel subunits expressed are A3, B1, and B3. Heterologous expression of the A3 subunit was cloned from testis and produced channels that are cGMP sensitive and selective. It is possible that these channels are involved in a cGMP-stimulates calcium influx into the sperm. However a more extensive characterization of the channel has not been accomplished due to low success rate of detecting channel activity. Since A3 subunit knock-out mice are fertile, CNG channels could be involved in some form of motility control and even in chemotactic swimming behavior or in the acrosomal exocytosis. However, a receptor-type GC in mammalian sperm has yet to be identified. Mouse sperm express other channels such as CatSper1. Male sterility can be accomplished by disrupting the CatSper1 gene; additionally, the cAMP-induced calcium influx is abolished in mutant mice. CNG channels and CatSper are unrealted because CatSper lacks a cAMP/cGMP-binding site but does need additional subunits to become functional. It is possible that CNG and CatSper subunits assemble to form calcium-permeable and cyclic nucleotide-sensitive ion channels.

In Kidney
cGMP-sensitive channels have been analyzed in renal inner medullary collecting duct cells, which influence the body’s electrolyte and fluid balance. CNG channel activity is controlled by the interaction between cGMP-dependent protein kinase and G1 protein. In the cells from an inner medullary collecting duct, the channel exhibits cation selectivity unit conductance, calcium permeability, and pharmacology very similar to cyclic nucleotide-gated ion channels.

In Gonads
There has been identification of cyclic nucleotide-gated ion channel subunits A2, A4, and B1 in a neuronal cell line that secrets gonadotropin-releasing hormone (GnRH). The three subunits make up the CNG channels on chemosensitive cilia of OSNs. In high extracellular calcium, the unit conductance of CNG channels in rods and OSNs are significantly smaller than those measured in the neuronal line. It seems doubtful that CNG channels would create large unit conductance.

Structure
As a member of the family of voltage-dependent potassium ion channels, cyclic nucleotide-gated ion channels are made up of 4 subunits situated around a central pore. Each subunit includes six transmembrane segments (S1-S6), a reentrant P-loop, and intracellular amino- and carboxy-terminal regions.

Cyclic Nucleotide Binding Domain
A cyclic nucleotide binding domain is an intracellular domain located in the C-terminal and has a similar sequence to other cyclic nucleotide-binding proteins. The domain is believed to have a ß-roll and two α-helices. A ligand initially binds to the ß-roll by following an opening allosteric transition involving the movement to an α-helix toward the ß-roll. The α-helix is flexible in closed channels. When an α-helix of a CNGA1 subunit is in close proximity to another α-helix, they form intersubunit disulfide bonds. This occurs mainly in closed channels, inhibiting movement of the α-helix towards the ß-roll. When a ligand binds to the ß-roll, the now bind ligand stabilizes the movement of the α-helix toward the ß-roll of each subunit, pulling the α-helices away from each other.

C-Linker
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 cyclic nucleotide-gated ion channels are closed, the cytoplasmic ends of the S6 helices are in close proximity 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 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 a ion selectivity filter that changes structure in the open conformation. The four identical subunits sacrifice a single P region loop from selectivity filter in the open state.

Family Of CNG Channels
In vertebrates, six members of cyclic nucleotide-gated ion channel gene family were identified. These genes are grouped based on sequence similarity into two subtypes, CNGA and CNGB.

Selectivity and Sensitivity of Ligands

 * cNMP-Binding Site
 * C-Linker Region
 * NH2-Terminal Region
 * Pore

Significance in Plants

 * Role in plant immunity

Division of the Workload
We decided that we would start by doing individual research to come up with different areas within the topic to focus on. We then chose the areas that we deemed the most significant and well-documented. From there we will continue our research more in depth to expand on each area. This will require both individual and group efforts.