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The mammalian cyclic nucleotide-gated channels (CNG channels) play an important role in many signal transduction pathways, especially in the visual and olfactory sensory systems.
After the surface receptors of competent cells receive light signals or chemical signals, the level of cyclic nucleotides in the cells will change, thereby regulating the opening or closing of CNG channels.
The electrical signal generated by the CNG channel activates the downstream neurons, so mammals can feel the changes in light and smell from the outside [1].
Mutations in the CNG gene can induce various diseases such as Retinitis pigmentosa, Achromatopsia, and olfactory disorders.
Although the CNG channel is a member of the voltage-gated ion channel superfamily, it is not regulated by changes in membrane potential.
Instead, it senses changes in cyclic nucleotide levels (cGMP or cAMP) in the cytoplasm, and then opens or closes [2].
In mammals, natural CNG channels are heterotetramers formed by CNGA (A1, A2, A3, A4) and CNGB (B1, B3) subunits.
Among them, the A1, A2, and A3 subunits can each form a functional homotetramer, so they are often used to study the ligand regulation and ion selectivity of CNG channels [3].
CNG channel is a non-selective cation channel, which can permeate various cations such as Na+, K+ and Ca2+, among which it has a higher affinity for Ca2+.
The binding of Ca2+ to the ion conduction path of the channel will effectively hinder the permeation of monovalent cations (referred to as calcium blockade).
Previous studies reported the structure of C.
elegans CNG channel (TAX-4) in open (cGMP binding) and closed (no cGMP binding) states, revealing the gating mechanism of CNG channels [3, 4].
However, the ion selectivity and calcium blocking mechanism of CNG channels are not clear.
On March 1, 2021, Jiang Youxing’s research group from the University of Texas Southwestern Medical Center published an article Structural mechanisms of gating and selectivity of human rod CNGA1 channel in Neuron, analyzing the opening and closing of human CNGA1 homotetramers The three-dimensional structure of cryo-electron microscopy of the state reveals the gate position that is different from the classical voltage-gated ion channel.
By comparing the ion binding state of CNGA1 under different conditions, it revealed the binding site of Ca2+ in the selective filter (SF).
The researchers first obtained a homogeneous protein sample through expression optimization, and then used cryo-electron microscopy to analyze the open and closed structure of the human CNGA1 homotetramer.
CNGA1 is composed of a transmembrane region (S1-S6) and a cytoplasmic gating device.
Once the cytoplasmic gating device is combined with cGMP, a significant conformational change will occur.
This conformational change is amplified and transmitted to the transmembrane region to control the channel Switch.
The structural analysis of the Pore domain (S5-S6) showed that after the binding of cGMP, the two conserved hydrophobic residues F389 and V393 on S6 flipped their side chains to the outside of the channel hole, triggering the opening of the channel, thus forming the central gate.
Control (Figure 1b).
This is also consistent with the conclusion drawn by TAX-4.
The high-quality density map of Pore domain clearly shows the electron density of the ions bound in the selective filter (SF).
Comparison of the ion binding modes under five different conditions reveals that there are two Ca2+ binding sites in the filter.
Point (1, 2); And compared with Na+ and K+, Ca2+ has stronger binding capacity and specificity. Previous mutation and electrophysiological analysis showed that the conservative acidic amino acid E365 in SF is very important for calcium binding and calcium blockade.
Therefore, it is speculated that E365 forms the main calcium ion binding site at the outer entrance of the CNG channel.
However, structural analysis shows that there is no ion density at E365.
Since E365 does not form a calcium binding site, how does it work? In order to explain this problem, the researchers mutated E365 to Q and analyzed the binding mode of Ca2+.
They found that there was almost no electron density at calcium binding sites 1 and 2, indicating that although E365 does not directly form external calcium ion binding sites, it does It is very important for the binding of calcium ions at sites 1 and 2 (Figure 2).
The E365Q mutation will also turn the CNG channel into a voltage-gated channel with outward rectification.
Interestingly, the structure of E365Q shows that Q365 has two different conformations (blocking conformation and conducting conformation), which induce voltage gating.
In general, the mechanism of central gating is explained by comparing the structure of CNG channel closed and open state.
The analysis of the ion binding mode in the filter clarified the binding site of calcium ions, and explained the mechanism of calcium blockage and calcium permeability.
Although CNGA1 homotetramer is used as a CNG channel research model, its structural analysis helps us understand the gating and ion selective mechanism of CNG channels.
However, in the natural state of the body, the CNGA subunit and the CNGB subunit form a heterotetramer, which has a different regulatory mechanism from that of the homotetramer in vitro.
It is possible to analyze the structure of the heterotetramer in the true natural state.
Help us better understand the CNG channel. Original link: https://doi.
org/10.
1016/j.
neuron.
2021.
02.
007 Plate maker: Qijiang Reference 1.
Bradley, J.
, Reisert, J.
, and Frings, S.
(2005).
Regulation of cyclic nucleotide-gated channels.
Curr Opin Neurobiol 15, 343-349.
2.
Varnum, MD, and Zagotta, WN (1996).
Subunit interactions in the activation of cyclic nucleotide-gated ion channels.
Biophys J 70, 2667-2679.
3.
Kaupp, UB, and Seifert, R.
(2002).
Cyclic nucleotide-gated ion channels.
Physiol Rev 82, 769-824.
4.
Li, M.
, Zhou, X.
, Wang, S.
, Michailidis, I.
, Gong, Y.
, Su, D.
, Li, H.
, Li, X.
, and Yang, J.
(2017).
Structure of a eukaryotic cyclic-nucleotide-gated channel.
Nature 542, 60-65.
5.
Zheng, X.
, Fu, Z.
, Su, D.
, Zhang, Y.
, Li, M.
, Pan, Y.
, Li, H.
, Li, S.
, Grassucci, RA, Ren, Z.
, et al.
(2020).
Mechanism of ligand activation of a eukaryotic cyclic nucleotide-gated channel.
Nat Struct Mol Biol 27, 625-634.
After the surface receptors of competent cells receive light signals or chemical signals, the level of cyclic nucleotides in the cells will change, thereby regulating the opening or closing of CNG channels.
The electrical signal generated by the CNG channel activates the downstream neurons, so mammals can feel the changes in light and smell from the outside [1].
Mutations in the CNG gene can induce various diseases such as Retinitis pigmentosa, Achromatopsia, and olfactory disorders.
Although the CNG channel is a member of the voltage-gated ion channel superfamily, it is not regulated by changes in membrane potential.
Instead, it senses changes in cyclic nucleotide levels (cGMP or cAMP) in the cytoplasm, and then opens or closes [2].
In mammals, natural CNG channels are heterotetramers formed by CNGA (A1, A2, A3, A4) and CNGB (B1, B3) subunits.
Among them, the A1, A2, and A3 subunits can each form a functional homotetramer, so they are often used to study the ligand regulation and ion selectivity of CNG channels [3].
CNG channel is a non-selective cation channel, which can permeate various cations such as Na+, K+ and Ca2+, among which it has a higher affinity for Ca2+.
The binding of Ca2+ to the ion conduction path of the channel will effectively hinder the permeation of monovalent cations (referred to as calcium blockade).
Previous studies reported the structure of C.
elegans CNG channel (TAX-4) in open (cGMP binding) and closed (no cGMP binding) states, revealing the gating mechanism of CNG channels [3, 4].
However, the ion selectivity and calcium blocking mechanism of CNG channels are not clear.
On March 1, 2021, Jiang Youxing’s research group from the University of Texas Southwestern Medical Center published an article Structural mechanisms of gating and selectivity of human rod CNGA1 channel in Neuron, analyzing the opening and closing of human CNGA1 homotetramers The three-dimensional structure of cryo-electron microscopy of the state reveals the gate position that is different from the classical voltage-gated ion channel.
By comparing the ion binding state of CNGA1 under different conditions, it revealed the binding site of Ca2+ in the selective filter (SF).
The researchers first obtained a homogeneous protein sample through expression optimization, and then used cryo-electron microscopy to analyze the open and closed structure of the human CNGA1 homotetramer.
CNGA1 is composed of a transmembrane region (S1-S6) and a cytoplasmic gating device.
Once the cytoplasmic gating device is combined with cGMP, a significant conformational change will occur.
This conformational change is amplified and transmitted to the transmembrane region to control the channel Switch.
The structural analysis of the Pore domain (S5-S6) showed that after the binding of cGMP, the two conserved hydrophobic residues F389 and V393 on S6 flipped their side chains to the outside of the channel hole, triggering the opening of the channel, thus forming the central gate.
Control (Figure 1b).
This is also consistent with the conclusion drawn by TAX-4.
The high-quality density map of Pore domain clearly shows the electron density of the ions bound in the selective filter (SF).
Comparison of the ion binding modes under five different conditions reveals that there are two Ca2+ binding sites in the filter.
Point (1, 2); And compared with Na+ and K+, Ca2+ has stronger binding capacity and specificity. Previous mutation and electrophysiological analysis showed that the conservative acidic amino acid E365 in SF is very important for calcium binding and calcium blockade.
Therefore, it is speculated that E365 forms the main calcium ion binding site at the outer entrance of the CNG channel.
However, structural analysis shows that there is no ion density at E365.
Since E365 does not form a calcium binding site, how does it work? In order to explain this problem, the researchers mutated E365 to Q and analyzed the binding mode of Ca2+.
They found that there was almost no electron density at calcium binding sites 1 and 2, indicating that although E365 does not directly form external calcium ion binding sites, it does It is very important for the binding of calcium ions at sites 1 and 2 (Figure 2).
The E365Q mutation will also turn the CNG channel into a voltage-gated channel with outward rectification.
Interestingly, the structure of E365Q shows that Q365 has two different conformations (blocking conformation and conducting conformation), which induce voltage gating.
In general, the mechanism of central gating is explained by comparing the structure of CNG channel closed and open state.
The analysis of the ion binding mode in the filter clarified the binding site of calcium ions, and explained the mechanism of calcium blockage and calcium permeability.
Although CNGA1 homotetramer is used as a CNG channel research model, its structural analysis helps us understand the gating and ion selective mechanism of CNG channels.
However, in the natural state of the body, the CNGA subunit and the CNGB subunit form a heterotetramer, which has a different regulatory mechanism from that of the homotetramer in vitro.
It is possible to analyze the structure of the heterotetramer in the true natural state.
Help us better understand the CNG channel. Original link: https://doi.
org/10.
1016/j.
neuron.
2021.
02.
007 Plate maker: Qijiang Reference 1.
Bradley, J.
, Reisert, J.
, and Frings, S.
(2005).
Regulation of cyclic nucleotide-gated channels.
Curr Opin Neurobiol 15, 343-349.
2.
Varnum, MD, and Zagotta, WN (1996).
Subunit interactions in the activation of cyclic nucleotide-gated ion channels.
Biophys J 70, 2667-2679.
3.
Kaupp, UB, and Seifert, R.
(2002).
Cyclic nucleotide-gated ion channels.
Physiol Rev 82, 769-824.
4.
Li, M.
, Zhou, X.
, Wang, S.
, Michailidis, I.
, Gong, Y.
, Su, D.
, Li, H.
, Li, X.
, and Yang, J.
(2017).
Structure of a eukaryotic cyclic-nucleotide-gated channel.
Nature 542, 60-65.
5.
Zheng, X.
, Fu, Z.
, Su, D.
, Zhang, Y.
, Li, M.
, Pan, Y.
, Li, H.
, Li, S.
, Grassucci, RA, Ren, Z.
, et al.
(2020).
Mechanism of ligand activation of a eukaryotic cyclic nucleotide-gated channel.
Nat Struct Mol Biol 27, 625-634.
