Neuron Jiang Youxing's group reveals the working mechanism of CNGA1/B1 ion channels in human rod cells

Responsible Editor | Xi The mammalian cyclic nucleotide-gated channels (CNG channels) play an important role in the signal transduction process of the visual and olfactory sensory systems
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After light changes and odor molecules are recognized by receptors on the cell membrane of cones/rods and olfactory sensory neurons, the levels of cyclic nucleotides in the cells change, which then regulate the opening or closing of CNG channels [1]
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After binding to cyclic nucleotides (cGMP or cAMP) in the cytoplasm, the CNG channel undergoes a conformational change, thereby opening the ion conduction pathway [2]
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In mammals, the CNG ion channel family contains 6 homologous components: 4 A subunits (A1–A4) and 2 B subunits (B1, B3)
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Among them, the A1, A2, and A3 subunits can each form a functional homotetramer, which is often used to study the physiological characteristics of CNG channels
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However, the natural CNG ion channel is a heterotetramer composed of A and B subunits [3]
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Although CNGB subunits alone cannot form functional ion channels, they endow CNG ion channels with unique functional characteristics in their natural state
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The natural ion channels CNGA1/B1 in rod cells have different physiological characteristics from the homotetramer CNGA1
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CNGA1/B1 has a weaker calcium blocking phenomenon; in addition to being fully activated by cGMP, CNGA1/B1 can also be partially activated by high concentrations of cAMP; L-cis-Diltiazem can effectively block CNGA1/B1, and has a blocking effect on CNGA1 But it is very weak [3,4] (Figure 1)
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Although the structure of human CNGA1 homotetramer has been resolved [5], the molecular mechanism of the lack of structural information of CNGB1 leading to these unique physiological properties is still unclear
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 On October 25, 2021, Jiang Youxing's research group from the University of Texas Southwestern Medical Center published an article Structural mechanisms of assembly, permeation, gating, and pharmacology of native human rod CNG channel in Neuron, analyzing human rod cells The three-dimensional structure of CNGA1/B1 ion channels in different states (Apo, cGMP-bound open, cAMP-bound closed and L-cis-Diltiazem-blocked) reveals that CNG ion channels in their natural state have unique ion penetration, gating regulation, The pharmacological characteristics and the structural basis of the 3:1 ratio assembly
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The researchers first co-expressed CNGA1 and CNGB1 subunits in a special way, purified a homogeneously assembled CNGA1:CNGB1 heterotetramer in a ratio of 3:1, and then used cryo-electron microscopy to analyze its three-dimensionality in different states.
Structure
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CNGA1/B1 has an overall structure similar to CNGA1 homotetramer
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The unique amino acid residues of B1 in the ion conduction pathway attenuate the calcium blockage and single channel conductance of CNGA1/B1
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Below the CNBD domain of CNGA1/B1, the CLZ domains of three A1 subunits form a 3-helix coiled coil structure, interacting with the α-helix of one B1 subunit, revealing the ratio of A1:B1 to 3:1 The structural basis for assembly (Figure 2A)
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In the CNGA1/B1 heterotetramer in the closed state of Apo, the gated amino acid side chains of A1 and B1 are located in the middle of the pore, blocking the passage of ions; but in the open state of cGMP binding, only two A1 subunits are gated The amino acid side chain flips to the outside of the channel, while the gated amino acid side chains of the other A1 and B1 subunits only move outwards slightly, without flipping, which results in an asymmetric channel opening
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Compared with the symmetrically opened CNGA1 homotetramer, CNGA1/B1 has a narrower ion conduction path (Figure 2B)
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 The cGMP-bound samples showed two different open states
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These two states have similar asymmetric open pores, but the CNBD domain of the B1 subunit is in a different conformation
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Compared with the closed state structure, the CNBD domain of the four subunits in CNGA1/B1 undergoes conformational changes after binding to cGMP, but the ability to transmit this change and ultimately cause the change of the ion channel spiral S6 is different, which causes Although all four subunits can bind ligands, only the gates of two A subunits enter the open state (Figure 3)
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The activation efficiency of cAMP on CNGA1/B1 is very low.
Although the three-dimensional structure of cAMP binding can be resolved, it is in the closed state.
Therefore, the structural mechanism of cAMP activation is unclear
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 In order to reveal the structural basis of the unique pharmacomechanical properties of CNG ion channels in the natural state, the author also analyzed the structure of CNGA1/B1 and L-cis-Diltiazem complex
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L-cis-Diltiazem is combined in the center of the open pore.
The B1 subunit does not provide any special interaction, but the unique asymmetric gated structure of CNGA1/B1 creates a perfect open space for L-cis-Diltiazem.
-cis-Diltiazem is stabilized by hydrophobic interactions, which also explains why CNGA1 homotetramer cannot bind to L-cis-Diltiazem
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In general, by analyzing the structure of CNGA1/B1 ion channels in different states, the structural basis of its unique assembly method, ion penetration, gating mechanism and pharmacological properties are revealed
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In the closed state, CNGA1/B1 heterotetramer and CNGA1 homotetramer have similar structures; the combination of cGMP and CNGA1/B1 causes conformational changes, but the four subunits have different ability to transmit conformational changes and cause changes in channel spiral S6 , Resulting in the asymmetric gate opening of CNGA1/B1, and this asymmetric gate is the structural basis of its unique pharmacological properties
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