Allosteric signaling in C-linker and cyclic nucleotide-binding domain of HCN2 channels

Pfleger, Christopher; Kusch, Jana; Kondapuram, Mahesh; Schwabe, Tina; Sattler, Christian; Benndorf, Klaus; Gohlke, Holger

doi:10.1016/j.bpj.2021.01.017

Cited by 10 publications

(11 citation statements)

References 76 publications

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“…Under these model assumptions, the phenomenon of subunit cooperativity would be confined to the level of the CNBDs. This interpretation gets nice support from recent computational studies on the allosteric signaling within the CNBD and the C-linker of structurally related HCN2 channels [ 38 ]. The authors applied MD simulations and a rigidity-theory-based approach and identified complex interactions to mediate the cAMP effect.…”

Section: Discussionsupporting

confidence: 59%

Subunit promotion energies for channel opening in heterotetrameric olfactory CNG channels

et al. 2022

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Cyclic nucleotide-gated (CNG) ion channels of olfactory sensory neurons contain three types of homologue subunits, two CNGA2 subunits, one CNGA4 subunit and one CNGB1b subunit. Each subunit carries an intracellular cyclic nucleotide binding domain (CNBD) whose occupation by up to four cyclic nucleotides evokes channel activation. Thereby, the subunits interact in a cooperative fashion. Here we studied 16 concatamers with systematically disabled, but still functional, binding sites and quantified channel activation by systems of intimately coupled state models transferred to 4D hypercubes, thereby exploiting a weak voltage dependence of the channels. We provide the complete landscape of free energies for the complex activation process of heterotetrameric channels, including 32 binding steps, in both the closed and open channel, as well as 16 closed-open isomerizations. The binding steps are specific for the subunits and show pronounced positive cooperativity for the binding of the second and the third ligand. The energetics of the closed-open isomerizations were disassembled to elementary subunit promotion energies for channel opening, ΔΔGfpn, adding to the free energy of the closed-open isomerization of the empty channel, E0. The ΔΔGfpn values are specific for the four subunits and presumably invariant for the specific patterns of liganding. In conclusion, subunit cooperativity is confined to the CNBD whereas the subunit promotion energies for channel opening are independent.

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Section: Discussionsupporting

confidence: 59%

Subunit promotion energies for channel opening in heterotetrameric olfactory CNG channels

et al. 2022

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“…30 Allosteric effects due to ligand binding are described with the free-energy measure Δ G i ,CNA (see ESI eqn (S4)†). 59–61 The per-residue Δ G i ,CNA values from individual trajectories correlate well for each system, indicating robust and consistent results were obtained across the independent MD trajectories (Table S4†). We performed receiver operating characteristic (ROC) analyses to evaluate whether the pockets identified in our MD simulations comprise more residues with larger Δ G i ,CNA values than other surface regions (Fig.…”

Section: Resultsmentioning

confidence: 55%

Combining structural and coevolution information to unveil allosteric sites

et al. 2023

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“…Similar to previous measurements, ligand binding was quantified by the fluorescence intensity of a fluorescent cAMP derivative binding to the channels in the excised patch membrane ( Biskup et al, 2007 ; Kusch et al, 2010 ). As cAMP derivative, 8-AHT-Cy3B-cAMP (f1cAMP) was used ( Otte et al, 2019 ; Pfleger et al, 2021 ), which contains a different dye moiety and a longer linker compared to the originally used cAMP derivative 8-AET-Dy547-cAMP (fcAMP) ( Kusch et al, 2010 ) (see also Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

Uncoupling of Voltage- and Ligand-Induced Activation in HCN2 Channels by Glycine Inserts

et al. 2022

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Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are tetramers that generate electrical rhythmicity in special brain neurons and cardiomyocytes. The channels are activated by membrane hyperpolarization. The binding of cAMP to the four available cyclic nucleotide-binding domains (CNBD) enhances channel activation. We analyzed in the present study the mechanism of how the effect of cAMP binding is transmitted to the pore domain. Our strategy was to uncouple the C-linker (CL) from the channel core by inserting one to five glycine residues between the S6 gate and the A′-helix (constructs 1G to 5G). We quantified in full-length HCN2 channels the resulting functional effects of the inserted glycines by current activation as well as the structural dynamics and statics using molecular dynamics simulations and Constraint Network Analysis. We show functionally that already in 1G the cAMP effect on activation is lost and that with the exception of 3G and 5G the concentration-activation relationships are shifted to depolarized voltages with respect to HCN2. The strongest effect was found for 4G. Accordingly, the activation kinetics were accelerated by all constructs, again with the strongest effect in 4G. The simulations reveal that the average residue mobility of the CL and CNBD domains is increased in all constructs and that the junction between the S6 and A′-helix is turned into a flexible hinge, resulting in a destabilized gate in all constructs. Moreover, for 3G and 4G, there is a stronger downward displacement of the CL-CNBD than in HCN2 and the other constructs, resulting in an increased kink angle between S6 and A′-helix, which in turn loosens contacts between the S4-helix and the CL. This is suggested to promote a downward movement of the S4-helix, similar to the effect of hyperpolarization. In addition, exclusively in 4G, the selectivity filter in the upper pore region and parts of the S4-helix are destabilized. The results provide new insights into the intricate activation of HCN2 channels.

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Allosteric signaling in C-linker and cyclic nucleotide-binding domain of HCN2 channels

Cited by 10 publications

References 76 publications

Subunit promotion energies for channel opening in heterotetrameric olfactory CNG channels

Subunit promotion energies for channel opening in heterotetrameric olfactory CNG channels

Combining structural and coevolution information to unveil allosteric sites

Uncoupling of Voltage- and Ligand-Induced Activation in HCN2 Channels by Glycine Inserts

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