Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel

Schmidpeter, Philipp A. M.; Rheinberger, Jan; Nimigean, Crina M.

doi:10.1038/s41467-020-20104-4

Cited by 13 publications

(9 citation statements)

References 79 publications

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“…The A′ helix has been shown to rearrange after cyclic-nucleotide binding to promote the pore opening, which was further corroborated in the new cryo-EM structures of a eukaryotic CNG channel TAX-4 and a human rod photoreceptor CNGA1 channel 11 , 30 , 44 – 47 . Our models display an upward movement of the C-linker after cyclic nucleotide binding similar to other CNBD family channels 44 , 46 , 48 , 49 . However, a counterclockwise rotation of the A′–B′ helix around the central axis of the pore after cAMP binding (Supplementary Movies 1 and 2 ) was also revealed in our tmFRET measurements and modeling.…”

Section: Discussionsupporting

confidence: 63%

Electromechanical coupling mechanism for activation and inactivation of an HCN channel

et al. 2021

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Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET, patch-clamp fluorometry and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removing cAMP produced a largely rigid-body rotation of the C-linker relative to the transmembrane domain, bringing the A’ helix of the C-linker in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not the presence of cAMP. These results suggest that — in contrast to electromechanical coupling for channel activation — the A’ helix serves to couple the S4-helix movement for channel inactivation, which is likely a conserved mechanism for CNBD-family channels.

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Section: Discussionsupporting

confidence: 63%

Electromechanical coupling mechanism for activation and inactivation of an HCN channel

et al. 2021

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“…The second conformation (109647 particles, ~9%) displayed global rearrangements in the cytosolic domains in agreement with the proposed ligand gating movements in both SthK and CNG channels in general (Fig. 2a, b ) 17 , 23 , 28 , 33 , 34 . The CNBD adopts the same structure as the isolated, activated CNBD of SthK solved by X-ray crystallography 35 , where the terminal C-helix moves towards the binding pocket by ~10 Å to contact the bound cAMP and to close over it like a lid (Supplementary Fig.…”

Section: Resultssupporting

confidence: 84%

“…Previous efforts to structurally determine functional states along the ligand-activation pathway for cyclic nucleotide-modulated channels (CNG and HCN channels) have so far been limited to only end states [14][15][16]28,33,43,44 . Here, we were able to stabilize several on-pathway gating intermediates and obtained cryo-EM structures of these otherwise high-energy, meta-stable conformations.…”

Section: Discussionmentioning

confidence: 99%

Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel

et al. 2022

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Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the intracellular gate, pointing to an inhibitory VSD at rest. We propose that in wild-type SthK, depolarization leads to such VSD displacements resulting in release of inhibition. In summary, we report conformational transitions along the activation pathway that reveal allosteric couplings between key sites integrating to open the intracellular gate.

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“…This can be achieved with a stopped-flow fluorometric ion flux assay (Posson et al, 2018), which measures the rate of fluorescence quenching of a fluorophore as a quenching ion (usually thallium) enters the liposomes through ion channels. This technique has been successfully applied to proton-gated (Rusinova et al, 2014), calcium-gated (Posson et al, 2015), cyclic nucleotide-gated (Schmidpeter et al, 2020), and pentameric ligand-gated ion channels (Menny et al, 2017;Tong et al, 2019). We anticipate that the stopped-flow ion flux assay, together with innovative methods to make asymmetric proteoliposomes, will provide novel insights into the mechanism of lipid regulation of ion channels.…”

Section: Functional Measurements Of Lipid Regulationmentioning

confidence: 99%

Druggable Lipid Binding Sites in Pentameric Ligand-Gated Ion Channels and Transient Receptor Potential Channels

2022

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Lipids modulate the function of many ion channels, possibly through direct lipid-protein interactions. The recent outpouring of ion channel structures by cryo-EM has revealed many lipid binding sites. Whether these sites mediate lipid modulation of ion channel function is not firmly established in most cases. However, it is intriguing that many of these lipid binding sites are also known sites for other allosteric modulators or drugs, supporting the notion that lipids act as endogenous allosteric modulators through these sites. Here, we review such lipid-drug binding sites, focusing on pentameric ligand-gated ion channels and transient receptor potential channels. Notable examples include sites for phospholipids and sterols that are shared by anesthetics and vanilloids. We discuss some implications of lipid binding at these sites including the possibility that lipids can alter drug potency or that understanding protein-lipid interactions can guide drug design. Structures are only the first step toward understanding the mechanism of lipid modulation at these sites. Looking forward, we identify knowledge gaps in the field and approaches to address them. These include defining the effects of lipids on channel function in reconstituted systems using asymmetric membranes and measuring lipid binding affinities at specific sites using native mass spectrometry, fluorescence binding assays, and computational approaches.

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Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel

Cited by 13 publications

References 79 publications

Electromechanical coupling mechanism for activation and inactivation of an HCN channel

Electromechanical coupling mechanism for activation and inactivation of an HCN channel

Gating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel

Druggable Lipid Binding Sites in Pentameric Ligand-Gated Ion Channels and Transient Receptor Potential Channels

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