A Secondary Structural Transition in the C-helix Promotes Gating of Cyclic Nucleotide-regulated Ion Channels

Puljung, Michael C.; Zagotta, William N.

doi:10.1074/jbc.m113.464123

Cited by 33 publications

(47 citation statements)

References 46 publications

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“…The ligand-induced movement of the C-helix is widely thought to initiate the conformational changes that lead to opening of the channel pore, but the structural evidence in support of this hypothesis is equivocal (10,(24)(25)(26)(27)(28)(29). The crystal structure of the HCN2 carboxyl-terminal region in the absence of ligand shows little difference from the cyclic nucleotide-bound structure (12).…”

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confidence: 93%

Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

Puljung¹,

DeBerg

Zagotta³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Binding of 3′,5′-cyclic adenosine monophosphate (cAMP) to hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels regulates their gating. cAMP binds to a conserved intracellular cyclic nucleotide-binding domain (CNBD) in the channel, increasing the rate and extent of activation of the channel and shifting activation to less hyperpolarized voltages. The structural mechanism underlying this regulation, however, is unknown. We used double electron-electron resonance (DEER) spectroscopy to directly map the conformational ensembles of the CNBD in the absence and presence of cAMP. Site-directed, double-cysteine mutants in a soluble CNBD fragment were spin-labeled, and interspin label distance distributions were determined using DEER. We found motions of up to 10 Å induced by the binding of cAMP. In addition, the distributions were narrower in the presence of cAMP. Continuouswave electron paramagnetic resonance studies revealed changes in mobility associated with cAMP binding, indicating less conformational heterogeneity in the cAMP-bound state. From the measured DEER distributions, we constructed a coarse-grained elastic-network structural model of the cAMP-induced conformational transition. We find that binding of cAMP triggers a reorientation of several helices within the CNBD, including the C-helix closest to the cAMP-binding site. These results provide a basis for understanding how the binding of cAMP is coupled to channel opening in HCN and related channels.hyperpolarization-activated ion channels | allosteric regulation

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confidence: 93%

Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

Puljung¹,

DeBerg

Zagotta³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…In addition to these initial structural insights about possible conformational changes of the mHCN2 CNBD upon ligandbinding, extensive structural analysis with transition metal ion FRET experiments support the 'coil-to-helix' transition of helix αF′ and a change in the length of helix αC upon ligand-binding (Taraska et al, 2009;Puljung and Zagotta, 2013). Furthermore, in intact CNGA1 channels, homologous to HCN channels, it was shown that stabilization of helix αC promoted channel opening by metal coordination by a pair of histidine residues (Puljung and Zagotta, 2013). In the same study, transition metal ion FRET results suggest movement of the N-terminal region of helix αC closer to the β roll core of the CNBD upon cyclic nucleotide binding.…”

Section: Structure Of Hcn2 Cnbd In the Ligand-free Statementioning

confidence: 99%

“…Minor structural differences include the overall α-helical content caused by the absence of helix αF′ and the shortening of helix αC (by five residues) in the apo-state. However, crystal packing interactions in the region of helix αC or putative ligand replacement by bromide ions located in the binding pocket may have forced the conformation of helix αC in the apo-state structure, and therefore this structure may not represent the actual apo-state conformation in solution (Taraska et al, 2009;Puljung and Zagotta, 2013). In addition to these initial structural insights about possible conformational changes of the mHCN2 CNBD upon ligandbinding, extensive structural analysis with transition metal ion FRET experiments support the 'coil-to-helix' transition of helix αF′ and a change in the length of helix αC upon ligand-binding (Taraska et al, 2009;Puljung and Zagotta, 2013).…”

Section: Structure Of Hcn2 Cnbd In the Ligand-free Statementioning

confidence: 99%

“…However, crystal packing interactions in the region of helix αC or putative ligand replacement by bromide ions located in the binding pocket may have forced the conformation of helix αC in the apo-state structure, and therefore this structure may not represent the actual apo-state conformation in solution (Taraska et al, 2009;Puljung and Zagotta, 2013). In addition to these initial structural insights about possible conformational changes of the mHCN2 CNBD upon ligandbinding, extensive structural analysis with transition metal ion FRET experiments support the 'coil-to-helix' transition of helix αF′ and a change in the length of helix αC upon ligand-binding (Taraska et al, 2009;Puljung and Zagotta, 2013). Furthermore, in intact CNGA1 channels, homologous to HCN channels, it was shown that stabilization of helix αC promoted channel opening by metal coordination by a pair of histidine residues (Puljung and Zagotta, 2013).…”

Section: Structure Of Hcn2 Cnbd In the Ligand-free Statementioning

confidence: 99%

“…Based on first models (Craven and Zagotta, 2006;Rehmann et al, 2007) and their new data, Zagotta and co-workers proposed a recent model for ligand-induced conformational changes of the CNBD in HCN channels (Taraska et al, 2009;Puljung and Zagotta, 2013): i. In the absence of cyclic nucleotides the channel pore is closed (unbound/closed state) and the αC region adopts a more flexible conformation rather than a stable α-helix.…”

Section: Model For Ligand-induced Conformational Changes Of Cnbds In mentioning

confidence: 99%

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Structural snapshot of cyclic nucleotide binding domains from cyclic nucleotide-sensitive ion channels

Schünke

Stoldt²

2013

Biological Chemistry

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Cyclic nucleotide-binding domains (CNBDs) that are present in various channel proteins play crucial roles in signal amplification cascades. Although atomic resolution structures of some of those CNBDs are available, the detailed mechanism by which they confer cyclic nucleotide-binding to the ion channel pore remains poorly understood. In this review, we describe structural insights about cyclic nucleotide-binding-induced conformational changes in CNBDs and their potential coupling with channel gating.

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Patch Fluorometry

Zheng

Yang

2013

Encyclopedia of Life Sciences

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Patch fluorometry is a biophysical technique that combines the power of patch clamping and optical recording, with the aim of correlating local conformational rearrangements in ion channel protein with the channel gating process. This is achieved through simultaneous recordings of fluorescence and current signals from the same population of ion channels in a membrane patch. The method can be applied to studies of ion channel structure–function relationship as well as membrane protein dynamics. Unlike nuclear magnetic resonance or other biophysical methods for structural study, patch fluorometry provides real‐time dynamic structural information from ion channels in their native membrane environment whereas they carry out their functions. Recent advances greatly expand the scope of this powerful method, allowing researchers to follow structural changes in channel protein with unprecedented spatial and temporal resolutions and accuracy. Key Concepts: Ion channels achieve their cell‐sensor functions through changes in conformation in response to physical or chemical stimuli. Conformational changes in an ion channel protein do not always alter the current, and are thus invisible to patch clamp recording. Fluorescence emission is highly sensitive to the local environment of fluorophores. Site‐directed fluorophores serve as sensors for local structural changes. Patch fluorometry allows direct correlation between channel protein conformational changes and the functional states of the channel.

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A Secondary Structural Transition in the C-helix Promotes Gating of Cyclic Nucleotide-regulated Ion Channels

Cited by 33 publications

References 46 publications

Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

Double electron–electron resonance reveals cAMP-induced conformational change in HCN channels

Structural snapshot of cyclic nucleotide binding domains from cyclic nucleotide-sensitive ion channels

Patch Fluorometry

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