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J Gen Physiol
2018 Jun 04;1506:821-834. doi: 10.1085/jgp.201812023.
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Ligand binding and activation properties of the purified bacterial cyclic nucleotide-gated channel SthK.
Schmidpeter PAM
,
Gao X
,
Uphadyay V
,
Rheinberger J
,
Nimigean CM
.
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Cyclic nucleotide-modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide-gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide-modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels.
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Figure 1. SthK topology and purification details. (A) Cartoon representation of the HCN1 channel structure (Lee and MacKinnon, 2017; Protein Data Bank accession no. 5U6O) as a model to highlight the domain architecture of the homologous SthK channel. One subunit of the channel is shown with the S1–S4 in blue, S5–S6 in purple, the C-linker in yellow, the CNBD in green, and the remaining three subunits in gray. The figure was prepared using PyMol (http://www.pymol.org). (B) Elution profile from size-exclusion chromatography (Superdex 200 10/300 GL) for SthK in the presence (solid line) and absence (dashed line) of cAMP; the inset shows an SDS-PAGE analysis of purified SthK (lane 1) and the results of cross-linking with 0.12% glutaraldehyde, confirming tetrameric assembly of SthK (lane 2). BenchMark Prestained Protein Ladder (Life Technologies) was used to estimate the molecular weight. (C) A representative negative-stain EM micrograph (bar, 141 nm) is shown to illustrate the quality of the final protein sample. A few representative particles used for 2-D classification are highlighted (small box) and enlarged in the inset (top, right; bar, 10 nm). (D) The resulting 2-D classes (box size, 248 Å) from negative-stain images are shown.
Figure 2. Characterization of SthK channel activity. (A) Normalized 86Rb+ flux through SthK (reconstituted in 5:3:2 DOPC:POPG:cardiolipin) in the presence of 200 µM cAMP (red squares), cGMP (blue circles), both cAMP and cGMP (black triangles), and without cyclic nucleotides (open triangles). Flux through empty liposomes (open circles) is shown as a reference. All flux values are normalized to the maximum uptake recorded in the presence of valinomycin. Bars represent means ± SEM for three separate experiments. (B) SthK channel activity represented by the maximum achieved 86Rb+ uptake through SthK reconstituted in liposomes made from 3:1 POPE:POPG (PE:PG) or 5:3:2 DOPC:POPG:cardiolipin (PC:PG:CA), as indicated, in the presence of 200 µM cAMP. Bars represent means ± SEM for three separate experiments. (C) Representative single-channel recordings of SthK in 0.1 mM intracellular cAMP. (D) I-V relationship of SthK single-channel current amplitude at 100 µM cAMP. (E) Open probability of SthK at 100 mV as a function of the cAMP concentration. Fitting the data with Eq. 10 yields an apparent activation constant of EC50 = 17 µM and a Hill coefficient of nH = 3. The number of repeats is indicated beside each data point. (F) Open probability of SthK in the presence of 500 µM cAMP as function of the membrane potential. The line indicates the fit according to Eq. 13, leading to z = 0.8 and Vhalf = 87 mV, Pomax = 0.65, and Pomin= 0.05. Symbols and error bars in D–F represent means ± SEM from at least three separate bilayers.
Figure 3. Kinetics of SthK activation by cAMP and inhibition by cGMP. (A) Fluorescence decay traces as a measure of channel activity in the stopped-flow Tl+ flux assay after incubating the SthK liposomes with 200 µM cAMP for 15 ms (blue), 100 ms (red), and 5 s (green). 0 µM cAMP is in black. The corresponding flux rates (Eq. 9) as a function of activation time are shown in D. (B) Fluorescence decay traces from SthK liposomes incubated for 2.5 s with cAMP over a range of concentrations (0, 25, 50, 100, and 400 µM from top to bottom). The corresponding flux rates as a function of the cAMP concentration are shown in E. (C) Fluorescence decay traces from SthK liposomes incubated with 200 µM cAMP and increasing concentrations of cGMP for 2.5 s (0 µM [green], 25 µM [blue], 100 µM [turquoise], 500 µM [yellow], and 1,000 µM [red]). No cyclic nucleotides (black). The corresponding flux rates as a function of the cGMP concentration are shown in F. (D) The activation time course is best fitted with a sum of two exponentials (line through symbols) with time constants of τ1 ≈ 0.05 s (amplitude 0.45) and τ2 ≈ 1.04 s (amplitude 0.55). (E) A fit with Eq. 10 yields an EC50 of ∼100 μM (line through symbols) and a Hill coefficient of 2.2 ± 0.3. (F) The apparent inhibition constant was determined using Eq. 11 (line through symbols) and yields an IC50 of 40 µM, from which the Ki of 6 µM was calculated (Eq. 12; Table 1). (D–F) Rates were normalized to the maximum value, and symbols and error bars represent means ± SEM for three experiments.
Figure 4. cGMP inhibits cAMP-dependent channel activity. (A) Representative single-channel traces of SthK at 100 mV in the presence of 100 µM cAMP (top trace). Addition of 1 mM cGMP (middle trace) significantly, and reversibly, reduces channel activity. Application of only cGMP (bottom trace) leads to low but nonzero channel activity. (B) Inhibition of preactivated SthK (by 100 µM cAMP) with increasing concentrations of cGMP. All data analyzed are from recordings at 100 mV. Data are fitted using Eq. 11, giving an IC50 of 16 µM. Symbols and error bars represent means ± SEM for three experiments.
Figure 5. Interaction of cAMP and cGMP with SthK. (A) Titration of 0.1 µM fcAMP (closed circles) or fcGMP (open circles) with SthK in A8-35; lines represent fits according to Eq. 4, giving Kd values of 0.4 µM and 2 µM, respectively. The data presented are from a single measurement; averaged values from three separate titrations are listed in Table 1. (B) Representative titration of the SthK · fcAMP complex with increasing concentrations of cAMP (closed circles) or cGMP (open circles). The Kd values for the unlabeled cyclic nucleotides obtained from these titrations according to Eq. 6 are 50 ± 8 µM for cAMP and 19 ± 4 µM for cGMP. Averaged values from three competition experiments are given in Table 1. (C and D) Overlay of the CNBDs of HCN2 (apo [blue], 5JON [Goldschen-Ohm et al., 2016]) and SthK in complex with cAMP (orange, 4D7T [Kesters et al., 2015]) in C, and the same HCN2 and SthK in complex with cGMP (cyan, 4D7S [Kesters et al., 2015]) in D. The figures were prepared using PyMol (http://www.pymol.org). The ligands cAMP (C) and cGMP (D) are shown in stick representation in CPK colors. Only E421 is shown to indicate an interaction with cAMP (C) but not with cGMP (D).
Accili,
From funny current to HCN channels: 20 years of excitation.
2002, Pubmed
Accili,
From funny current to HCN channels: 20 years of excitation.
2002,
Pubmed
Akimoto,
A mechanism for the auto-inhibition of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel opening and its relief by cAMP.
2014,
Pubmed
Altieri,
Structural and energetic analysis of activation by a cyclic nucleotide binding domain.
2008,
Pubmed
Benndorf,
Gating by cyclic GMP and voltage in the alpha subunit of the cyclic GMP-gated channel from rod photoreceptors.
1999,
Pubmed
,
Xenbase
Biel,
Hyperpolarization-activated cation channels: from genes to function.
2009,
Pubmed
Brams,
Family of prokaryote cyclic nucleotide-modulated ion channels.
2014,
Pubmed
,
Xenbase
Brelidze,
Absence of direct cyclic nucleotide modulation of mEAG1 and hERG1 channels revealed with fluorescence and electrophysiological methods.
2009,
Pubmed
Carvalho-de-Souza,
Nonsensing residues in S3-S4 linker's C terminus affect the voltage sensor set point in K+ channels.
2018,
Pubmed
,
Xenbase
Cer,
IC50-to-Ki: a web-based tool for converting IC50 to Ki values for inhibitors of enzyme activity and ligand binding.
2009,
Pubmed
Chen,
Identification and characterization of small molecules as potent and specific EPAC2 antagonists.
2013,
Pubmed
Cheng,
Mechanism for selectivity-inactivation coupling in KcsA potassium channels.
2011,
Pubmed
Chiu,
The structure of the prokaryotic cyclic nucleotide-modulated potassium channel MloK1 at 16 A resolution.
2007,
Pubmed
Clayton,
Structural basis of ligand activation in a cyclic nucleotide regulated potassium channel.
2004,
Pubmed
Craven,
CNG and HCN channels: two peas, one pod.
2006,
Pubmed
Cukkemane,
Cooperative and uncooperative cyclic-nucleotide-gated ion channels.
2011,
Pubmed
Cukkemane,
Subunits act independently in a cyclic nucleotide-activated K(+) channel.
2007,
Pubmed
DiFrancesco,
Characterization of single pacemaker channels in cardiac sino-atrial node cells.
,
Pubmed
Flynn,
Structure and rearrangements in the carboxy-terminal region of SpIH channels.
2007,
Pubmed
,
Xenbase
Gauss,
Molecular identification of a hyperpolarization-activated channel in sea urchin sperm.
1998,
Pubmed
Goldschen-Ohm,
Structure and dynamics underlying elementary ligand binding events in human pacemaking channels.
2016,
Pubmed
Gordon,
Localization of regions affecting an allosteric transition in cyclic nucleotide-activated channels.
1995,
Pubmed
,
Xenbase
Gordon,
Altered ligand specificity by protonation in the ligand binding domain of cyclic nucleotide-gated channels.
1996,
Pubmed
,
Xenbase
Heginbotham,
Functional reconstitution of a prokaryotic K+ channel.
1998,
Pubmed
Hite,
Structural Titration of Slo2.2, a Na+-Dependent K+ Channel.
2017,
Pubmed
Ingólfsson,
Screening for small molecules' bilayer-modifying potential using a gramicidin-based fluorescence assay.
2010,
Pubmed
James,
Structural insights into the mechanisms of CNBD channel function.
2018,
Pubmed
James,
CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel.
2017,
Pubmed
Karpen,
Gating kinetics of the cyclic-GMP-activated channel of retinal rods: flash photolysis and voltage-jump studies.
1988,
Pubmed
Kaupp,
Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel.
1989,
Pubmed
,
Xenbase
Kaupp,
Cyclic nucleotide-gated ion channels.
2002,
Pubmed
Kawate,
Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins.
2006,
Pubmed
Kesters,
Structure of the SthK carboxy-terminal region reveals a gating mechanism for cyclic nucleotide-modulated ion channels.
2015,
Pubmed
Kimanius,
Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2.
2016,
Pubmed
Komatsu,
Functional reconstitution of a heteromeric cyclic nucleotide-gated channel of Caenorhabditis elegans in cultured cells.
1999,
Pubmed
Kowal,
High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer.
2018,
Pubmed
Kowal,
Ligand-induced structural changes in the cyclic nucleotide-modulated potassium channel MloK1.
2014,
Pubmed
Kraemer,
Dynamic interaction of cAMP with the Rap guanine-nucleotide exchange factor Epac1.
2001,
Pubmed
Kusch,
Interdependence of receptor activation and ligand binding in HCN2 pacemaker channels.
2010,
Pubmed
,
Xenbase
Lander,
Appion: an integrated, database-driven pipeline to facilitate EM image processing.
2009,
Pubmed
LeMasurier,
KcsA: it's a potassium channel.
2001,
Pubmed
Lee,
Structures of the Human HCN1 Hyperpolarization-Activated Channel.
2017,
Pubmed
Li,
Structure of a eukaryotic cyclic-nucleotide-gated channel.
2017,
Pubmed
Livermore,
Lipids of the Spirochaetales: comparison of the lipids of several members of the genera Spirochaeta, Treponema, and Leptospira.
1974,
Pubmed
Lolicato,
Tetramerization dynamics of C-terminal domain underlies isoform-specific cAMP gating in hyperpolarization-activated cyclic nucleotide-gated channels.
2011,
Pubmed
,
Xenbase
Ludwig,
A family of hyperpolarization-activated mammalian cation channels.
1998,
Pubmed
Nache,
Gating of cyclic nucleotide-gated (CNGA1) channels by cGMP jumps and depolarizing voltage steps.
2006,
Pubmed
,
Xenbase
Nimigean,
A radioactive uptake assay to measure ion transport across ion channel-containing liposomes.
2006,
Pubmed
Ohi,
Negative Staining and Image Classification - Powerful Tools in Modern Electron Microscopy.
2004,
Pubmed
Paoletti,
C-Linker of cyclic nucleotide-gated channels controls coupling of ligand binding to channel gating.
1999,
Pubmed
Posson,
Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies.
2018,
Pubmed
Robinson,
Hyperpolarization-activated cation currents: from molecules to physiological function.
2003,
Pubmed
Rusinova,
Regulation of ion channel function by the host lipid bilayer examined by a stopped-flow spectrofluorometric assay.
2014,
Pubmed
Santoro,
Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain.
1998,
Pubmed
,
Xenbase
Saponaro,
Structural basis for the mutual antagonism of cAMP and TRIP8b in regulating HCN channel function.
2014,
Pubmed
Scheres,
RELION: implementation of a Bayesian approach to cryo-EM structure determination.
2012,
Pubmed
Suloway,
Automated molecular microscopy: the new Leginon system.
2005,
Pubmed
Tang,
Transfer of voltage independence from a rat olfactory channel to the Drosophila ether-à-go-go K+ channel.
1997,
Pubmed
Thompson,
Molecular mechanism of pH sensing in KcsA potassium channels.
2008,
Pubmed
Varnum,
Molecular mechanism for ligand discrimination of cyclic nucleotide-gated channels.
1995,
Pubmed
Vishnuvardhan Reddy,
Spirochaeta sphaeroplastigenens sp. nov., a halo-alkaliphilic, obligately anaerobic spirochaete isolated from soda lake Lonar.
2013,
Pubmed
Voss,
DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy.
2009,
Pubmed
Voss,
A toolbox for ab initio 3-D reconstructions in single-particle electron microscopy.
2010,
Pubmed
Wang,
An exact mathematical expression for describing competitive binding of two different ligands to a protein molecule.
1995,
Pubmed
Wilkinson,
Quantitative analysis of protein-protein interactions.
2004,
Pubmed
Xu,
Structural basis for the cAMP-dependent gating in the human HCN4 channel.
2010,
Pubmed
,
Xenbase
Yellen,
The voltage-gated potassium channels and their relatives.
2002,
Pubmed
Yu,
Overview of the voltage-gated sodium channel family.
2003,
Pubmed
Zagotta,
Structural basis for modulation and agonist specificity of HCN pacemaker channels.
2003,
Pubmed
Zhou,
Gating of HCN channels by cyclic nucleotides: residue contacts that underlie ligand binding, selectivity, and efficacy.
2007,
Pubmed
,
Xenbase